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    UNITED NATIONS ENVIRONMENT PROGRAMME
    INTERNATIONAL LABOUR ORGANISATION
    WORLD HEALTH ORGANIZATION


    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY



    ENVIRONMENTAL HEALTH CRITERIA 188





    Nitrogen Oxides

    (Second Edition)



    This report contains the collective views of an international group of
    experts and does not necessarily represent the decisions or the stated
    policy of the United Nations Environment Programme, the International
    Labour Organisation, or the World Health Organization.


    First draft prepared by Drs J.A. Graham, L.D. Grant, L.J. Folinsbee,
    D.J. Kotchmar and J.H.B. Garner, US Environmental Protection Agency



    Published under the joint sponsorship of the United Nations
    Environment Programme, the International Labour Organisation, and the
    World Health Organization, and produced within the framework of the
    Inter-Organization Programme for the Sound Management of Chemicals.


    World Health Organization
    Geneva, 1997

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    WHO Library Cataloguing in Publication Data

    Nitrogen oxides - 2nd ed.

    (Environmental health criteria ; 188)

    1.Nitrogen dioxide                 2.Nitrogen oxides
    I.Series

    ISBN 92 4 157188 8                 (NLM Classification: WA 754)
    ISSN 0250-863X

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    CONTENTS

    ENVIRONMENTAL HEALTH CRITERIA FOR NITROGEN OXIDES

    Preamble

    1. SUMMARY

         1.1. Nitrogen oxides and related compounds
               1.1.1. Atmospheric transport
               1.1.2. Measurement
               1.1.3. Exposure
         1.2. Effects of atmospheric nitrogen species, particularly
               nitrogen oxides, on vegetation
         1.3. Health effects of exposures to nitrogen dioxide
               1.3.1. Studies of the effects of nitrogen compounds on
                       experimental animals
                       1.3.1.1    Biochemical and cellular mechanisms of
                                  action of nitrogen oxides
                       1.3.1.2    Effects on host defence
                       1.3.1.3    Effects of chronic exposure on the
                                  development of chronic lung disease
                       1.3.1.4    Potential carcinogenic or co-carcinogenic
                                  effects
                       1.3.1.5    Age susceptibility
                       1.3.1.6    Influence of exposure patterns
               1.3.2. Controlled human exposure studies on nitrogen
                       oxides
               1.3.3. Epidemiology studies on nitrogen dioxide
               1.3.4. Health-based guidance values for nitrogen dioxide

    2. PHYSICAL AND CHEMICAL PROPERTIES, AIR SAMPLING AND ANALYSIS,
         TRANSFORMATIONS AND TRANSPORT IN THE ATMOSPHERE

         2.1. Introduction
               2.1.1. The nomenclature and measurement of atmospheric
                       nitrogen species
         2.2. Nitrogen species and their physical and chemical properties
               2.2.1. Nitrogen oxides
                       2.2.1.1    Nitric oxide
                       2.2.1.2    Nitrogen dioxide
                       2.2.1.3    Nitrous oxide
                       2.2.1.4    Other nitrogen oxides
               2.2.2. Nitrogen acids
                       2.2.2.1    Nitric acid
                       2.2.2.2    Nitrous acid
               2.2.3. Ammonia
               2.2.4. Ammonium nitrate
               2.2.5. Peroxyacetyl nitrate
               2.2.6. Organic nitrites and nitrates

         2.3. Sampling and analysis methods
               2.3.1. Nitric oxide
                       2.3.1.1    Nitric oxide continuous methods
                       2.3.1.2    Passive samplers for NO
                       2.3.1.3    Calibration of NO analysis methods
                       2.3.1.4    Sampling considerations for NO
               2.3.2. Nitrogen dioxide
                       2.3.2.1    Chemiluminescence (NO + O3)
                       2.3.2.2    Chemiluminescence (luminol)
                       2.3.2.3    Laser-induced fluorescence and tuneable
                                  diode laser absorption spectrometry
                       2.3.2.4    Wet chemical methods
                       2.3.2.5    Other methods
                       2.3.2.6    Passive samplers
                       2.3.2.7    Calibration
               2.3.3. Total reactive odd nitrogen
               2.3.4. Peroxyacetyl nitrate
               2.3.5. Other organic nitrates
               2.3.6. Nitric acid
               2.3.7. Nitrous acid
               2.3.8. Dinitrogen pentoxide and nitrate radicals
               2.3.9. Particulate nitrate
               2.3.10. Nitrous oxide
               2.3.11. Summary
         2.4. Transport and transformation of nitrogen oxides in the air
               2.4.1. Introduction
               2.4.2. Chemical transformations of oxides of nitrogen
                       2.4.2.1    Nitric oxide, nitrogen dioxide and ozone
                       2.4.2.2    Transformations in indoor air
                       2.4.2.3    Formation of other oxidized nitrogen
                                  species
               2.4.3. Advection and dispersion of atmospheric nitrogen
                       species
                       2.4.3.1    Transport of reactive nitrogen species
                                  in urban plumes
                       2.4.3.2    Air quality models
                       2.4.3.3    Regional transport
         2.5. Conversion factor for nitrogen dioxide
         2.6. Summary

    3. SOURCES, EMISSIONS AND AIR CONCENTRATIONS

         3.1. Introduction
         3.2. Sources of nitrogen oxides
               3.2.1. Sources of NOx emission
                       3.2.1.1    Fuel combustion
                       3.2.1.2    Biomass burning
                       3.2.1.3    Lightning
                       3.2.1.4    Soils
                       3.2.1.5    Oceans

               3.2.2. Removal from the ambient environment
               3.2.3. Summary of global budgets for nitrogen oxides
         3.3. Ambient concentrations of nitrogen oxides
               3.3.1. International comparison studies of NOx
                       concentrations
               3.3.2. Example case studies of NOx and NO2
                       concentrations
         3.4. Occurrence of nitrogen oxides indoors
               3.4.1. Indoor sources
                       3.4.1.1    Gas-fuelled cooking stoves
                       3.4.1.2    Unvented gas space heaters and water
                                  heaters
                       3.4.1.3    Kerosene space heaters
                       3.4.1.4    Wood stoves
                       3.4.1.5    Tobacco products
               3.4.2. Removal of nitrogen oxides from indoor environments
         3.5. Indoor concentrations of nitrogen oxides
               3.5.1. Homes without indoor combustion sources
               3.5.2. Homes with combustion appliances
               3.5.3. Homes with combustion space heaters
               3.5.4. Indoor nitrous acid concentrations
               3.5.5. Predictive models for indoor NO2 concentration
         3.6. Human exposure
         3.7. Exposure of plants and ecosystems

    4. EFFECTS OF ATMOSPHERIC NITROGEN COMPOUNDS (PARTICULARLY NITROGEN
         OXIDES) ON PLANTS

         4.1. Properties of NOx and NHy
               4.1.1. Adsorption and uptake
               4.1.2. Toxicity, detoxification and assimilation
               4.1.3. Physiology and growth aspects
               4.1.4. Interactions with climatic conditions
               4.1.5. Interactions with the habitat
               4.1.6. Increasing pest incidence
               4.1.7. Conclusions for various atmospheric nitrogen
                       species and mixtures
                       4.1.7.1    NO2
                       4.1.7.2    NO
                       4.1.7.3    NH3
                       4.1.7.4    NH4+ and NO3- in wet and occult
                                  deposition
                       4.1.7.5    Mixtures
               4.1.8. Appraisal
                       4.1.8.1    Representativity of the data
               4.1.9. General conclusions
         4.2. Effects on natural and semi-natural ecosystems
               4.2.1. Effects on freshwater and intertidal ecosystems
                       4.2.1.1    Effects of nitrogen deposition on
                                  shallow softwater lakes

                       4.2.1.2    Effects of nitrogen deposition on lakes
                                  and streams
               4.2.2. Effects on ombrotrophic bogs and wetlands
                       4.2.2.1    Effects on ombrotrophic (raised) bogs
                       4.2.2.2    Effects on mesotrophic fens
                       4.2.2.3    Effects on fresh- and saltwater marshes
               4.2.3. Effects on species-rich grasslands
                       4.2.3.1    Effects of nitrogen on calcareous
                                  grasslands
                       4.2.3.2    Critical loads for nitrogen in
                                  calcareous grasslands
                       4.2.3.3    Comparison with other semi-natural
                                  grasslands
               4.2.4. Effects on heathlands
                       4.2.4.1    Effects on inland dry heathlands
                       4.2.4.2    Effects of nitrogen on inland wet
                                  heathlands
                       4.2.4.3    Effects of nitrogen on arctic and alpine
                                  healthlands
                       4.2.4.4    Effects on herbs of matgrass swards
               4.2.5. Effects of nitrogen deposition on forests
                       4.2.5.1    Effects on forest tree species
                       4.2.5.2    Effects on tree epiphytes, ground
                                  vegetation and ground fauna of forests
               4.2.6. Effects on estuarine and marine ecosystems
               4.2.7. Appraisal and conclusions

    5. STUDIES OF THE EFFECTS OF NITROGEN OXIDES ON EXPERIMENTAL ANIMALS

         5.1. Introduction
         5.2. Nitrogen dioxide
               5.2.1. Dosimetry
                       5.2.1.1    Respiratory tract dosimetry
                       5.2.1.2    Systemic dosimetry
               5.2.2. Respiratory tract effects
                       5.2.2.1    Host defence mechanisms
                       5.2.2.2    Lung biochemistry
                       5.2.2.3    Pulmonary function
                       5.2.2.4    Morphological studies
               5.2.3. Genotoxicity, potential carcinogenic or
                       co-carcinogenic effects
               5.2.4. Extrapulmonary effects
         5.3. Effects of mixtures containing nitrogen dioxide
         5.4. Effects of other nitrogen oxide compounds
               5.4.1. Nitric oxide
                       5.4.1.1    Endogenous formation of NO
                       5.4.1.2    Absorption of NO
                       5.4.1.3    Effects of NO on pulmonary function,
                                  morphology and host lung defence
                                  function

                       5.4.1.4    Metabolic effects
                       5.4.1.5    Haematological changes
                       5.4.1.6    Biochemical mechanisms for nitric oxide
                                  effects: reaction with iron and effects
                                  on enzymes and nucleic acids
               5.4.2. Nitric acid
               5.4.3. Nitrates
         5.5. Summary of studies of the effects of nitrogen compounds on
               experimental animals

    6. CONTROLLED HUMAN EXPOSURE STUDIES OF NITROGEN OXIDES

         6.1. Introduction
         6.2. Effects of nitrogen dioxide
               6.2.1. Nitrogen dioxide effects on pulmonary function and
                       airway responsiveness to bronchoconstrictive agents
                       6.2.1.1    Nitrogen dioxide effects in healthy
                                  subjects
                       6.2.1.2    Nitrogen dioxide effects on asthmatics
                       6.2.1.3    Nitrogen dioxide effects on patients
                                  with chronic obstructive pulmonary
                                  disease
                       6.2.1.4    Age-related differential susceptibility
               6.2.2. Nitrogen dioxide effects on pulmonary host defences
                       and bronchoalveolar lavage fluid biomarkers
               6.2.3. Other classes of nitrogen dioxide effects
         6.3. Effects of other nitrogen oxide compounds
         6.4. Effects of nitrogen dioxide/gas or gas/aerosol mixtures on
               lung function
         6.5. Summary of controlled human exposure studies of oxides of
               nitrogen

    7. EPIDEMIOLOGICAL STUDIES OF NITROGEN OXIDES

         7.1. Introduction
         7.2. Methodological considerations
               7.2.1. Measurement error
               7.2.2. Misclassification of the health outcome
               7.2.3. Adjustment for covariates
               7.2.4. Selection bias
               7.2.5. Internal consistency
               7.2.6. Plausibility of the effect
         7.3. Studies of respiratory illness
               7.3.1. Indoor air studies
                       7.3.1.1    St Thomas' Hospital Medical School
                                  Studies (United Kingdom)
                       7.3.1.2    Harvard University - Six Cities Studies
                                  (USA)
                       7.3.1.3    University of Iowa Study (USA)

                       7.3.1.4    Agricultural University of Wageningen
                                  (The Netherlands)
                       7.3.1.5    Ohio State University Study (USA)
                       7.3.1.6    University of Dundee (United Kingdom)
                       7.3.1.7    Harvard University - Chestnut Ridge
                                  Study (USA)
                       7.3.1.8    University of New Mexico Study (USA)
                       7.3.1.9    University of Basel Study (Switzerland)
                       7.3.1.10   Yale University Study (USA)
                       7.3.1.11   Freiburg University Study (Germany)
                       7.3.1.12   McGill University Study (Canada)
                       7.3.1.13   Health and Welfare Canada Study (Canada)
                       7.3.1.14   University of North Carolina Study (USA)
                       7.3.1.15   University of Tucson Study (USA)
                       7.3.1.16   Hong Kong Anti-Cancer Society Study
                                  (Hong Kong)
                       7.3.1.17   Recent studies
               7.3.2. Outdoor studies
                       7.3.2.1    Harvard University - Six City Studies
                                  (USA)
                       7.3.2.2    University of Basel Study (Switzerland)
                       7.3.2.3    University of Wuppertal Studies
                                  (Germany)
                       7.3.2.4    University of Tubigen (Germany)
                       7.3.2.5    Harvard University - Chestnut Ridge
                                  Study (USA)
                       7.3.2.6    University of Helsinki Studies (Finland)
                       7.3.2.7    Helsinki City Health Department Study
                                  (Finland)
                       7.3.2.8    Oulu University Study (Finland)
                       7.3.2.9    Seth GS Medical College Study (India)
         7.4. Pulmonary function studies
               7.4.1. Harvard University - Six City Studies (USA)
               7.4.2. National Health and Nutrition Examination Survey
                       Study (USA)
               7.4.3. Harvard University - Chestnut Ridge Study (USA)
               7.4.4. Other pulmonary function studies
         7.5. Other exposure settings
               7.5.1. Skating rink exposures
         7.6. Occupational exposures
         7.7. Synthesis of the evidence for school-age children
               7.7.1. Health outcome measures
               7.7.2. Biologically plausible hypothesis
               7.7.3. Publication bias
               7.7.4. Selection of studies
                       7.7.4.1    Brief description of selected studies
                       7.7.4.2    Studies not selected for quantitative
                                  analysis
               7.7.5. Quantitative analysis

         7.8. Synthesis of the evidence for young children
         7.9. Summary

    8. EVALUATION OF HEALTH AND ENVIRONMENT RISKS ASSOCIATED WITH
         NITROGEN OXIDES

         8.1. Sources and exposure
         8.2. Evaluation of the effects of atmospheric nitrogen species
               on the environment
               8.2.1. Guidance values - critical levels for air
                       concentrations of nitrogen oxides
               8.2.2. Environment-based guidance values - critical loads
                       for total nitrogen deposition
         8.3. Evaluation of health risks associated with nitrogen oxides
               8.3.1. Concentration-response relationships
               8.3.2. Subpopulations potentially at risk
               8.3.3. Derivation of health-based guidance values

    9. CONCLUSIONS AND RECOMMENDATIONS FOR PROTECTION OF HUMAN HEALTH
         AND THE ENVIRONMENT

    10. FURTHER RESEARCH

    REFERENCES

    RESUME

    RESUMEN
    

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    FIGURE 1

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    WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR NITROGEN OXIDES

     Members 

    Dr K. Bentley*, Health and Environment Policy Section, Department
         of Community Services and Health, Canberra ACT, Australia

    Dr S. Dobson, Institute of Terrestrial Ecology, Monks Wood
         Experimental Station, Abbots Ripton, Huntingdon, Cambridgeshire,
         United Kingdom

    Dr L. van der Eerden, Centre "De Bom"  Wageningen, The Netherlands

    Dr L. Folinsbee, Health Effects Research Laboratory, US Environmental
         Protection Agency, Research Triangle Park, North Carolina, USA
          (Rapporteur)

    Dr L. Grant*, National Center for Environmental Assessment, US
         Environmental Protection Agency, Research Triangle Park, North
         Carolina, USA

    Mr L. Heiskanen, Health and Environment Policy Section, Department of
         Community Services and Health, Canberra ACT, Australia

    Mr G.M. Johnson, CSIRO, Division of Coal and Energy Technology, Centre
         for Pollution Assessment and Control, North Ryde, NSW, Australia

    Dr J. Kagawa, Professor of Hygiene and Public Health, Tokyo Women's
         Medical College, Shinjuku-ku, Tokyo, Japan

    Dr R.R. Khan, Ministry of Environment and Forests, Paryavaran Bhawan,
         New Delhi, India

    Dr D.B. Menzel, University of California, Department of Community &
         Environment and Medicine, California, USA

    Dr L. Neas, Department of Environmental Health, Environmental
         Epidemiology Program, Harvard School of Public Health, Boston,
         Massachusetts, USA

    Dr S.E. Paulson, Department of Atmospheric Sciences, University of
         California, Los Angeles, California, USA

    Dr P.J.A. Rombout, Department for Inhalation Toxicology, National
         Institute of Public Health and Environmental Hygiene, Bilthoven,
         The Netherlands  (Chairman)

               

    *  Invited, but unable to attend

    Dr W. Tyler, Veterinary Anatomy and Cell Biology, University of
         California, California, USA

    Dr K. Victorin, Karolinska Institute, Institute of Environmental
         Medicine, Stockholm, Sweden

    Dr A. Woodward, Department of Community Medicine, University of
         Adelaide, Adelaide, Australia

    Dr R. Ye, Deputy Director, National Environmental Protection Agency,
         Xizhimennei Nanziaojie, Beijing, People's Republic of China

     Observers

    Professor M. Moore, National Research Centre for Environmental
         Toxicology, Nathan, Australia

    Dr M. Pain, Department of Thoracic Medicine, Royal Melbourne Hospital,
         Melbourne VIC, Australia

    Dr P. Psaila-Savona, WA Department of Health, Perth WA, Australia

    Mr B. Taylor, Policy and Planning Group, Public and Planning Group,
         Public Health Commission, Wellington, New Zealand

    Mr B. Saxby, AGL Gas Companies, North Sydney NSW, New  Zealand

     Secretariat

    Dr B.H. Chen, International Programme on Chemical Safety, World Health
         Organization, Geneva, Switzerland  (Secretary)

    Dr M. Younes, WHO European Centre for Environment & Health, Bilthoven,
         The Netherlands

    ENVIRONMENTAL HEALTH CRITERIA FOR NITROGEN OXIDES

         A WHO Task Group on Environmental Health Criteria for Nitrogen
    Oxides met in Melbourne, Australia from 14 to 18 November 1994.  The
    meeting was hosted by the Clean Air Society of Australia and New
    Zealand and the Victorian Departments of Health and Environment,
    Australia.  Dr B.H. Chen, IPCS, opened the meeting and welcomed the
    participants on behalf of the Director, IPCS, and the three IPCS
    cooperating organizations (UNEP/ILO/WHO).  The Task Group reviewed and
    revised the draft criteria monograph and made an evaluation of the
    risks for human health and the environment from exposure to nitrogen
    oxides. 

         The first draft of this monograph was prepared by Drs J.A.
    Graham, L.D. Grant, L.J. Folinsbee, D.J. Kotchmar and J.H.B. Garner,
    US EPA.  Drs W.G. Ewald, T.B. McMullen and B.E. Tilton, US EPA,
    contributed to the preparation of the first draft.  The second draft
    was prepared by Dr L.D. Grant incorporating comments received
    following the circulation of the first draft to the IPCS Contact
    Points for Environmental Health Criteria.  Drs R. Bobbink, L. Van der
    Eerden and S. Dobson prepared the final text of the environmental
    section.  Mr G.M. Johnson contributed to the final text of the
    chemistry section.

         Dr B.H. Chen and Dr P.G. Jenkins, both members of the IPCS
    Central Unit, were responsible for the overall scientific content and
    technical editing, respectively.

         The efforts of all who helped in the preparation and finalization
    of the document are gratefully acknowledged.

         Financial support for this Task Group meeting was provided by the
    Department of Community Services and Health, Australia, Victorian
    Departments of Health and Environment, Australia, and the Clean Air
    Society of Australia and New Zealand.

    ABBREVIATIONS

    ADP       adenosine diphosphate
    AM        alveolar macrophages
    AQG       Air Quality Guidelines
    BAL       bronchoalveolar lavage
    BHPN       N-bis (2-hydroxypropyl) nitrosamine
    CI        confidence interval
    CLM       chemiluminescence method
    COPD      chronic obstructive pulmonary disease
    ECD       electron capture detection
    FEF       forced expiratory flow
    FEV       forced expiratory volume
    FTIR      Fourier transformed infrared
    FVC       forced vital capacity
    GC        gas chromatography
    GDH       glutamate dehydrogenase
    (c)GMP    (cyclic) guanosine monophosphate
    GS        glutamine synthetase
    HNO2      nitrous acid
    HNO3      nitric acid
    LIF       laser-induced fluorescence
    MS        mass spectrometry
    N2        nitrogen (elemental)
    NH3       ammonia
    NH4+      ammonium ion
    NHy       the sum of NH3 and NH4+
    NiR       nitrate reductase
    NK        natural killer
    NO        nitric oxide
    NO2       nitrogen dioxide
    NO2-      nitrite ion
    NO3-      nitrate ion
    N2O       nitrous oxide
    N2O5      nitrogen pentoxide
    NOx       nitric oxide plus nitrogen dioxide
    NOy       gas-phase oxidized nitrogen species (except nitrous oxide)
    NPSH      non-protein sulfhydryl
    NR        nitrate reductase
    O3        ozone
    PAN       peroxyacetyl nitrate
    PBzN      peroxybenzoyl nitrate
    PEF       peak expiratory flow
    PFC       plaque-forming cell
    PMN       polymorphonuclear leukocyte
    ppb       parts per billion (10-9)
    ppm       parts per million (10-6)
    ppt       parts per trillion (10-12)
    pptv      parts per trillion (by volume)
    PSD       passive sampling device

    Raw       airway resistance
    ROC       reactive organic carbon
    RUBISCO   ribulose 1,5-biphosphate carboxylase
    SD        standard deviation
    SES       socioeconomic status
    SGaw      specific airway conductance
    SO2       sulfur dioxide
    SOy       sulfur oxides
    SPM       suspended particulate matter
    SRaw      specific airway resistance
    TDLAS     tuneable diode laser absorption spectrometry
    TSP       total suspended particulate
    VOC       volatile organic carbon

    1.  SUMMARY

    1.1  Nitrogen oxides and related compounds

         Nitrogen oxides can be present at significant concentrations in
    ambient air and in indoor air.  The types and concentrations of
    nitrogenous compounds present can vary greatly from location to
    location, with time of day, and with season.  The main sources of
    nitrogen oxide emissions are combustion processes.  Fossil fuel power
    stations, motor vehicles and domestic combustion appliances emit
    nitrogen oxides, mostly in the form of nitric oxide (NO) and some
    (usually less than about 10%) in the form of nitrogen dioxide (NO2). 
    In the air, chemical reactions occur that oxidize NO to NO2 and other
    products. There are also biological processes that liberate nitrogen
    species from soils, including nitrous oxide (N2O).  Emissions of N2O
    can cause perturbation of the stratospheric ozone layer.

         Human health may be affected when significant concentrations of
    NO2 or other nitrogenous species, such as peroxyacetyl nitrate (PAN),
    nitric acid (HNO3), nitrous acid (HNO2), and nitrated organic
    compounds, are present.  In addition, nitrates and HNO3 may cause
    health effects and significant effects on ecosystems when deposited on
    the ground.

         The sum of NO and NO2 is generally referred to as NOx.  Once
    released into the air, NO is oxidized to NO2 by available oxidants
    (particularly ozone, O3).  This happens rapidly under some conditions
    in outdoor air; in indoor air, it is generally a much slower process.
    Nitrogen oxides are a controlling precursor of photochemical oxidant
    air pollution resulting in ozone and smog formation; interactions of
    nitrogen oxides (except N2O) with reactive organic compounds and
    sunlight form ozone in the troposphere and smog in urban areas.

         NO and NO2 may also undergo reactions to form a range of other
    oxides of nitrogen, both in indoor and outdoor air, including HNO2,
    HNO3, nitrogen trioxide (NO3), dinitrogen pentoxide (N2O5), PAN
    and other organic nitrates.  The complex range of gas-phase nitrogen
    oxides is referred to as NOy.  The partitioning of oxides of nitrogen
    among these compounds is strongly dependent on the concentrations of
    other oxidants and on the meteorological history of the air.

         HNO3 is formed from the reaction of OH- and NO2.  It is a
    major sink for active nitrogen and also a contributor to acidic
    deposition.  Potential physical and chemical sinks for HNO3 include
    wet and dry deposition, photolysis, reaction with OH radicals, and
    reaction with gaseous ammonia to form ammonium nitrate aerosol.

         PANs are formed from the combination of organic peroxy radicals
    with NO2.  PAN is the most abundant organic nitrate in the
    troposphere and can serve as a temporary reservoir for reactive
    nitrogen, which may be regionally transported.

         The NO3 radical, a short-lived NOy species that is formed in
    the troposphere primarily by the reaction of NO2 with O3, undergoes
    rapid photolysis in daylight or reaction with NO.  Appreciable
    concentrations are observed during the night.

         N2O5 is primarily a night-time constituent of ambient air as it
    is formed from the reaction of NO3 and NO2.  In ambient air, N2O5
    reacts heterogeneously with water to form HNO3, which in turn is
    deposited.

         N2O is ubiquitous because it is a product of natural biological
    processes in soil.  It is not known, however, to be involved in any
    reactions in the troposphere.  N2O participates in upper atmospheric
    reactions contributing to stratospheric ozone (O3) depletion and is
    also a relatively potent greenhouse gas that contributes to global
    warming.

    1.1.1  Atmospheric transport

         The transport and dispersion of the various nitrogenous
    species in the lower troposphere is dependent on both meteorological
    and chemical parameters.  Advection, diffusion and chemical
    transformations combine to dictate the atmospheric residence times. 
    In turn, atmospheric residence times help determine the geographic
    extent of transport of given species.  Surface emissions are dispersed
    vertically and horizontally through the atmosphere by turbulent mixing
    processes that are dependent to a large extent on the vertical
    temperature structure and wind speed.

         As the result of meteorological processes, NOx emitted in the
    early morning hours in an urban area typically disperses vertically
    and moves downwind as the day progresses.  On sunny summer days, most
    of the NOx will have been converted to HNO3 and PAN by sunset, with
    concomitant formation of ozone.  Much of the HNO3 is removed by
    deposition as the air mass is transported, but HNO3 and PAN carried
    in layers aloft (above the nighttime inversion layer but below a
    higher subsidence inversion) can potentially be transported long
    distances in oxidant-laden air masses.

    1.1.2  Measurement

         There are a number of methods available to measure airborne
    nitrogen-containing species.  This document briefly covers
    methodologies currently available or in general use for  in situ

    monitoring of airborne concentrations in both ambient and indoor
    environments.  The species considered are NO, NO2, NOx, total
    reactive odd nitrogen (NOy), PAN and other organic nitrates, HNO3,
    HNO2, N2O5, the nitrate radical, NO3-, and N2O.

         Measuring concentrations of nitrogen oxides is not trivial. 
    While a straightforward, widely available method exists for measuring
    NO (the chemiluminescent reaction with ozone), this is an exception
    for nitrogen oxides.  Chemiluminescence is also the most common
    technique used for NO2; NO2 is first reduced to NO.  Unfortunately,
    the catalyst typically used for the reduction is not specific, and has
    various conversion efficiencies for other oxidized nitrogen compounds. 
    For this reason, great care must be taken in interpreting the results
    of the common chemiluminescence analyser in terms of NO2, as the
    signal may include many other compounds.  Additional difficulties
    arise from nitrogen oxides that may partition between the gaseous and
    particulate phases both in the atmosphere and in the sampling
    procedure.

    1.1.3  Exposure

         Human and environmental exposure to nitrogen oxides varies
    greatly from indoors to outdoors, from cities to the countryside, and
    with time of day and season.  The concentrations of NO and NO2
    typically present outdoors in a range of urban situations are
    relatively well established. The concentrations encountered indoors
    depend on the specific details of the nature of combustion appliances,
    chimneys and ventilation.  When unvented combustion appliances are
    used for cooking or heating, indoor concentrations of nitrogen oxides
    typically greatly exceed those existing outside.  Recent research has
    shown in these circumstances that HNO2 can reach significant
    concentrations. One report showed that HNO2 can represent over 10% of
    the concentrations usually reported as NO2.

    1.2  Effects of atmospheric nitrogen species, particularly nitrogen
         oxides, on vegetation

         Most of earth's biodiversity is found in (semi-)natural
    ecosystems, both in aquatic and terrestrial habitats.  Nitrogen is the
    limiting nutrient for plant growth in many (semi-)natural ecosystems. 
    Most of the plant species from these habitats are adapted to nutrient-
    poor conditions, and can only compete successfully on soils with low
    nitrogen levels.

         Human activities, both industrial and agricultural, have greatly
    increased the amount of biologically available nitrogen compounds,
    thereby disturbing the natural nitrogen cycle.  Various forms of
    nitrogen pollute the air: mainly NO, NO2 and ammonia (NH3) as dry
    deposition; and nitrate (NO3-) and ammonium (NH4+) as wet
    deposition.  NHy refers to the sum of NH3 and NH4+.  Another

    contribution is from occult deposition (fog and clouds).  There are
    many more nitrogen-containing air pollutants (e.g., N2O5, PAN, N2O,
    amines), but these are neglected here, either because their
    contribution to the total nitrogen deposition is believed to be small,
    or because their concentrations are probably far below effect
    thresholds.

         Nitrogen-containing air pollutants can affect vegetation
    indirectly, via photochemical reaction products, or directly after
    being deposited on vegetation, soil or water surface.  The  indirect
    pathway is largely neglected here although it includes very relevant
    processes, and should be taken into account when evaluating the entire
    impact of nitrogen-containing air pollutants: NO2 is a precursor for
    tropospheric O3, which acts both as a phytotoxin and a greenhouse
    gas.

         The impacts of increased nitrogen deposition upon biological
    systems can be the result of direct uptake by foliage or uptake via
    the soil.  At the level of individual plants, the most relevant
    effects are injury to the tissue, changes in biomass production and
    increased susceptibility to secondary stress factors.  At the
    vegetation level, deposited nitrogen acts as a nutrient; this results
    in changes in competitive relationships between species and loss of
    biodiversity.  The critical loads for nitrogen depend on (i) the type
    of ecosystem; (ii) the land use and management in the past and
    present; and (iii) the abiotic conditions (especially those that
    influence the nitrification potential and immobilization rate in the
    soil).

         Adsorption on the outer surface of the leaves takes place and may
    damage wax layers of the cuticle, but the quantitative relevance for
    the field situation has not yet been proved.  Uptake of NOx and NH3
    is driven by the concentration gradient between atmosphere and
    mesophyll.  It generally, but not always, is directly determined by
    stomatal conductance and thus depends on factors influencing stomatal
    aperture.  There is increasing evidence that foliar uptake of nitrogen
    reduces the uptake of nitrogen by the roots.  Uptake and exchange of
    ions through the leaf surface is a relatively slow process, and thus
    is only relevant if the surface remains wet for longer periods.

         NO is only slightly soluble in water, but the presence of other
    substances can alter the solubility.  NO2 has a higher solubility,
    while that of NH3 is much higher.  NO2- (the primary reaction
    product of NOx), NH3 and NH4+ are all highly phytotoxic, and could
    well be the cause of adverse effects of nitrogen-containing air
    pollutants.  The free radical *N=O may play a role in the phytotoxicity
    of NO.

         More-than-additive effects (synergism) have been found in nearly
    all studies concerning SO2 plus NO2.  With other NO2 mixtures (NO,
    O3 and CO2), interactive effects are the exception rather than the
    rule.

         When climatic conditions and supply of other nutrients allow
    biomass production, both NOx and NHy result in growth stimulation at
    low concentrations and growth reduction at higher concentrations. 
    However, the exposure level at which growth stimulation turns into
    growth inhibition is much lower for NOx than for NHy.

         Evidence exists that plants are more sensitive at low light
    intensity (e.g., at night and in winter) and at low temperatures (just
    above 0C).  NOx and NHy can increase the sensitivity of plants to
    frost, drought, wind and insect damage.

         An interaction exists between soil chemistry and sensitivity of
    vegetation to nitrogen deposition; this is related to pH and nitrogen
    availability.

         The relative contribution of NO and NO2 to the NOx effect on
    plants is unclear.  The vast majority of information is on effects of
    NO2 but available information on NO suggests that NO and NO2 have
    comparable phytotoxic effects.

         Air quality guidelines refer to thresholds for adverse effects. 
    Two different types of effect thresholds exist: critical levels (CLEs)
    and critical loads (CLOs).  The critical level is defined as the
    concentration in the atmosphere above which direct adverse effects on
    receptors, such as plants, ecosystems or materials, may occur
    according to present knowledge.  The critical load is defined as a
    quantitative estimate of an exposure (deposition) to one or more
    pollutants below which significant harmful effects on specified
    sensitive elements of the environment do not occur according to
    present knowledge.

         According to current practice, critical levels have been derived
    from assessment of the lowest exposure concentrations causing adverse
    effects on physiology or growth of plants (biochemical effects were
    excluded), using a graphical method.

         To include the impact of NO, a critical level for NOx is
    proposed instead of one for NO2; for this purpose it has been assumed
    that NO and NO2 act in an additive manner.  A strong case can be made
    for the provision of critical levels for short-term exposure. However,
    currently there are insufficient data to provide these with sufficient
    confidence.  Current evidence suggests a critical level of about
    75 g/m3 for NOx as a 24-h mean.

         The critical level for NOx (NO and NO2 added in ppb and
    expressed as NO2 in g/m3) is considered to be 30 g/m3 as an
    annual mean.

         Information on organisms in the environment is almost exclusively
    restricted to plants, with minimum data on soil fauna.  This
    evaluation and guidance values are, therefore, expressed in terms of
    nitrogen species effects on vegetation.  However, it is expected that
    plants will form the most sensitive component of natural systems and
    that the effect on biodiversity of plant communities is a sensitive
    indicator of effects on the whole ecosystem.

         Critical loads are derived from empirical data and steady-state
    soil models.  Estimated critical loads for total nitrogen deposition
    in a variety of natural aquatic and terrestrial ecosystems are given. 
    Possible differential effects of deposited nitrogen species (NOx and
    NHy) are insufficiently known to differentiate between nitrogen
    species for critical load estimation.

         The great majority of ecosystems for which there is sufficient
    information to estimate critical loads are from temperate climates. 
    The few arctic and montane ecosystems included, which might be
    expected to be representative of higher latitudes, have the least
    reliable basis.  There is no information on tropical ecosystems and
    little on estuarine or marine ecosystems in any climatic zone. 
    Nutrient-poor tropical ecosystems such as rain forests and mangrove
    swamps are likely to be adversely affected by nitrogen deposition. 
    The lack of both deposition data and effect thresholds make it
    impossible to make risk assessments for these climatic regions.

         The most sensitive ecosystems (ombrotrophic bogs, shallow soft-
    water lakes and arctic and alpine heaths) for which effects thresholds
    can be estimated show critical loads of 5-10 kg N.ha-1.year-1 based
    on decreased biological diversity in plant communities.  A more
    average value for the limited range of ecosystems studied is 15-20 kg
    N.ha-1.year-1, which applies to forest trees.

         The atmospheric chemistry of nitrogen oxides includes the
    capacity for ozone generation in the troposphere, ozone depletion in
    the stratosphere, and contribution to global warming as greenhouse
    gases.  Nitrogen oxides and ammonia contribute to soil acidification
    (along with sulfur oxides) and thereby to increased bioavailability of
    aluminium.

         The phytotoxic effects of nitrogen oxides on plants have little
    direct relevance to crop plants when concentrations marginally exceed
    the critical level. However, the role of NOx in the generation of
    ozone and other phytotoxic substances, e.g., organic nitrates leads to

    crop loss. Nitrogen deposited on growing crops will represent a very
    small increase in total available nitrogen compared to that added as
    fertilizer.

    1.3  Health effects of exposures to nitrogen dioxide

         A large number of studies designed to evaluate the health effects
    of NOx have been conducted.  Of the NOx compounds, NO2 has been
    most studied.  The discussion in this section focuses on NO2, NO,
    HNO2 and HNO3, while nitrates are mentioned briefly.

    1.3.1  Studies of the effects of nitrogen compounds on experimental
           animals

         Extrapolating animal data to humans has both qualitative and
    quantitative components.  As summarized below, NO2 causes a
    constellation of effects in several animal species; most notably,
    effects on host defence against infectious pulmonary disease, lung
    metabolism/biochemistry, lung function and lung structure.  Because of
    basic physiological, metabolic and structural similarities in all
    mammals (laboratory animals and humans), the commonality of the
    observations in several animal species leads to a reasonable
    conclusion that NO2 could cause similar types of effects in humans. 
    However, because of the differences between mammalian species, exactly
    what exposures would actually cause these effects in humans is not yet
    known.  That is the topic of quantitative extrapolation.  Limited
    modelling research on the dosimetric aspect (i.e., the dose to the
    target tissue/cell that actually causes toxicity) of quantitative
    extrapolation suggests that the distribution of the deposition of NO2
    within the respiratory tract of animals and humans is similar,
    without yet providing adequate values to use for animal-to-human
    extrapolation.  Unfortunately, very little information is available on
    the other key aspect of extrapolation, species sensitivity (i.e., the
    response of the tissues of different species to a given dose).  Thus,
    from currently available animal studies, we know which human health
    effects NO2 may cause. We are unable to assert with great confidence
    the effects that are  actually caused by a given inhaled dose of
    NO2.

         With the above issues in mind, the animal toxicology database
    for NO2 is summarized below according to major classes of effects
    and topics of special interest.  Although it is clear that the
    effects of NO2 exposure extend beyond the confines of the lung, the
    interpretation of these systemic effects relative to potential human
    risk is not clear.  Therefore they are not summarized further here,
    but are discussed in later chapters.  Although interactions of NO2
    and other co-occurring pollutants, such as O3 and sulfuric acid
    (H2SO4), can be quite important, especially if synergism occurs, the
    database does not yet allow conclusions that enable assessment of
    real-world potential interactions.

    1.3.1.1  Biochemical and cellular mechanisms of action of nitrogen
             oxides

         NO2 acts as a strong oxidant.  Unsaturated lipids are readily
    oxidized with peroxides as the dominant product.  Both ascorbic acid
    (vitamin C) and alpha-tocopherol (vitamin E) inhibit the peroxidation
    of unsaturated lipids.  When ascorbic acid is sealed within bilayer
    liposomes, NO2 rapidly oxidizes the sealed ascorbic acid.  The
    protective effects of alpha-tocopherol and ascorbic acid in animals
    and humans are due to the inhibition of NO2 oxidation.  NO2 also
    oxidizes membrane proteins.  The oxidation of either membrane lipids
    or proteins results in the loss of cell permeability control.  The
    lungs of NO2-exposed humans and experimental animals have larger
    amounts of protein within the lumen.  The recruitment of inflammatory
    cells and the changes in the lung are due to these events.

         The oxidant properties of NO2 also induce the peroxide
    detoxification pathway of glutathione peroxidase, glutathione
    reductase and glucose-6-phosphate dehydrogenase. Following NO2
    exposure the increase in the peroxide detoxification pathway in
    animals follows an exposure-response relationship.

         The mechanism of action of NO is less clear.  NO is readily
    oxidized to NO2 and peroxidation then occurs.  Because of the
    concurrent exposure to some NO2 in NO exposures, it is difficult to
    discriminate NO effects from NO2.  NO functions as an intracellular
    second messenger modulating a wide variety of essential enzymes, and
    it inhibits its own production (e.g., negative feedback).  NO
    activates guanylate cyclase which in turn increases intracellular cGMP
    levels.  A possible mechanism of action of nitrates may be through the
    release of histamine from mast cell granules.  Acidic nitrogenous air
    pollutants, particularly HNO3, may act by alteration of intracellular
    pH.

         PAN decomposes in water, generating hydrogen peroxide.  Little is
    known of the mechanism of action, but oxidative stress is likely for
    PAN and its congeners.

         Inorganic nitrates may act through alterations in intracellular
    pH.  Nitrate ion is transported into alveolar type 2 cells acidifying
    the cell.  Nitrate also mobilizes histamine from mast cells.  HNO2
    could also act to alter intracellular pH, but this mechanism is
    unclear.

         The mechanisms of action of the other nitrogen oxides are
    unknown.

         Acute exposure to NO2 at a concentration of 750 g/m3 (0.4 ppm)
    can result in lipid peroxidation.  NO2 can oxidize polyunsaturated

    fatty acids in cell membranes as well as functional groups of proteins
    (either soluble proteins in the cell, such as enzymes, or structural
    proteins, such as components of cell membranes).  Such oxidation
    reactions (mediated by free radicals) are a mechanism by which NO2
    exerts direct toxicity on lung cells.  This mechanism of action is
    supported by animal studies showing the importance of lung antioxidant
    defences, both endogenous (e.g., maintenance of lung glutathione
    levels) and exogenous (e.g., dietary vitamins C and E), in protecting
    against the effects of NO2.  Many studies have suggested that various
    enzymes in the lung, including glutathione peroxidase, superoxide
    dismutase and catalase, may also serve to defend the lung against
    oxidant attack.

    1.3.1.2  Effects on host defence

         Although the primary function of the respiratory tract is to
    ensure an efficient exchange of gases, this organ system also provides
    the body with a first line of defence against inhaled viable and non-
    viable airborne agents.  An extensive database clearly shows that
    exposure to NO2 can result in the dysfunction of these host defences,
    increasing susceptibility to infectious respiratory disease.  The
    host-defence parameters affected by NO2 include the functional and
    biochemical activity of cells in lungs, alveolar macrophages (AMs),
    immunological competence, susceptibility to experimentally induced
    respiratory infections, and the rate of mucociliary clearance.

         Alveolar macrophages are affected by NO2.  These cells
    are responsible for maintaining the sterility of the pulmonary
    region, clearing particles from this region, and participating in
    immunological functions.  Functional changes that have been reported
    include the following: the suppression of phagocytic ability and
    stimulation of lung clearance at 560 g/m3 (0.3 ppm) 2 h/day for
    13 days; a decrease in bactericidal activity at 4320 g/m3 (2.3 ppm)
    for 17 h; and a decreased response to migration inhibition factor at
    3760 g/m3 (2.0 ppm) 8 h/day, 5 days/week for 6 months.  The
    morphological appearance of these defence cells changes after chronic
    exposure to NO2.

         The importance of host defences becomes evident when animals have
    to cope with laboratory-induced pulmonary infections.  Animals exposed
    to NO2 succumb to bacterial or viral infection in a concentration-
    dependent manner.  Mortality also increases with increased NO2
    concentration or duration of exposure.  After acute exposure,
    effects are observed at concentrations as low as 3760 g/m3 (2 ppm). 
    Exposure to concentrations as low as 940 g/m3 (0.5 ppm) will cause
    effects in the infectivity model after 6 months.

         Both humoral and cell-mediated defence systems are changed by
    NO2 exposure.  In the cases in which the immune system has been
    investigated, effects have been observed after short-term exposure to

    concentrations > 9400 g/m3 (5 ppm).  The effects are complex
    since the direction of the change (i.e., increase or decrease) is
    dependent upon NO2 concentration and the length of exposure.

    1.3.1.3  Effects of chronic exposure on the development of chronic
             lung disease

         Humans are chronically exposed to NO2.  Therefore, such
    exposures in animals have been studied rather extensively, typically
    using morphological and/or morphometric methods.  This research has
    generally shown that a variety of pulmonary structural and correlated
    functional alterations occur.  Some of these changes may be reversible
    when exposure ceases.

         Pulmonary function may be altered following chronic NO2 exposure
    of experimental animals. Impaired gas exchange occurred following
    exposure to 7520 g/m3 (4.0 ppm) NO2 for four months and this was
    reflected in decreased arterial O2 tension, impaired physical
    performance and increased anaerobic metabolism.

         Although NO2 produces morphological changes in the respiratory
    tract, the database is sometimes confusing due to quantitative and
    qualitative variability in responsiveness between, and even within,
    species.  The rat, the most commonly used experimental animal in
    morphological assessments of exposure, appears to be relatively
    resistant to NO2.  Short-term exposures to concentrations of
    9400 g/m3 (5.0 ppm) or less generally have little effect in the
    rat, where similar exposures in the guinea-pig may result in some
    centriacinar epithelial damage.

         Longer-term exposures result in lesions in some species with
    concentrations as low as 560 to 940 g/m3 (0.3 to 0.5 ppm).  These
    are characterized by epithelial remodelling similar to that described
    above, but with the involvement of more proximal airways and
    thickening of the interstitium. Many of these changes, however, will
    resolve even with continued exposure, and long-term exposures to
    levels above about 3760 g/m3 (2.0 ppm) are required for more
    extensive and permanent changes in the lungs.  Some effects are
    relatively persistent (e.g., bronchiolitis), whereas others tend to be
    reversible and limited even with continued exposure.  In any case, it
    seems that for either short- or long-term exposure, the response is
    more dependent upon concentration than duration of exposure.

         There is substantial evidence that long-term exposure of several
    species of laboratory animals to high concentrations of NO2 results
    in morphological lung lesions.  Destruction of alveolar walls, an
    essential additional criterion for human emphysema, has been reliably
    reported in lungs from animals in a limited number of studies.  The

    lowest NO2 concentration for the shortest exposure duration that will
    result in emphysematous lung lesions cannot be determined from these
    published studies.

    1.3.1.4  Potential carcinogenic or co-carcinogenic effects

         NO2 has been shown to be mutagenic in  Salmonella bacteria, but
    was not mutagenic in one study with a mammalian cell culture.  Other
    studies using cell cultures have demonstrated sister chromatid
    exchanges (SCE) and DNA single strand breaks.  No genotoxic effects
    have been demonstrated  in vivo concerning lymphocytes, spermatocytes
    or bone marrow cells, but two inhalation studies with high
    concentrations (50 760 and 56 400 g/m3, 27 and 30 ppm) for 3 h and
    16 h, respectively, have demonstrated such effects in lung cells.

         Literature searches revealed no published reports of NO2 studies
    using classical whole-animal chronic bioassays for carcinogenesis. 
    Research with mice having spontaneously high tumour rates was
    equivocal.  In one study, NO2 at 18 800 g/m3 (10 ppm) slightly
    enhanced the incidence of lung adenomas in a sensitive strain of mice
    (A/J).  Although several co-carcinogenesis investigations have been
    undertaken, conclusions are precluded because of problems with
    methodology and interpretation. Reports on whether NO2 facilitates
    the metastasis of tumours to the lung are also inadequate to form
    conclusions.  Other investigations have centred on whether NO2 could
    produce nitrates and nitrites that, by reacting with amines in the
    body, could produce nitrosamines.  A few studies suggest that
    nitrosamines are formed in animals treated with high doses of amines
    and exposed to NO2, but other studies have indicated that nitrosamine
    formation is unlikely.

    1.3.1.5  Age susceptibility

         Investigations into age dependency are inadequate and results so
    far are equivocal.

    1.3.1.6  Influence of exposure patterns

         Several animal toxicological studies have elucidated the
    relationships between concentration (C) and duration (T) of exposure,
    indicating that the relationship is complex.  Most of this research
    has used the infectivity model.  Early C  T studies demonstrated that
    concentration had more impact on mortality than did duration of
    exposure.  An evaluation of the toxicity of NO2 exposures cannot be
    delineated by C  T relationships.

    1.3.2  Controlled human exposure studies on nitrogen oxides

         Human responses to a variety of oxidized nitrogen compounds have
    been evaluated.  By far, the largest database and the one most

    suitable for risk assessment is that available for controlled
    exposures to NO2.  The database on human responses to NO, HNO3
    vapour, HNO2 vapour and inorganic nitrate aerosols is not as
    extensive. A number of sensitive or potentially sensitive subgroups
    have been examined, including adolescent and adult asthmatics, older
    adults, and patients with chronic obstructive pulmonary disease (COPD)
    and pulmonary hypertension.  Exercise during exposure increases the
    total uptake and alters the distribution of the deposited inhaled
    material within the lung. The relative proportion of NO2 deposited in
    the lower respiratory tract is also increased by exercise.  This may
    increase the effects of the above compounds in people who exercise
    during exposure.

         As is typical with human biological response to inhaled particles
    and gases, there is variability in the biological response to NO2. 
    Healthy individuals tend to be less responsive to the effects of NO2
    than  individuals with lung disease.  Asthmatics are clearly the most
    responsive group to NO2 that has been studied to date.  Individuals
    with COPD may be more responsive than healthy individuals, but they
    have limited capacity to respond to NO2 and thus quantitative
    differences between COPD patients and others are difficult to assess. 
    Sufficient information is not available at present to evaluate whether
    age and sex play a role in the response to NO2.

         Healthy subjects can detect the odour of NO2, in some cases at
    concentrations below 188 g/m3 (0.1 ppm).  Generally, NO2 exposure
    did not increase respiratory symptoms in any of the subject groups
    tested.

         NO2 causes decrements in lung function, particularly increased
    airway resistance in resting healthy subjects at 2-h concentrations as
    low as 4700 g/m3 (approx.2.5 ppm).  Available data are insufficient
    to determine the nature of the concentration-response relationship.

         Exposure to NO2 results in increased airway responsiveness to
    bronchoconstrictive agents in exercising healthy, non-smoking subjects
    exposed to concentrations as low as 2800 g/m3 (approx.1.5 ppm) for
    1 h or longer.

         Exposure of asthmatics to NO2 causes, in some subjects,
    increased airway responsiveness to a variety of provocative mediators,
    including cholinergic and histaminergic chemicals, SO2 and cold air. 
    The presence of these responses appears to be influenced by the
    exposure protocol, particularly whether or not the exposure includes
    exercise.  These responses may begin at concentrations as low as
    380 g/m3 (0.2 ppm).  A meta-analysis suggests that effects may occur
    at even lower concentrations.  However, an unambiguous concentration-
    response relationship is observed between 350 to 1150 g/m3
    (approx.0.2 to 0.6 ppm).

         The implications of this overall trend are unclear, but increased
    airway responsiveness could potentially lead to increased response to
    aeroallergens or temporary exacerbation of asthma, possibly leading to
    increased medication usage or even increased hospital admissions.

         Modest increases in airway resistance may occur in COPD patients
    from brief exposure (15-60 min) to concentrations of NO2 as low as
    2800 g/m3 (approx.1.5 ppm), and decrements in spirometric measures
    of lung function (3 to 8% change in FEV1 (forced expiratory volume
    in 1 second)) may also be observed with longer exposures (3 h) to
    concentrations as low as 600 g/m3 (approx.0.3 ppm).

         Exposure to NO2 at levels above 2800 g/m3 (approx.1.5 ppm) may
    alter the numbers and types of inflammatory cells in the distal
    airways or alveoli.  NO2 may alter the functioning of cells within
    the lungs and production of mediators that may be important in lung
    host defences.  The constellation of changes in host defences,
    alterations in lung cells and their activities, and changes in
    biochemical mediators is consistent with the epidemiological findings
    of increased host susceptibility associated with NO2 exposure.

         In studies on mixtures of NO2 with other pollutants, NO2 has
    not been observed to increase responses to other co-occurring
    pollutant(s) beyond that which would be observed for the other
    pollutant(s) alone.  A notable exception is the observation that
    pre-exposure to NO2 enhanced the ozone-induced change in airway
    responsiveness in healthy exercising subjects during a subsequent
    ozone exposure.  This observation suggests the possibility of delayed
    or persistent responses to NO2.

         Within an NO2 concentration range that may be of interest with
    regard to risk evaluation (i.e., 100-600 g/m3), the characteristics
    of the concentration-response relationship for acute changes in lung
    function, airway responsiveness to bronchoconstricting agents or
    symptoms cannot be determined from the available data.

         On the basis of an effect at 400 g/m3 and the possibility of
    effects at lower levels, based on a meta analysis, a one-hour average
    daily maximum NO2 concentration of 200 g/m3 (approx.0.11 ppm) is
    recommended as a short-term guideline.

         NO is acknowledged as an important endogenous second messenger
    within several organ systems.  Inhaled NO concentrations above
    6000 g/m3 (approx.5 ppm) can cause vasodilation in the pulmonary
    circulation without affecting the systemic circulation.  The lowest
    effective concentration has not been established.  Information on
    pulmonary function and lung host defences consequent to NO exposure
    are too limited for any conclusions to be drawn at this time.

    Relatively high concentrations (> 40 000 g/m3) have been used in
    clinical applications for brief periods (< 1 h) without reported
    adverse reactions.

         Nitric acid levels in the range of 250-500 g/m3 (97-194 ppb)
    may cause some pulmonary function responses in adolescent asthmatics,
    but not in healthy adults.

         Limited information on HNO2 suggests that it may cause eye
    inflammation at 760 g/m3 (0.40 ppm).  There are currently no
    published data on human pulmonary responses to HNO2.

         Limited data on inorganic nitrates suggest that there are no lung
    function effects of nitrate aerosols at concentrations of 7000 g/m3
    or less.

    1.3.3  Epidemiology studies on nitrogen dioxide

         Epidemiological studies on the health effects of nitrogen oxides
    have mainly focused on NO2.  Many indoor and outdoor epidemiological
    studies designed to evaluate the health effects of NO2 have been
    conducted.  Two health outcome measurements of NO2 exposure are
    generally considered: lung function measurements and respiratory
    symptoms and diseases.

         The evidence from individual studies of the effect of NO2 on
    lower respiratory symptoms and disease in school-aged children is
    somewhat mixed.  The consistency of these studies was examined and
    the evidence synthesized in a combined quantitative analysis
    (meta-analysis) of the subject studies.  Most of the indoor studies
    showed increased lower respiratory morbidity in children associated
    with long-term exposure to NO2.  Mean weekly NO2 concentrations
    in bedrooms in studies reporting NO2 levels were predominantly
    between 15 and 122 g/m3 (0.008 and 0.065 ppm).  Combining the
    indoor studies as if the end-points were similar gives an estimated
    odds ratio of 1.2 (95% confidence limits of 1.1 and 1.3) for the effect
    per 28.3 g/m3 (0.015 ppm) increase of NO2 on lower respiratory
    morbidity.  This suggests that, subject to assumptions made for the
    combined analysis, an increase of about 20% in the odds of lower
    respiratory symptoms and disease corresponds to each increase of
    28.3 g/m3 (0.015 ppm) in estimated 2-week average NO2
    exposure.  Thus, the combined evidence is supportive for the effects
    of estimated exposure to NO2 on lower respiratory symptoms and
    disease in children aged 5 to 12 years.
    
         In individual indoor studies of infants 2 years of age or younger,
    no consistent relationship was found between estimates of NO2
    exposure and the prevalence of respiratory symptoms and disease.  Based

    on a meta-analysis of these indoor infant studies, subject to the
    assumptions made for the meta-analysis, the combined odds ratio for the
    increase in respiratory disease per increase of 28.2 g/m3 (0.015 ppm)
    NO2 was 1.09 with a 95% confidence interval of 0.95 to 1.26, where
    mean weekly NO2 concentrations in bedrooms were predominantly between
    9.4 and 94 g/m3 (0.005 and 0.050 ppm) in studies reporting levels.
    The increase in risk was very small and was not reported consistently
    by all studies.  We cannot conclude that the evidence suggests an effect
    in infants comparable to that seen in older children.  The reasons for
    these age-related differences are not clear.

         The measured NO2 studies gave a higher estimated odds ratio than
    the surrogate estimates, which is consistent with a measurement error
    effect.  The effect of having adjusted for covariates such as
    socioeconomic status, smoking and sex was that those studies that
    adjusted for a particular covariate found larger odds ratios than
    those that did not.

         Although many of the epidemiological studies that involved
    measured NO2 levels used measurements over only 1 or 2 weeks, these
    levels were used to characterize children's exposures over a much
    longer period.  The standard respiratory symptom questionnaire used by
    most of these studies summarizes information on health status over an
    entire year.  The 28.2 g/m3 (0.015 ppm) difference in NO2 levels
    used in the meta-analyses relates to a difference in the household
    annual average exposure between gas and electric cooking stoves.
    Some studies measured NO2 levels only in the winter and may have
    overestimated annual average exposures.  This would tend to have
    underestimated the health effect of a 28.2 g/m3 (0.015 ppm)
    difference in the annual NO2 exposure.  A study based on a household
    annual average exposure measured in both the winter and summer found a
    stronger health effect than many of the other studies.  The true
    biologically relevant exposure period is unknown, but these exposures
    extended over a lengthy period up to the entire lifetime of the child.

         The association between outdoor NO2 and respiratory health is
    not clear from current research.  There is some evidence that the
    duration of respiratory illness may be increased at higher ambient
    NO2 levels.  A major difficulty in the analysis of outdoor studies is
    distinguishing possible effects of NO2 from those of other associated
    pollutants.

         Several uncertainties need to be considered in interpreting the
    above studies and meta-analysis.  Error in measuring exposure is
    potentially one of the most important methodological problems in
    epidemiological studies of NO2.  Although there is evidence that
    symptoms are associated with indicators of NO2 exposure, the quality
    of these exposure estimates may be inadequate to determine a
    quantitative relationship between exposure and symptoms.  Most of the
    studies that measured NO2 exposure did so only for periods of 1 to

    2 weeks and reported the values as averages.  Few of the studies
    attempted to relate the observed effects to the pattern of exposure
    (e.g., transient NO2 peaks). Furthermore, measured NO2 concentration
    may not be the biologically relevant dose; estimating actual exposure
    requires knowledge of pollutant species, levels and related human
    activity patterns.  However, only very limited activity and aerometric
    data are available that examine such factors.  The extrapolation to
    possible patterns of ambient exposure is difficult.  In addition,
    although the level of similarity and common elements between the
    outcome measures in the NO2 studies provide some confidence in their
    use in the quantitative analysis, the symptoms and illnesses combined
    are to some extent different and could indeed reflect different
    underlying processes.  Thus, caution is necessary in interpreting the
    meta-analysis results.

         Other epidemiological studies have attempted to relate some
    measure of indoor and/or outdoor NO2 exposure to changes in pulmonary
    function.  These changes were marginally significant.  Most studies
    did not find any effects, which is consistent with controlled human
    exposure study data.  However, there is insufficient epidemiological
    evidence to draw any conclusions about the long- or short-term effects
    of NO2 on pulmonary function.

         On the basis of a background level of 15 g/m3 (0.008 ppm) and
    the fact that significant adverse health effects occur with an
    additional level of 28.2 g/m3 (0.015 ppm) or more, an annual
    guideline value of 40 g/m3 (0.023 ppm) is proposed.  This value will
    avoid the most severe exposures.  The fact that a no-effect level for
    subchronic or chronic NO2 exposure concentrations has not yet been
    determined should be emphasized.

    1.3.4  Health-based guidance values for nitrogen dioxide

         On the basis of human controlled exposure studies, the
    recommended short-term guidance value is for a one-hour average NO2
    daily maximum concentration of 200 g/m3 (0.11 ppm).  The recommended
    long-term guidance value, based on epidemiological studies of
    increased risk of respiratory illness in children, is 40 g/m3
    (0.023 ppm) annual average.

    2.  PHYSICAL AND CHEMICAL PROPERTIES, AIR SAMPLING AND ANALYSIS,
        TRANSFORMATIONS AND TRANSPORT IN THE ATMOSPHERE

    2.1  Introduction

         Nitrogen oxides are produced by combustion processes and are
    emitted to the air mainly as NO together with some NO2.  Natural
    biological processes and lightning also emit NO and N2O.  In the
    atmosphere nitrogen oxides undergo complex chemical and photochemical
    reactions; NO is oxidized to NO2 and other products and eventually to
    HNO3 and nitrates.  Nitrogenous species are removed from the air to
    the ground by wet and dry deposition processes.  Oxidized nitrogen
    compounds can have impacts on human health and the environment, and
    are important to the formation of photochemical smog and tropospheric
    ozone.

         In this chapter the properties of nitrogen compounds are briefly
    described and techniques for their sampling and analysis outlined. 
    Atmospheric chemical reactions that cause the oxidation of NO to NO2
    and the production of ozone, organic nitrates and HNO3 are described.
    The differences between night-time and day-time chemistry and the
    composition of the atmosphere are discussed.  The nature of the
    nitrogen species and their chemical reactions in urban regions, in
    chimney plumes such as those from power stations, in air advected away
    from urban regions and in rural and remote areas are described.  The
    role of nitrogen oxides in photochemical smog production and the
    effects of nitrous oxide on stratospheric ozone are briefly discussed.

    2.1.1  The nomenclature and measurement of atmospheric nitrogen
           species

         There are several methods available for determining nitrogen
    species, but many of these techniques are nonspecific.

         To denote various mixtures of nitrogen species, the terms NOx,
    NOy and NOz are often employed.  It is customary to refer to the sum
    of NO and NO2 emitted from a source as NOx, the unit of measure for
    NOx being the NO2 mass equivalent of the NO plus NO2.

         The term NOy is frequently used to denote the sum of the gas
    phase oxidized nitrogen species (except N2O) and NOz to denote the
    sum of NOy plus the oxidized nitrogen present as particulate matter. 
    Measurement of NOz requires a combination of particulate and gas
    phase sampling and analysis.

         A confusion arises because one of the most commonly used methods
    for determining NO2 in ambient air (thermal conversion of NO2 to NO
    and measurement of the resultant NO by chemiluminescent reaction with
    O3) is nonspecific and responds to several gaseous species in
    addition to NO2.  These include organic nitrogen compounds and,

    depending on the converter, HNO3, although HNO3 can be readily lost
    to the sampling system.  Therefore, depending on the composition of
    the air being sampled, the results from this type of instrument can be
    representative of NOy rather than NOx (or NO2) concentrations. 
    This technique is used in most routine determinations of ambient NOx
    and NO2 concentrations but the discrepancy between these values and
    true NOx and NO2 can be considerable for air in which the pollutant
    emissions have undergone substantial exposure to sunlight.

         Nitrous oxide is ubiquitous in the atmosphere because it is a
    product of biological processes in soil as well as anthropogenic
    activities.  It is not involved to any appreciable extent in chemical
    reactions in the lower atmosphere, but it is an active "greenhouse"
    gas.  In the stratosphere N2O forms NO by reaction with excited
    oxygen atoms, and this NO then acts to deplete the stratospheric O3
    concentration.

         Although NO3, dinitrogen trioxide (N2O3), dinitrogen tetroxide
    (N2O4), and N2O5 may play a role in atmospheric chemical reactions
    leading to the transformation, transport, and ultimate removal of
    nitrogen compounds from ambient air, they are present in very low
    concentrations, even in polluted environments.

         NH3 is generated during decomposition of nitrogenous matter in
    natural ecosystems and may be locally produced in high concentrations
    by human activities such as intensive animal husbandry and feedlots. 
    Under suitable conditions NH3 can react with oxidized nitrogen
    species to form ammonium nitrate aerosol.

    2.2  Nitrogen species and their physical and chemical properties

         There are seven oxides of nitrogen that may be present in ambient
    air, namely: NO, NO2, N2O, NO3, N2O3, N2O4 and N2O5.  In
    addition these can be present as HNO2, HNO3 and various organic
    nitrogen species, such as PAN, other organic nitrates and particles
    containing oxidized nitrogen compounds (particularly adsorbed nitric
    acid).  Of these species, NO and NO2 are the ones most often measured
    and are present in the greatest concentrations in urban and industrial
    air.

         The chemical and physical properties of individual nitrogen
    species are given below and are summarized in Table 1.

        Table 1.  Some physical and thermodynamic properties of oxides of nitrogen and other nitrogen compoundsa
                                                                                                                                              

    Oxide               Relative         Melting point    Boiling point     Solubility in water            Thermodynamic functions
                        molecular        (C)b,c,d        (C)b,c           at 0C (cm3 per 100 g)b        (Ideal gas, 1 atm, 25C)
                        mass (g/mol)                                                                                                          
                                                                                                           Enthalpy of      Entropy
                                                                                                           formation        (cal/mol-deg)
                                                                                                           (kcal/mol)
                                                                                                                                              

    NO                  30.01            -163.6           -151.8            7.34                              21.58            50.35

    NO2                 46.01            -11.2            21.2              Reacts with H2O forming            7.91            57.34
                                                                            HNO2 and HNO3

    N2O                 44.01            -90.8            -88.5             130.52                            19.61            52.55

    N2O3                76.01            -102             47                Reacts with H2O forming           19.80            73.91
                                                          (decomposes)      HNO2

    N2O4                92.02            -11.3            21.2              Reacts with H2O forming            2.17            72.72
                                                                            HNO2 and HNO3

    N2O5                108.01           30               3.24              Reacts with H2O forming            2.7             82.8
                                                          (decomposes)      HNO2

    HNO2                47.01            -                -                 -                                  -                -

    HNO3                63.01            -42              83                                                 -32.1             63.7
                                                                                                                                              

    Table 1.  (Con't)
                                                                                                                                              

    Oxide               Relative         Melting point    Boiling point     Solubility in water            Thermodynamic functions
                        molecular        (C)b,c,d        (C)b,c           at 0C (cm3 per 100 g)b        (Ideal gas, 1 atm, 25C)
                        mass (g/mol)                                                                                                          
                                                                                                           Enthalpy of      Entropy
                                                                                                           formation        (cal/mol-deg)
                                                                                                           (kcal/mol)
                                                                                                                                              

    PAN                 121.06           -                -                 -                                  -                -
    (CH3COOONO2)

    NH4NO3              80.04            169.6            210 at            118.3 g/100 cm3                  -87.37            36.11
                                                          11 torr           H2O at 0C
                                                                                                                                              

    a  Adopted from: US EPA (1993)
    b  Matheson Gas Data Book (Matheson Company, 1966)
    c  Handbook of Chemistry and Physics (Weast et al., 1986)
    d  At 0C and 1 atm pressure
        2.2.1  Nitrogen oxides

    2.2.1.1  Nitric oxide

         NO is a colourless, odourless gas that is only slightly soluble
    in water.  It is a by-product of combustion processes, arising from
    (i) high temperature oxidation of molecular nitrogen from the
    combustion air, and (ii) from oxidation of nitrogen present in certain
    fuels such as coal and heavy oil.

    2.2.1.2  Nitrogen dioxide

         NO2 is a reddish-orange-brown gas with a characteristic pungent
    odour.  The boiling point is 21.1C, but the low partial pressure of
    NO2 in the atmosphere prevents condensation.  NO2 is corrosive and
    highly oxidizing.  About 5 to 10% by volume of the total emissions of
    NOx from combustion sources is usually in the form of NO2, although
    substantial variations from one source type to another have been
    observed.

         In the atmosphere, photochemical reactions involving ozone
    and organic compounds convert NO to NO2.  NO2 is an efficient
    absorber of light over a broad range of ultraviolet (UV) and visible
    wavelengths.  Because of its brown colour, NO2 can contribute to
    discoloration and reduced visibility of polluted air.  Photolysis of
    NO2 by sunlight produces NO and an oxygen atom, which usually adds to
    an oxygen molecule to produce ozone.

    2.2.1.3  Nitrous oxide

         N2O is a colourless gas with a slight odour at high
    concentrations.  It is emitted to the atmosphere as a trace component
    from some combustion sources and from the consumption of nitrate by
    an ubiquitous group of denitrification bacteria that use nitrate as
    their terminal electron acceptor in the absence of oxygen (Delwiche,
    1970; Brezonik, 1972; Keeney, 1973; Focht & Verstraete, 1977).  At
    atmospheric concentrations N2O has no significant physiological
    effects in humans, although at higher concentrations it is employed as
    an anaesthetic.

         N2O does not play a significant role in atmospheric reactions in
    the lower troposphere.  In the stratosphere it reacts with singlet
    oxygen to produce NO, which participates in O3 decomposition in
    the stratosphere.  These reactions are of concern because of the
    possibility that increasing N2O concentrations resulting from fossil
    fuel use, and also from denitrification of excess fertilizer, may
    contribute to a decrease in stratospheric O3 (Council for
    Agricultural Science and Technology, 1976; Crutzen, 1976) with
    consequent potential for adverse impacts on ecosystems and human

    health.  Also of concern is the fact that N2O absorbs long-wave
    radiation, and therefore serves as a radiatively important greenhouse
    gas that may contribute to global warming.

    2.2.1.4  Other nitrogen oxides

         Other nitrogen oxides can be present in trace quantities in the
    air.  NO3 has been identified in laboratory systems containing
    NO2/O3, NO2/O and N2O5 as an important reactive transient
    (Johnston, 1966).  It is likely to be present in photochemical smog. 
    In the presence of sunlight, NO3 is rapidly converted to either NO or
    NO2 (Wayne et al., 1991).  Nitrogen trioxide is highly reactive
    towards both NO and NO2.  Its expected concentration in polluted air
    is very low (about 10-6 g/m3).  However, traces of NO3 may play an
    important role in atmospheric chemistry, especially at night when it
    may serve as a reservoir for NOx (Wayne et al., 1991).  In the
    atmosphere N2O3 is in equilibrium with NO and NO2.  It reacts with
    water to form HNO2.  N2O4 is the dimer of NO2, formed in
    equilibrium with NO2 molecules, and it readily dissociates to NO2. 
    N2O5 can be a trace night-time component of the air because it is
    formed by a reaction between NO2 and NO3.  Since NO3 can exist in
    appreciable quantities only in the absence of sunlight, N2O5 is only
    important at night, when its reaction with water can be a significant
    source of nitric acid.

    2.2.2  Nitrogen acids

    2.2.2.1  Nitric acid

         HNO3 is the most oxidized form of nitrogen.  In the gaseous
    state it is colourless.  It is photochemically stable in the
    troposphere.  HNO3 is volatile, so that at typical concentrations and
    temperatures in the atmosphere the vapour does not coalesce into
    aerosol and is not retained on particles unless the aerosol contains
    reactants such as sodium chloride or ammonium salts to react with the
    acid, when it produces particulate nitrates (Wolff, 1984).

         In the aqueous phase (e.g., rain drops), HNO3 dissociates to
    form the nitrate ion (NO3-).  Because nitrate is chemically
    unreactive in dilute aqueous solution, nearly all of the
    transformations involving nitrate in natural waters result from
    biochemical pathways.  The nitrate salts of all common metals are
    quite soluble.

    2.2.2.2  Nitrous acid

         HNO2 is formed when NO and NO2 are present in the atmosphere,
    as a result of their reaction with water.  In sunlight, the dominant

    pathway for HNO2 formation is the reaction of NO with hydroxyl
    radicals.  During the daytime, atmospheric concentrations of HNO2 are
    limited by the photolysis of HNO2 to produce NO and hydroxyl radical.

         Nitrous acid is a weak reducing agent and is oxidized to nitrate
    only by strong chemical oxidants and by nitrifying bacteria.

    2.2.3  Ammonia

         NH3 is the completely reduced form of nitrogen.  It is a
    colourless gas with a pungent odour.  It is extremely soluble in
    water, forming ammonium (NHy+) and hydroxyl (OH-) ions.  In the
    atmosphere, NH3 has been reported to be converted into NOx by
    reaction with hydroxyl radicals (Soederlund & Svensson, 1976).  In the
    stratosphere, NH3 can be dissociated by irradiation with sunlight at
    wavelengths below 230 nm (McConnell, 1973).

    2.2.4  Ammonium nitrate

         Gas-phase ammonia reacts with nitric acid to form ammonium
    nitrate (NH4NO3).  Ammonium nitrate is a solid at room temperature. 
    Like ammonia, it is very soluble in water and hence will be absorbed
    by any water droplets present.  Thus it readily forms an aerosol in
    the atmosphere.  Pathways to aerosol formation include nucleation and
    condensation on existing particles.  The presence of NH4NO3
    particles can result in a visible haze.

    2.2.5  Peroxyacetyl nitrate

         Of the various peroxy nitrates found in ambient air, peroxyacetyl
    nitrate (CH3COOONO2), or PAN, is found at the highest concentrations.
    PAN undergoes a temperature-dependent decomposition to its precursors,
    NO2 and acetyl peroxy radicals.  At low ambient temperatures PAN
    can have a substantial lifetime in the atmosphere (Cox & Roffey, 1977).
    In polluted air PAN concentrations can reach several parts per billion.

    2.2.6  Organic nitrites and nitrates

         A wide variety of organic nitrites (RNO2) and nitrates (RNO3),
    where R denotes CH3, CH2CH3, benzyl, etc., may be found in ambient
    air.  Some of these are emitted directly while others are formed by
    photochemical reactions in the atmosphere.

    2.3  Sampling and analysis methods

         This section outlines methods for measuring nitrogen-containing
    species in the atmosphere.  The main focus is on methodologies
    currently available and in general use for monitoring concentrations
    in both ambient and indoor air.

         Table 2 summarizes sampling and analytical methods for selected
    species and addresses relevant characteristics, including the type of
    method (i.e.,  in situ, remote, active, passive, continuous or
    integrative), the stage of development of the method, sampling
    duration, precision, accuracy and detection limits.

    2.3.1  Nitric oxide

    2.3.1.1  Nitric oxide continuous methods

         Nitric oxide reacts rapidly with O3 to give NO2 in an excited
    electronic stage.  The transition of excited NO to the grand state can
    be accompanied by the emission of light in the red-infrared spectral
    range.  When this chemiluminescent reaction occurs under controlled
    conditions, the intensity of the emitted light is proportional to the
    concentration of the NO reactant.  This provides the basis of the
    chemiluminescence method (CLM) for analysis of NO.  This method is a
    continuous technique and is the most commonly used method for
    measuring NO in ambient air.  Commercial instruments for measuring NO
    and NO2 are available with detection limits of approximately 5 ppb
    and response times of the order of minutes.  CLM measurement of NO2
    can also be accomplished by firstly converting the NO2 of the sample
    to NO.  This is discussed in section 2.3.2.1.

         Other NO analytical methods include laser-induced fluorescence
    (LIF) (Bradshaw et al., 1985), absorption spectroscopy (e.g., tuneable
    diode laser absorption spectroscopy, TDLAS) and passive samplers.

    2.3.1.2  Passive samplers for NO

         Passive samplers are used for air with higher-than-typical
    ambient concentrations, which may be found indoors or in the
    workplace.  They are often used to obtain data at a large number of
    sites.  Sampling typically lasts a few hours.

         The Palmes tube is a passive sampler that relies on diffusion of
    an analyte molecule through a quiescent diffusion path of known length
    and cross-sectional area to a reactive surface where the molecule is
    captured by chemical reaction (Palmes et al., 1976).  The Palmes tube
    does not measure NO directly.  Two tubes are required; the first one
    has reactive grids coated with triethanolamine (TEA) to collect NO2,
    the second tube is similar but has an additional reactive surface
    coated with chromic acid to convert NO to NO2, which is in turn
    collected by the TEA-coated grids.  The NO concentration of the air is
    determined from the difference in the results from the two tubes.  The
    data is corrected for the effects of the different diffusivities of NO
    and NO2 molecules. To ensure reliable results, contact between the
    chromic-acid-coated surface and the TEA-coated grids for longer than
    24 h must be avoided.  Analysis of the material contained in the TEA

        Table 2.  Selected instruments and methods for determining oxides of nitrogen in ambient air (from: Sickles, 1992)
                                                                                                                                              

    Species        Methodsa   Typeb      Development  Sample                Performance           Comments                 References
                                         stagec       duration                                
                                                                 Precision  Accuracy   MDLd
                                                                                                                                              

    NO             CLM        I, A, C      C          5 min      < 10%      < 20%      < 9 ppb    -                    Finlayson-Pitts &
                   (NO + O3)                                                                                           Pitts (1986)

                   TP-LIF     I, A, C      R          30 sec     -          16%        10 ppt     -                    Bradshaw et al. (1985);
                                                                                                                       Davis et al. (1987)

                   TDLAS      I, A, C      R, C       60 sec     -          -          0.5 ppb    40-m path length     NASA (1983)

                   PSD        I, P, IN     C          24 h       -          -          70 ppb-he

    NO2            CLM        I, A, C      C          5 min      10%        20%        9 ppb      Commonly used        Finlayson-Pitts &
                   (NO + O3)                                                                      method; many         Pitts (1986)
                                                                                                  interferences

                   CLM        I, A, C      R          < 100 sec  20 ppt     30%        10-25 ppt  Uses thermal or      Helas et al. (1987);
                   (NO + O3)                                                                      photolytic           Fehsenfeld et al.
                                                                                                  converters           (1987)

                   CLM        I, A, C      C          100 sec    0.6 ppb    -          10 ppt     Interferences:
                   (Luminol)                                                                      PAN, HNO2, O3

                   TP-LIF     I, A, C      R          2 min      20 ppt     16%        12 ppt     -                    Davis (1988)

                   TDLAS      I, A, C      R, C       60 sec     -          15%        100 ppt    150-m path length    NASA (1983)

                   DOAS       R, A, C      R, C       12 min     -          10%        4 ppb      800-m path length    Platt & Perner (1983)

                   Bubbler    I, A, IN     RM         24 h       6 ppb      10%        8 ppbe                          Purdue & Hauser (1980)
                                                                                                                                              

    Table 2.  (Con't)
                                                                                                                                              

    Species        Methodsa   Typeb      Development  Sample                Performance           Comments                 References
                                         stagec       duration                                 
                                                                 Precision  Accuracy   MDLd
                                                                                                                                              

                   TEA        I, A, IN     L          24 h       15%        10%        0.2 ppbe   Interferences:       Sickles et al. (1990)
                   filter                                                                          PAN and HNO2f

                   Guaiacol   I, A, IN     L          1 h        4%         -          0.1 ppbe   Stability of         Buttini et al. (1987)
                   Denuder                                                                        extract uncertain

                   DPA        I, A, IN     L          8 h        8%         -          0.1 ppbe   DPA may volatilize;  Lipari (1984)
                   Cartridge                                                                      interferences:
                                                                                                  HNO2 and PAN

                   TEA PSD    I, P, IN     L          24 h       30%        -          30 ppb-he  Similar to Palmes
                                                                                                  Tube; interferences
                                                                                                  as abovef

    NOy           CLM        I, A, C      R          10 sec     -          15%        10 ppt     CO with Au           Fahey et al. (1986)
                   (NO + O3)                                                                      reducing catalyst

    PAN            GC-ECD     I, A, IN     R, RM      15 min     -          30%        10 ppte    Sensitivity can be   Vierkorn-Rudolph
                                                                                                  enhanced by using    et al. (1985)
                                                                                                  cryogenic sampling
                                                                                                  and capillary
                                                                                                  columns

                   GC-CLM     I, A, IN     L          -          -          -          -          CLM (NO + O3) and
                                                                                                  (Luminol) reported

    Other organic  GC-ECD/MS  I, A, C      R          24 h       -          -          1 ppte     Sample collected     Atlas (1988)
    Nitrates                                                                                      on charcoal
                                                                                                                                              

    Table 2.  (Con't)
                                                                                                                                              

    Species        Methodsa   Typeb      Development  Sample                Performance           Comments                 References
                                         stagec       duration                                
                                                                 Precision  Accuracy   MDLd
                                                                                                                                              

    NHO3           Filter     I, A, IN     R, RM      24 h       10%        20%        8 ppte     May be nylon or      Finlayson-Pitts &
                                                                                                  calcium chloride     Pitts (1986)
                                                                                                  impregnated filter;
                                                                                                  subject to
                                                                                                  artifactsf

                   Denuder    I, A, IN     R, RM      24 h       8%         -          8 ppte     Not subject to       Sickles (1987);
                                                                                                  above artifactsf     Sickles et al. (1989)

                   TDLAS      I, A, C      R, C       5 min      -          20%        100 ppt    150-m path length    NASA (1983)

    HNO2           Denuder    I, A, IN     R, RM      24 h       15%        -          10 ppte    Annular denuder      Sickles et al. (1989);
    
                                                                                                  preferredf           Vossler et al. (1988)

                   LIF        I, A, C      R          15 min     -          -          20 ppt     OH detected
                                                                                                  following photo-
                                                                                                  fragmentation

                   DOAS       R, A, C      R, C       12 min     -          30%        600 ppt    800-m path length    Biermann et al. (1988)
                                                                                                                                              

    Table 2.  (Con't)
                                                                                                                                              

    Species        Methodsa   Typeb      Development  Sample                Performance           Comments                 References
                                         stagec       duration                                
                                                                 Precision  Accuracy   MDLd
                                                                                                                                              

    NO3            DOAS       R, A, C      R, C       12 min     -          15%        20 ppt     800-m path length    Platt & Perner (1983)

    Particulate    Denuder/   I, A, IN     R, RM      24 h       10%        -          40 ng/m3e  Use of denuders      Vossler et al. (1988)
    NO3            Filter(s)                                                                      avoids artifacts;
                                                                                                  denuders collect
                                                                                                  HNO3 and NH3;
                                                                                                  teflon and nylon
                                                                                                  filters used

    N2O            GC-ECD     I, A, IN     R, RM      15 min     3%         -          20 ppbe    -
                                                                                                                                              

    a  CLM (NO + O3) = Chemiluminescent using NO + O3 reaction       b  I = In situ
       TP-LIF = Two-photon laser-induced                                A = Active
       TDLAS = Tuneable diode laser absorption spectroscopy             C = Continuous
       TTFMS = Two-tone frequency modulated spectroscopy                P = Passive
       PSD = Passive sampling device                                    IN = Integrative
       CLM (Luminol) = Chemiluminescent using reaction with Luminol     R = Remote
       DOAS = Differential optical absorption spectroscopy
       DIAL = Differential absorption lidar                          c  C = Commercially available
       TEA = Triethanolamine                                            R = Research tool
       DPA = Diphenylamine                                              L = Laboratory prototype
       GC-ECD = Gas chromatography with electron capture detector       RM = Routine method
       CG-CLM = Gas chromatography with CLM detector
       LIF = Laser-induced fluorescence                              d  MDL  =  Minimum detection limit
       GC-MS = gas chromatography with mass spectrometer             e  Depends on the sampled air volume (i.e., flow rate and sampling
                                                                        duration)
                                                                     f  Uses ion chromatographic or colorimetric analytical finish
        is accomplished by extracting the grids into solution and analysing
    the extract for NO2- by the use of the spectrophotometric or ion
    chromatographic method (Miller, 1984).  The colorimetric analysis is
    calibrated by dilution of gravimetrically prepared nitrite solutions. 
    The Palmes Tube method was proposed for sampling occupational
    exposures where the dosage does not exceed 25 ppm for 8 h (i.e.,
    200 ppm-h).  The reliability of this method for measuring NO in the
    field at the parts-per-billion or parts-per-million level remains to
    be demonstrated.

         A badge-type sampler similar to the Palmes tube has been devised
    by Yanagisawa & Nishimura (1982).  This device uses a series of
    12 layers of chromium-trioxide-impregnated glass fibre to oxidize NO
    to NO2.  This technique is claimed to be more sensitive by
    approximately a factor of 10 than the Palmes tube and to have a lower
    limit dosage of 0.07 ppm-h.

    2.3.1.3  Calibration of NO analysis methods

         Calibration of CLM, TP-LIF and TDLAS measurement systems for NO
    all rely on compressed gas mixtures of known concentration being
    available.  Typically compressed gas mixtures are supplied in
    passivated aluminium/stainless steel gas bottles certified by the
    manufacturer and with NO diluted with N2 concentration in the rage of
    1 to 50 ppm (Schiff et al., 1983; Carroll et al., 1985; Bradshaw et
    al., 1985).  Calibrations are performed by dynamic dilution of the
    reference NO/N2 mixture with air to give NO concentrations within the
    range of 0.1 to 5 ppm.

         For passive NO samplers, only the analysis portion of the
    procedure is routinely calibrated (using gravimetrically prepared
    nitrite solution).

    2.3.1.4  Sampling considerations for NO

         Oxides of nitrogen are reactive species and exhibit various
    solubilities (Table 1).  The most inert materials (i.e. glass and
    TeflonTM) are recommended for use in sampling trains.  Since ambient
    air contains water vapour that may be sorbed on sampling lines,
    surface effects may influence the integrity of air samples containing
    the more reactive and more soluble NOy species.  In hot, humid
    conditions condensation in the sample lines of liquid water from the
    air can cause difficulties when analysis equipment is installed in an
    air-conditioned environment.  To minimize contamination of the system
    by dust and foreign matter, it is common practice to sample through an
    inert (teflon) sample inlet filter.  Of the NOy species, NO is
    probably the least susceptible to surface effects, whereas surface
    effects are very important in the sampling of HNO3.

         Nitric oxide reacts rapidly with O3 to form NO2.  In the
    presence of sunlight NO2 in air photolyses to yield NO and O3.  Thus
    in daylight NO, O3 and NO2 can exist simultaneously in ambient air
    in a condition known as a "photostationary state".  The relative
    amounts of the three species at any time are influenced by the
    intensity of the sunlight present at that moment.  Photolysis ceases
    when a sample is drawn into a dark sampling line, but NO and O3 can
    continue to react to form NO2.  Therefore residence times in sampling
    lines must be minimized to maintain the intensity of the NO/NO2 ratio
    of the sample.

    2.3.2  Nitrogen dioxide

         Airborne concentrations of NO2 can be determined by several
    methods including CLM, LIF, absorption spectroscopy, including
    differential optical absorption spectroscopy (DOAS) and TDLAS, bubbler
    and passive collection with subsequent wet chemical analysis.  The
    most common techniques are chemiluminescence and passive sampling.

    2.3.2.1  Chemiluminescence (NO + O3)

         Instruments discussed in this section do not detect NO2
    directly.  They sample continuously and rely on the conversion of some
    or all of the NO2 in the air sample to NO, followed by the CLM
    reaction of NO and O3. The NO2 concentration is calculated from the
    difference in the signal given by the sample after passing through the
    converter compared to that when the converter is by-passed.

         Several methods have been employed to reduce NO2 to NO (Kelly,
    1986).  They include catalytic reduction using heated molybdenum or
    stainless steel, reaction with carbon monoxide over a gold catalyst
    surface, reaction with iron sulfate at room temperature, reaction with
    carbon at 200C, and photolysis of NO2 to NO by light in the
    wavelength range of 320 to 400 nm.

         CLM instruments for the determination of NO2 are readily
    available commercially.  Field evaluation of nine instruments showed
    that the minimum detection limits (MDLs) ranged from 5 to 13 ppb
    (Michie et al., 1983; Holland & McElroy, 1986).

         Converters may be non-specific for NO2 and may convert
    several other nitrogen-containing compounds to NO, giving rise to
    overestimates for NO2 concentrations.  Using commercial instruments,
    Winer et al. (1974) found over 90% conversion of PAN, ethyl nitrate
    and ethyl nitrite to NO with a molybdenum converter, and similar
    responses to PAN and  n-propyl nitrate with a carbon converter.  With
    a stainless steel converter at 650C, Matthews et al. (1977) reported
    100% conversion for NO2, 86% for NH3, 82% for CH3NH2, 68% for HCN,
    1% for N2O and 0% for N2.  Using a commercial instrument, Joseph &
    Spicer (1978) found quantitative conversion of HNO3 to NO with a

    molybdenum converter at 350C.  Similar responses to PAN, methyl
    nitrate,  n-propyl nitrate,  n-butyl nitrate and HNO3, substantial
    response to nitrocresol, and no response to peroxybenzoyl nitrate
    (PBzN) were reported with a commercial instrument using a molybdenum
    converter at 450C (Grosjean & Harrison, 1985).  These results were
    confirmed for PAN and HNO3 by Rickman & Wright (1986) using
    commercial instruments with a molybdenum converter at 375C and a
    carbon converter at 285C.

         Interference from species that do not contain nitrogen have also
    been reported.  Joshi & Bufalini (1978), using a commercial instrument
    with a carbon converter, found significant apparent NO2 responses
    to phosgene, trichloroacetyl chloride, chloroform, chlorine (Cl2),
    hydrogen chloride, and photochemical reaction products of a
    perchloroethylene-NOx mixture.  Grosjean & Harrison (1985) reported
    substantial responses to photochemical reaction products of Cl2-NOx
    and Cl2-methanethiol mixtures and small negative responses to
    methanethiol, methyl sulfide, and ethyl sulfide.  Sickles & Wright
    (1979), using a commercial instrument with a molybdenum converter at
    450C, found small negative responses to 3-methylthiophene,
    methanethiol, ethanethiol, ethyl sulfide, ethyl disulfide, methyl
    disulfide, hydrogen sulfide, 2,5-dimethylthiophene, methyl sulfide
    and methyl ethyl sulfide, and negligible responses to thiophene,
    2-methylthiophene, carbonyl sulfide and carbon disulfide.

         Methods of sample trapping followed by batch measurement of NO
    and NO2 in the desorbed sample using a chemiluminescence instrument
    have been reported.  Gallagher et al. (1985) used cryosampling of
    stratospheric whole-air samples, and Braman et al. (1986) used
    copper(I) iodide coated denuder tubes to sample NO2 in ambient air.

    2.3.2.2  Chemiluminescence (luminol)

         A method for the direct chemiluminescence determination of NO2
    was reported by Maeda et al. (1980) and is based on the CLM reaction
    of gaseous NO2 with a surface wetted with an alkaline solution of
    luminol (5-amino-2,3-dihydro-1,4-phthalazinedione).  The light
    emission is strong at wavelengths between 380 and 520 nm.  The
    intensity of the light can be proportional to the NO2 concentration
    in the sampled air, and the NO2 concentration can be determined by
    calibration of the instrument with air of known NO2 concentration.

         Since the introduction of the luminol method by Maeda et al.
    (1980), improvements have been made to develop an instrument
    suitable for use in the field (Wendel et al., 1983), and additional
    modifications have been made recently to produce a continuous
    commercial instrument (Schiff et al., 1986).  Detection limits of 5 to
    30 ppt and a response time of seconds have been claimed, based on
    laboratory tests (Wendel et al., 1983; Schiff et al., 1986).  Recent
    laboratory evaluation of two instruments has revealed a detection

    limit (i.e., twice the standard deviation of the clean air response)
    of 5 ppt, and 95% rise and fall times of 110 and 15 seconds (Rickman
    et al., 1988).  Field tests of the same instruments have shown an
    operating precision of  0.6 ppb.

    2.3.2.3  Laser-induced fluorescence and tuneable diode laser
             absorption spectrometry

         Two newer techniques that show considerable promise for measuring
    NO2 specifically are photofragmentation/2-photon LIF and TDLAS.  The
    LIF and TDLAS techniques provide specific spectroscopic methods to
    measure NO2 directly and compare favourably to the sample photolysis-
    chemiluminescence technique (Fehsenfeld et al., 1990; Gregory et al,
    1990b).  For NO2 concentrations above 0.2 ppb, no interferences were
    found for TDLAS (Fehsenfeld et al., 1990).

    2.3.2.4  Wet chemical methods

         Most wet chemical methods for measuring NO2 involve the
    collection of NO2 in solution, followed by a colorimetric finish
    using an azo dye.  Many variations of this method exist, including
    both manual and automated versions.  These include the Griess-Saltzman
    method, the continuous Saltzman method, the alkaline guiacol
    method, the sodium arsenite method (manual or continuous), the
    triethanolamine-guaiacol-sulfite (TGS) method and the TEA method. 
    These methods have been reviewed by Purdue & Hauser (1980).

    2.3.2.5  Other methods

         Several other methods for the determination of NO2 have been
    reported.  Atmospheric pressure ionization mass spectrometry has been
    investigated for the continuous measurement of NO2 and SO2 in
    ambient air (Benoit, 1983).  Methods employing photothermal detection
    of NO2 have been reported (Poizat & Atkinson, 1982; Higashi et al.,
    1983; Adams et al., 1986).

         A portable, battery-powered analyser specific to NO2, which uses
    an electrochemical cell as the detector, is commercially available. 
    By careful selection and design of the cell, levels down to
    approximately 0.1 ppm (v/v) can be detected, although with
    uncertainties of approximately 20-50%.  The detection cell has a
    finite life, dependent on the time integral of the NO2 concentrations
    measured.  When the cell deteriorates, the instrument typically
    develops a gradual drift.

    2.3.2.6  Passive samplers

         Passive samplers are frequently used in industrial hygiene,
    indoor air and personal exposure studies and are less frequently used
    for ambient air analysis.  Namiesnik et al. (1984) have provided an
    overview of passive samplers.

         One type of passive NO2 sampler for ambient application is the
    nitration plate.  It is essentially an open petri dish containing
    TEA-impregnated filter paper.  Mulik & Williams (1986) have adapted
    the nitration plate concept by adding diffusion barriers in their
    design of a passive sampling device (PSD) for NO2 in ambient and
    personal exposure applications.  The device employs a TEA-coated
    cellulose filter paper, two 200-mesh stainless steel diffusion screens
    and two stainless steel perforated plates on each side of the coated
    filter to act as diffusion barriers and permit NO2 collection on both
    faces of the filter paper.  After sampling, the paper is removed
    from the PSD, extracted in water, and analysed for NO2- by
    ion chromatography.  A sensitivity of 0.03 ppm-h and a rate of
    2.6 cm3/second were claimed. Comparison of PSD results with
    chemiluminescence determinations of NO2 in laboratory tests at
    concentrations between 10 and 250 ppb showed a linear relation and
    high correlation (i.e.,  r = 0.996) (Mulik & Williams, 1987). 
    Interference from PAN and HNO2 would be expected (Sickles, 1987). 
    Results of TDLAS and triplicate daily PSD NO2 measurements in a
    13-day field study showed good agreement between the study average
    values but a correlation coefficient for daily results of only 0.47
    (Mulik & Williams, 1987; Sickles et al., 1990).  The Palmes tube
    described in section 2.3.1.2 has been used to sample air in the
    workplace and indoor environments to assess personal exposure to NO2
    (Palmes et al., 1976; Wallace & Ott, 1982).

    2.3.2.7  Calibration

         Calibration methods for NO2 use permeation tubes or gas-phase
    titration (GPT) to generate known concentrations of NO2. 
    Calibrations are performed dynamically using dilution with purified
    air.

         GPT employs the rapid, quantitative gas-phase reaction between
    NO, usually supplied as a known concentration from a gas cylinder, and
    O3 supplied from a stable O3 generator, to produce one NO2 molecule
    for each NO molecule consumed by reaction.  When O3 is added to
    excess NO in a titration system, the decrease in NO concentration
    (and O3) is equivalent to the increase in NO2 produced (US EPA,
    1987b).

         Use of cylinders of compressed gas containing NO2 for
    calibration purposes (Fehsenfeld et al., 1987; Davis, 1988) is unwise
    because of the uncertain stability of the NO2 concentrations
    delivered; this is a consequence of its relatively high boiling point.

    2.3.3  Total reactive odd nitrogen

         In this monograph, gas-phase total reactive odd nitrogen is
    represented by NOy.  Individual components comprising NOy are gas

    phase NO, NO2, NO3, N2O5, HNO2, HNO3, peroxynitric acid
    (HO2NO2), PAN, and other organic nitrates.  NH3 and N2O are not
    components of NOy.

         Researchers have successfully combined highly sensitive research-
    grade CLM NO detectors with catalytic converters that are sufficiently
    active to reduce most of the important gas phase NOy species to NO
    for subsequent detection (Helas et al., 1981; Dickerson, 1984; Fahey
    et al., 1986; Fehsenfeld et al., 1987).

    2.3.4  Peroxyacetyl nitrate

         Several methods have been used to measure the concentration of
    PAN in ambient air.  Roberts (1990) has provided an overview of many
    of these methods.  A well-developed method is gas chromatography using
    electron capture detection (GC-ECD) (Darley et al., 1963; Smith et
    al., 1972; Stephens & Price, 1973; Singh & Salas, 1983).

    2.3.5  Other organic nitrates

         Other organic nitrates (e.g., alkyl nitrates, peroxypropionyl
    nitrate and PBzN) can also be present in the atmosphere, but usually
    at lower concentrations than PAN (Fahey et al., 1986).  In general,
    similar methods for sampling, analysis and calibration may be used for
    other organic nitrates as are used for PAN (Stephens, 1969).  FTIR,
    GC-ECD and GC-MS may be used to measure these compounds.

    2.3.6  Nitric acid

         Several methods are available for the determination of HNO3
    concentrations in the atmosphere.  These include filtration (Okita et
    al., 1976; Spicer et al., 1978a), denuder tubes (Forrest et al., 1982;
    De Santis et al., 1985; Ferm, 1986), CLM (Joseph and Spicer, 1978) and
    absorption spectroscopy (Tuazon et al., 1978; Schiff et al., 1983;
    Biermann et al., 1988).  Many of these techniques carry significant
    uncertainties, which have been compared by Hering et al. (1988).

    2.3.7  Nitrous acid

         Available techniques for the measurement of HNO2 in ambient
    atmospheres employ denuders (Ferm & Sjodin, 1985), annular denuders
    (De Santis et al., 1985), CLM (Braman et al., 1986), PF/LIF (Rodgers &
    Davis, 1989), absorption spectroscopy (Tuazon et al., 1978; Biermann
    et al., 1988) and FTIR (Finlayson-Pitts & Pitts, 1986).

    2.3.8  Dinitrogen pentoxide and nitrate radicals

         N2O5 is readily reduced to NO at temperatures above 200C and
    may be measured nonspecifically as NO2 with CLM NO2 analysers
    (Bollinger et al., 1983; Fahey et al., 1986).

         Ambient concentrations of the NO3 radical have been measured
    using DOAS; concentrations between 1 and 430 ppt have been observed
    (Atkinson et al., 1986).

    2.3.9  Particulate nitrate

         Many methods are available for sampling ambient aerosols,
    including impactors, filtration, and filtration coupled with devices
    to remove particles larger than a specified size (e.g., elutriators,
    impactors and cyclones).

         Particulate nitrate samples are generally collected by
    filtration, extracted, and analysed directly or indirectly for nitrate
    by ion chromatography or colorimetry.

    2.3.10  Nitrous oxide

         The most commonly used analytical method for N2O employs GC-ECD. 
    It has a detection limit of 20 ppb (Thijsse, 1978) and a precision of
     3% at the background level of 330 ppb (Cicerone et al., 1978).

    2.3.11  Summary

         Gas-phase CLM instruments have replaced manual (wet) methods
    to a large extent in air quality monitoring network applications. 
    Gas-phase CLM measurement technology permits the determination of NO,
    NO2 and NOy in the low ppt range.  Although CLM NO detectors coupled
    with catalytic NO2 to NO converters are still not specific for NO2,
    they have proved to be useful for measuring NOy.  CLM NO detectors
    coupled with photolytic NO2 to NO converters have shown improved
    specificity for NO2.  Most ambient NO2 monitoring data reported are
    from the nonspecific thermal conversing technique.

         Passive samplers for NO2 have been used primarily for workplace
    and indoor applications, but hold promise for averaged ambient
    measurements as well.  GC-ECD is useful in the determination of PAN,
    other organic nitrates and N2O.

    2.4  Transport and transformation of nitrogen oxides in the air

    2.4.1  Introduction

         Oxides of nitrogen are transformed by and removed from the
    atmosphere by a complex web of reactions that are fundamental to the
    formation and destruction of ozone and other oxidants.  The
    predominant form of oxidized nitrogen (NO, NO2, HNO3, etc.)
    in the lower atmosphere varies, depending upon sunlight intensity,
    temperature, pollutant emissions, period of time since these emissions
    occurred and the meteorological history of an airmass.

    2.4.2  Chemical transformations of oxides of nitrogen

    2.4.2.1  Nitric oxide, nitrogen dioxide and ozone

         The dominant source of nitrogen oxides in the air is combustion
    processes (see chapter 3); 90-95% of these nitrogen oxides are usually
    emitted as NO and 5-10% as NO2.  NO may be oxidized to NO2 by
    atmospheric oxygen according to reaction 2-1:

              NO + NO + O2 -> 2 NO2                                   (2-1)

    However at low NO concentrations this reaction is slow and is
    important only when NO > 1 ppm (Bostrm C, 1993).  NO concentrations
    greater than 1 ppm are not frequently found in ambient air, but they
    may possibly occur in indoor air and in plumes from industrial sources
    (see Chapter 3).  When concentrations are below 1 ppm, NO is oxidized
    to NO2 by two types of reaction.  The first type of reaction is given
    in equations 2-2 to 2-4.  NO can react with O3:

              NO + O3 -> NO2 + O2                                     (2-2)

    Also O3 is formed when NO2 is photolysed, forming NO plus an O atom

              NO2 + hnu -> O + NO                                     (2-3)

    and O atoms react rapidly with O2 to form ozone:

                     M
              O + O2 -> O3                                            (2-4)

    Thus reactions 2-2, 2-3 and 2-4 recycle O3 rather than producing a
    net increase in O3 concentrations, where the "M" represents a third
    molecule such as N2, O2, etc., that absorbs excess vibrational
    energy from the newly formed O3 molecules.  However, a second
    oxidation path involving the reaction of organic species can lead to
    increases in O3 concentrations and in the conversion rate of NO to
    NO2 (2-9 and 2-10).  Organic compounds in the air are commonly
    referred to as VOC (volatile organic carbon), ROC (reactive organic
    carbon) and non-methane hydrocarbons (NmHC).  Urban areas are usually
    characterized by significant sources of both nitrogen oxides and ROC
    emissions.  With suitable atmospheric conditions this can lead to the
    formation of photochemical smog.  The smog-forming reactions are
    initiated by photolytic reactions which produce free radicals, for
    example:

    (i) the photolysis of O3

              O3 + hnu -> O2 + O*                                     (2-5)

    O* is an excited form of atomic oxygen, which can react with water to
    produce the hydroxyl radical (OH):

              O* + H2O -> 2OH                                       (2-6)


    (ii) the photolysis of aldehydes, which also results in the production
    of OH.  Aldehydes are emitted in motor vehicle exhaust and are
    produced in the air by reaction of ROC species with OH.  OH is the
    most important oxidizing agent in the lower atmosphere; it can react
    with all organic compounds, usually forming water and producing an
    organic radical.

         For a generalized organic compound, R-H (R = CH3, CHO, CH2CH3,
    etc.), the principal elements of the reaction sequence are:

              R-H + OH -> H2O + R                                   (2-7)

                     M
              R + O2 -> RO2 (fast)                                  (2-8)

    RO2 provides a pathway to oxidize NO to NO2 without destroying O3
    (unlike reaction 2-2):

              RO2 + NO -> NO2 + RO                                  (2-9)

    RO can undergo reactions that form additional HO2 or RO2.  HO2
    reacts with NO to form NO2 and regenerate OH:

              HO2 + NO -> NO2 + OH                                  (2-10)

         In the case of photochemical smog episodes, the quantity of NOx
    emitted into the air determines the ultimate quantity of O3 that may
    be produced.  The ROC concentration and sunlight intensity are the
    major determinates of the rates at which NO will be oxidized to
    produce net increases in NO2 and O3 concentrations.  Ozone
    production is terminated when NO and NO2 are consumed by reaction to
    form products such as HNO3 (see below), resulting in insufficient NO
    concentration for reactions 2-9 and 2-10 to proceed at significant
    rates.

         In large cities with sunny climates and poor dispersion of
    emissions (e.g., Los Angeles and Mexico City), O3 concentrations in
    excess of 200 ppb are not uncommon.

    2.4.2.2  Transformations in indoor air

         Oxides of nitrogen in indoor air arise from two sources: a)
    outdoor air; and b) indoor sources, such as combustion appliances and
    heaters.  Photochemical reactions do not take place under artificial
    lighting, so chemical transformations are limited by the amounts of
    oxidizing species (HO2, O3, etc.) that arrive in outdoor air, or are
    generated by combustion sources.

    2.4.2.3  Formation of other oxidized nitrogen species

         Oxidation products of NOx arising from tropospheric
    photochemical reactions include HNO3, HO2NO2, HNO2,
    peroxyacylnitrates (RC(O)O2NO2), N2O5, nitrate radical (NO3) and
    organic nitrates (RNO3).

         Fig. 1 shows a summary for the interconversion pathways for
    oxides of nitrogen.  These pathways govern urban and indoor air, as
    well as "clean" air, but the partitioning between the nitrogen oxide
    species varies according to the specific conditions characteristic of
    each type of airmass.

    a)  Nitric acid

         Nitric acid is a strong mineral acid that contributes to acidic
    deposition from the air.  In terms of atmospheric chemistry, HNO3 is
    a major sink for active nitrogen.  In daylight, HNO3 is formed by the
    reaction of NO2 with the OH radical:

                       M
              NO2 + OH -> HNO3                                       (2-11)

         This reaction is a chain-terminating step in the free radical
    chemistry that produces urban photochemical smog and it removes
    reactive nitrogen as well as the hydroxyl radical.  Reaction 2-11 is a
    relatively fast reaction that can produce significant amounts of HNO3
    over a period of a few hours.  At night, in polluted air containing
    significant ozone concentrations, the heterogeneous reaction between
    gaseous N2O5 and liquid water is thought to be a source of HNO3
    (N2O5 is produced from NO3 (see section 2.4.3.5) and NO2).  This
    pathway to HNO3 production is negligible during daytime, because the
    NO3 radical photolyses rapidly and is not present in sufficient
    quantities to react with NO2.  The NO3 radical can also abstract a
    hydrogen atom from certain organic compounds (such as aldehydes,
    dicarbonyls and cresols) to provide another night-time source of
    HNO3.

         Logan (1983) has estimated a lifetime of 1 to 10 days for
    HNO3 in the lower troposphere.  The primary removal mechanism is
    deposition.  The loss of HNO3 by rain-out is subject to precipitation
    frequency while the loss rate by dry deposition varies with the nature

    FIGURE 2

    of the ground and vegetation and atmospheric mixing characteristics of
    the boundary layer.  Chemical destruction mechanisms for HNO3 also
    exist, but their importance is not well understood and is suspected to
    be minor for the lower troposphere.

         In the presence of NH3, HNO3 may form the salt, ammonium
    nitrate:

              HNO3(g) + NH3(g) -> NH4NO3                             (2-12)

         Ammonium nitrate gas readily condenses to the particulate phase.
    Ammonium nitrate aerosol can be responsible for significant visibility
    reduction and particulate pollution, e.g., where nitric acid is
    produced in air from urban areas and this interacts with NH3 emitted
    from agricultural operations.

    b)  Nitrous acid

         HNO2 is produced from the reaction of NO and OH:

                      M
              NO + OH -> HNO2                                        (2-13)

         In indoor air other reactions (particularly surface reactions)
    may be important sources of nitrous acid.

         There have been a few measurements of nitrous acid in urban
    environments (Harris et al., 1982; Winer et al., l987).  Daytime
    levels of nitrous acid are expected to be low because it photolyses
    rapidly, yielding NO and OH.  This reaction probably serves as a
    source of OH radicals during the morning in urban regions, where
    nitrous acid may form (from NO, NO2 and H2O) and accumulate during
    the night-time hours.  Reaction 2-13 may lead to a build up of nitrous
    acid in urban air only during the late afternoon and evening hours
    when sunlight intensities are low but some OH radicals are still
    present.

    c)  Peroxynitric acid

         While peroxynitric acid (HO2NO2) has never been measured in the
    atmosphere, it is expected to be present in the upper troposphere. 
    Models suggest concentrations in the 10 to 100 ppt range at altitudes
    above 6 kilometres (Logan, 1983; Singh, 1987).  HO2NO2 is thermally
    unstable, so that boundary layer concentrations are expected to be
    extremely low (< 1 ppt).  Peroxynitric acid is formed through the
    combination of a hydroperoxy (HO2) radical with NO2.  In the upper
    troposphere, HO2NO2 is destroyed by photolysis or by reaction with
    OH radicals.

    d)  Peroxyacyl nitrates

         Peroxyacetyl nitrate (PAN) is the most abundant of this family of
    organic nitrates.  The second most abundant homologue, peroxypropionyl
    nitrate (PPN), is generally less than 10% of the PAN concentration,
    and species with higher relative molecular mass, such as PBzN, are
    expected to have even lower concentrations.  PAN is a strong oxidant
    and is known to be phytotoxic; it is formed from the reaction of
    acetylperoxy radical with NO:

              CH3C(O)OO + NO2 +M -> CH3C(O)O2NO2 +M                 (2-14)

         PAN is thermally unstable and so its lifetime is very dependent
    on ambient temperature.  For example, PAN lifetimes of about 5 and
    20 h have been calculated for 20C and 10C, respectively.

         In cold conditions PAN can serve as a reservoir for reactive
    nitrogen, which is liberated when the temperature of the air is
    increased.  PAN can be lost from the atmosphere by dry deposition over
    land, but it is very likely that a significant fraction of PAN
    produced in urban plumes can be transported into the regional
    environment.

    e)  Nitrate radical

         The nitrate (NO3) radical is a short-lived species formed mainly
    by the reaction of NO2 with O3, although other sources of NO3
    radicals exist (Wayne et al., 1991).

              NO2 + O3 -> NO3 + O2                                  (2-15)

         NO3 also reacts with NO2 to form N2O5

                        M
              NO2 + NO3 -> N2O5                                     (2-16)

         Nitrate radicals rapidly photolyse, resulting in a lifetime of
    about 5 seconds at midday.  They also react rapidly with NO, which
    limits their lifetime both during the day- and night-time hours.  At
    night if atmospheric NO concentrations are approximately 320 pptv,
    then the lifetime of NO3 radicals is similar to that at midday (about
    5 seconds).

         At night, NO3 concentrations range from about 0.3 ppt in clean
    tropospheric air to 70 ppt in urban areas (Biermann et al., 1988).  In
    clean background environments, it has been reported that measured NO3
    radical levels are significantly less than those predicted by the
    above reactions.  Several loss mechanisms have been suggested (Noxon
    et al., 1980; Platt et al., 1981): (i) NO3 radical reaction with

    organic compounds; (ii) heterogeneous losses of NO3 radicals and/or
    N2O5 on particle surfaces; (iii) reactions of NO3 radicals with
    H2O vapour; and (iv) reaction of NO3 radicals with NO.

    f)  Dinitrogen pentoxide

         N2O5 is formed from NO3 and NO2 (reaction 2-15).  Since NO3
    is present only at night, N2O5 is also primarily a night-time
    species.  N2O5 is thermally unstable, decomposing to NO3 and NO2
    (reaction 2-15).  At high altitudes in the troposphere, where
    temperatures are low, N2O5 can act as a temporary reservoir for
    NO3.  Dinitrogen pentoxide photolyses at wavelengths less than 330 nm
    to give NO3 and NO2.

         Dinitrogen pentoxide reacts heterogeneously with water to form
    HNO3.  This serves as the main night-time production mechanism for
    HNO3 and it provides an important route for removal of oxidized
    nitrogen from the atmosphere, since HNO3 is readily removed by dry
    and wet deposition.  Other atmospheric reactions of N2O5 include its
    reaction with gas-phase water to form HNO3 and possible reactions
    with aromatic VOCs such as naphthalene and pyrene (Pitts et al., 1985;
    Atkinson et al., 1986).  Nitroarenes appear to be the product of
    N2O5-aromatic reactions.

    2.4.3  Advection and dispersion of atmospheric nitrogen species

         The transport and dispersion of the various nitrogen species is
    dependent on both meteorological and chemical parameters.  Advection,
    diffusion and chemical transformations dictate the atmospheric
    residence time of a particular trace gas.  Nitrogenous species that
    undergo slow chemical changes in the troposphere and are not readily
    removed by depositional processes can have atmospheric lifetimes of
    several months.  Gases with lifetimes of the order of months can be
    dispersed over continental scales and possibly even over an entire
    hemisphere.  At the other extreme are gases that undergo rapid
    chemical transformation and/or depositional losses limiting their
    atmospheric residence times to a few hours or less.  Dispersion of
    these short-lived species may be limited to only a few kilometres from
    their point of emission.

         Surface emissions are dispersed vertically and horizontally
    through the atmosphere by turbulent mixing processes.  These processes
    are dependent to a large extent on the vertical temperature structure
    of the boundary layers and on wind speed.  In the vertical dimension,
    transport occurs as follows (see also Fig. 2.):

    a)   the daytime and/or night-time mixed layer; this layer can extend
         from the surface up to a few hundred metres at night or to
         several thousand metres during the daytime;

    FIGURE 3

    b)   a layer that can exist during the night-time above a low level
         surface inversion and below the daytime mixing height; this layer
         generally is situated between 200 and 2000 m altitude;

    c)   the free troposphere; this transport zone is above the boundary
         layer mixing region.

         During the warm, summertime period, vertical mixing follows a
    fairly predictable diurnal cycle.  A surface inversion normally
    develops during the evening hours and persists throughout the night-
    time and morning period until broken by sunlight heating the surface
    of the earth.  While the inversion is in place, surface NOx emissions
    can lead to relatively high local concentrations because of restricted
    vertical dispersion.  Following the break-up of the night-time surface
    inversion, vertical mixing will increase and surface-based emissions
    will disperse to higher altitudes.  The depth of the vertical mixing
    during the daytime is often controlled by synoptic weather features. 
    Temperature inversions aloft, associated with high pressure systems,
    are common in many parts of the world.

         The dispersion processes described above, coupled with the
    chemical transformations of reactive nitrogen compounds, determine the
    distances oxidized nitrogen will be transported in the troposphere.  A
    reasonable understanding exists concerning the short-term (daylight
    hours) fate of NOx emitted in urban areas during the morning hours. 
    As described above, NOx emitted in the early morning hours in an
    urban area will disperse vertically and move downwind as the day
    progresses.  On sunny summer days, most of the NOx will have been
    converted to HNO3 and PAN by sunset.  Much of the HNO3 will be
    removed by depositional processes as the air mass moves along.  After
    dusk, an upper portion of the daytime mixed layer will be decoupled
    from the surface because of formation of a low-level radiation
    inversion.  Transport will continue in this upper level during the
    night-time hours and, although photochemical processes will cease,
    dark-phase chemical reactions can proceed.  Peroxyacetyl nitrate and
    HNO3, if carried along in this layer, can be transported long
    distances.

    2.4.3.1  Transport of reactive nitrogen species in urban plumes

         Overall removal rates for reactive nitrogen species during
    daytime at mid-latitudes have been measured or calculated for a few
    areas.  For example, in the plume from Boston, USA, after correction
    for dilution, removal rates ranged from 0.14 to 0.24 h-1 on 4 days
    (Spicer, 1982, Altshuller, 1986).  In Los Angeles and Detroit, the
    removal rate has been estimated to be 0.04-0.1 h-1 (Calvert, 1976;
    Chang et al., 1979; Kelly, 1987).  Formation and removal of HNO3 is
    thought to be the rate-controlling step for removal of reactive
    nitrogen.

    2.4.3.2  Air quality models

         Air quality models are mathematical descriptions of pollutant
    emissions, atmospheric transport, diffusion and chemical reactions
    of pollutants.  However, air quality models are very complex and
    difficult to test for validity.  Inputs include emissions, topography
    and meteorology of a region.  Air quality models represent an
    integration of knowledge for the chemistry and physics of the
    atmospheric system; they offer some predictive capability for the
    effectiveness of pollution control strategies.  Models have also been
    developed for indoor air.

    2.4.3.3  Regional transport

         Transport of reactive nitrogen species in regional air masses can
    involve several mechanisms.  Mesoscale phenomena, such as land-sea
    breeze circulations or mountain-valley wind flows, will transport
    pollutants over distances of ten to hundreds of kilometres.  On a
    larger scale, synoptic weather systems such as the migratory highs
    that cross the eastern USA and other areas of the world in the
    summertime influence air quality over many hundreds of kilometres. 
    The accumulation and fate of nitrogen compounds will differ somewhat
    between the mesoscale and synoptic systems.  Mountain-valley and land-
    water transport mechanisms have dual temporal scales because of their
    dependence on solar heating.  However, in the larger-scale synoptic
    systems, reactive nitrogen species can build up over multiday periods. 
    The residence time of air parcels within a slow-moving high pressure
    system can be as long as 6 days (Vukovich et al., 1977).

         In many cases, the transport mechanisms mentioned above are
    interrelated.  Mountain-valley or land-water breezes can dictate
    pollutant transport in the immediate vicinity of sources, but the
    eventual fate of reactive nitrogen species will be distribution into
    the synoptic system.

    2.5  Conversion factor for nitrogen dioxide

         1 ppm     = 1.88 mg/m3
         1 mg/m3   = 0.53 ppm

    2.6  Summary

         Combustion provides the major source of oxides of nitrogen in
    both indoor and outdoor air, producing mostly NO with some NO2.  The
    sum of NO and NO2 is generally referred to as NOx.  Once released
    into the air, NO is oxidized to NO2 by available oxidants,
    particularly O3, and by photochemical reactions involving reactive
    organic compounds.  This happens rapidly under some conditions in
    outdoor air; for indoor air, it is generally a much slower process. 
    Nitrogen oxides are a controlling precursor of ozone and smog

    formation; interactions of nitrogen oxides (except N2O) with reactive
    organic compounds and sunlight form ozone in the troposphere and smog
    in urban areas.

         In both indoor and outdoor air, NO and NO2 may undergo reactions
    to form a suite of other nitrogenous species including HNO2, HNO3,
    NO3, N2O5, PAN and other organic nitrates.  The complete suite of
    gas-phase nitrogen oxides is referred to as NOy.  The partitioning
    of nitrogen among these compounds is strongly dependent on the
    concentrations of other oxidants, sunlight exposure, the presence of
    reactive organic compounds and the meteorological history of the air.

         A sensitive, specific and reliable analytical method exists for
    measuring NO (by the chemiluminescent reaction with ozone), but this
    is an exception for NOy species.  Chemiluminescence is also the
    most common technique used for NO2, which is first reduced to NO. 
    Unfortunately, the method of reduction usually used is not specific
    for NO2, and it has various conversion efficiencies for other
    oxidized nitrogen compounds that may also be present in the air
    sample.  For this reason, care must be taken in interpreting the NO2
    values given by the common chemiluminescence analyser, as the signal
    may include responses from interfering compounds.  Additional
    difficulties arise from nitrogen species such as HNO3 that may
    partition between the gas and particulate phases both in the
    atmosphere and in the sampling procedure.

    3.  SOURCES, EMISSIONS AND AIR CONCENTRATIONS

    3.1  Introduction

         Oxides of nitrogen can have significant concentrations in ambient
    air and in indoor air.  The types and concentrations of nitrogenous
    compounds present can vary greatly from location to location, with
    time of day, and with the season.  The main sources of nitrogen oxides
    emissions are combustion processes.  Fossil fuel power stations, motor
    vehicles and domestic combustion appliances emit nitrogen oxides,
    mostly in the form of NO but with some (usually less than about 10%)
    in the form of NO2.  In the air chemical reactions occur which
    oxidize NO to NO2 and other products (chapter 2).  Also, there are
    biological processes in soils which liberate nitrogen species,
    including N2O.  Emissions of N2O can cause perturbation of the
    stratospheric ozone layer.

         Human health may be affected when significant concentrations of
    NO2 or other nitrogenous species, such as PAN, HNO3, HNO2 and
    nitrated organic compounds, are present.  In addition, nitrates and
    nitric acid can cause significant effects on ecosystems when deposited
    on the ground.

         Indoors, the use of combustion appliances for cooking and heating
    can give rise to greater NO and NO2 concentrations than are present
    outdoors, especially when the appliance is not vented to the outside. 
    Recent research has shown that in these circumstances nitrous acid can
    reach significant concentrations (Brauer et al., 1993).

         This chapter discusses both ambient and indoor sources
    of nitrogenous compounds, their emissions, and the resulting
    concentrations that may directly affect human health or participate
    in atmospheric chemical pathways leading to effects on human health
    and welfare.  Nitrogen-containing compounds are also of particular
    interest because of their secondary impacts.  For example, production
    of photochemical smog and ozone pollution depends on emissions to the
    air of nitrogen oxides together with volatile organic compounds. 
    Nitric acid, which is produced in the air by the reaction of hydroxyl
    radicals (OH*) with NO2, is one of the major components of acidic
    precipitation.  As well as being present in the gas phase, oxidized
    nitrogen can, by reaction and adsorption, become incorporated into
    aerosol particles.  Graedel et al. (1986) identified 20 inorganic
    nitrogen-containing species detectable in the atmosphere.  Near
    cities and urban regions the species usually present in greatest
    concentrations are NO and NO2, and these are the most reliably
    measured and frequently monitored nitrogen oxide species.

         Knowledge of emission patterns and concentrations of nitrogenous
    compounds is critically important for air quality planning and human
    health and environment risk assessments.  Because nitrogen oxides and

    their reaction products have lifetimes of several days in the
    atmosphere, they can be transported long distances by the wind and
    give rise to environmental impacts far from their source of emission.

    3.2  Sources of nitrogen oxides

         Combustion systems emit NO and NO2 and together these species
    are usually denoted as NOx.

         When NOx emissions are expressed in mass units, the mass is
    expressed as if all the NO had been converted to NO2.  Another
    convention adopted in some of the following sections is to report the
    emissions on a mass basis in terms of the nitrogen content.

    3.2.1  Sources of NOx emission

    3.2.1.1  Fuel combustion

         Annual production of NOx from combustion of fossil fuels is
    typically estimated from emission factors for various combustion
    processes, combined with worldwide consumption data for coal, oil and
    natural gas.  Logan (1983) provided a tabular summary of emission
    factors, which has been updated by the US National Acid Precipitation
    Assessment Program (Placet et al., 1991).  Owing to variations in
    process operating conditions, the emission factors must be considered
    to be uncertain by about  30%.  Table 3 provides a summary of global
    emission estimates for NOx according to fuel type.  The estimates of
    Logan (1983) are slightly higher than those of Ehhalt & Drummond
    (1982), the largest discrepancies being in emission estimates for the
    transportation sector.  The differences arise because Logan (1983)
    based estimates of emissions on fuel usage, while Ehhalt & Drummond
    (1982) scaled the totals somewhat indirectly by using world automobile
    population numbers.

         Dignon (1992) has assembled a database for mapping (with a
    resolution of one degree in latitude and longitude) and estimated
    global NOx and sulfur oxides emissions from their common principal
    anthropogenic source, i.e. fossil fuel combustion.  For 1980, the
    global total was estimated to be 22 million tonnes, as nitrogen. 
    Countries heading the list (in millions of tonnes of nitrogen per
    year) were: USA, 6.4; USSR, 4.4; China, 1.7; Japan, 0.80; and Federal
    Republic of Germany, 0.66.  An estimated 95% of NOx emissions from
    fossil fuel combustion originates in the northern hemisphere.

         For oceanic regions, shipping is a source of NOx emissions. 
    Aircraft also emit nitrogen oxides and this may be significant for the
    upper troposphere and stratosphere.

        Table 3.  Estimates of global emissions of nitrogen oxides (NOx) from combustion of fossil fuels and biomass (from: US EPA, 1993)a
                                                                                                                                              

    Source type            Annual consumption                             Emission factorsb             Global source strength
                           (106 tonnes, unless indicated otherwise)                                     (106 tonnes nitrogen/year)
                                                                                                                                              
                           (E & D)          (L)            (C et al.)     (E & D)        (L)            (E & D)         (L)        (C et al.)
                                                                                                                                              

    Fossil fuelsc

      Hard coal            2150             2696           -              1.0-2.8        2.7            3.9 (1.9-5.8)   6.4        -
      Lignite              810                             -              0.9-2.7                       1.6 (0.8-2.3)              -
      Light fuel oil       300              1.39           -              1.5-3.0        2.2d           0.7 (0.5-0.9)   3.1        -
      Heavy fuel oil       470                                            1.5-3.1                       1.1 (0.7-1.5)              -
      Natural gas          1.04             1.2  109 m3   -              0.6-3.0        1.9d           1.9 (0.6-3.1)   2.3        -
      Industrial sources   -                               -                                            -               1.2        -
      Automobiles          (4.1-5.4)        1.0  109 m3   -              0.9-1.2e       8.0d           4.3 (3.7-6.4)   8.0
                            1012 km

      Total                                                                                             13.5 (8.2-18.5) 19.9

    Biomass burningf

      Savanna              (6-14)  103     2000           1200           1.0            1.7            3.1 (1.8-4.3)   3.4        2.1
      Forest clearings     (2.7-6.7)  103  4100           2700           1.0-1.6        2.0            2.1 (0.8-3.4)   8.2        4.7
      Fuel wood            -                850            1100           -              0.5            2.0 (1-3)       0.4        0.5
      Agricultural waste   -                15             1900           -              1.6            4.0 (2-6)       0.02       3.3

      Total                                                                                             11.2 (5.6-16.4) 12.0       10.6
                                                                                                                                              

    a  Estimates according to Ehhalt & Drummond (1982) (E & D) and Logan (1983) (L). Ranges are given in parentheses.
    b  Emission factors refer to grams of nitrogen per kg of fuel consumed, unless indicated otherwise
    c  Petroleum refining and manufacture of nitric acid and cement; global emissions were obtained by scaling USA emissions for each
       industrial process
    d  Grams of nitrogen per m3 of fuel consumed
    e  Grams of nitrogen per km
    f  For biomass-burning, Crutzen et al. (1979) (C et al.) have given annual consumption rates differing somewhat from those of the other
       authors.  The data of Crutzen et al. (1979) and the resulting nitrogen oxides production rates are included for comparison
        3.2.1.2  Biomass burning

         Table 3 includes a breakdown of estimates for release of NOx
    from burning of biomass.  In natural fires and the burning of wood,
    temperatures are rarely high enough to cause oxidation of nitrogen
    molecules of the air.  The emissions are thereby more closely related
    to the fixed nitrogen content of the fuel.  Logan (1983) reviewed a
    number of experimental determinations of nitrogen emission factors
    that indicate yields are highest for grass and agricultural refuse
    fires (1.3 g nitrogen/kg fuel), less for prescribed forest fires
    (0.6 g nitrogen/kg fuel), and still lower for burning of fuel wood in
    stoves and fireplaces (0.4 g nitrogen/kg fuel).  The values roughly
    reflect differences in nitrogen content of the materials burned. 
    Biomass burning is mainly associated with agricultural practices in
    the tropics, which include plant, slash, and shift practices as well
    as natural or intentional burning of savanna vegetation at the end of
    the dry season.  Forest wildfires and use of wood as fuel make a
    lesser contribution.

    3.2.1.3  Lightning

         Thunderstorm activity has been viewed as a major NOx source
    since 1827, when Von Liebig proposed it as a natural mechanism for
    fixation of atmospheric nitrogen.  Electrical discharges in air
    generate NOx by thermal dissociation of nitrogen molecules due to
    ohmic heating inside the discharge channel and shockwave heating of
    the surroundings.  Laboratory studies by Chameides et al. (1977) and
    Levine et al. (1981) indicate an NOx yield of 6  1016 molecules per
    joule of spent energy.  Great uncertainties exist, however, about the
    total energy generated by lightning in the atmosphere.  Noxon (1976,
    1978) first studied the increase of NOx in the air during a
    thunderstorm.  His results provide the basis for many of the estimates
    shown in Table 4.  Reviews by Kowalczyk & Bauer (1981) Borucki &
    Chameides (1984) and Albritton et al. (1984) provide a best estimate
    of annual generation by lightning: 1 million tonnes of NOx in North
    America and 13 million tonnes globally (Placet et al., 1991).

    3.2.1.4  Soils

         The biochemical release of NOx from soils is poorly understood,
    and the flux estimates must be viewed with caution.  Both rely on the
    observations by Galbally & Roy (1978), who used the flux box method in
    conjunction with chemiluminescence detection of NOx.  They found
    average fluxes of 5.7 and 12.6 g nitrogen/m2*h on ungrazed and
    grazed pastures, respectively, where NO was the main product.  More
    recent measurements of Slemr & Seiler (1984) indicate that the release
    of NOx from soils depends critically on the temperature and moisture

    content of the soil, which in turn complicates the estimate of the
    global emissions.  Slemr & Seiler (1984) also found an average release
    rate of 20 g nitrogen/m2 per h for uncovered natural soils, evenly
    divided between NO and NO2. Grass coverage reduced the escape flux,
    whereas fertilization enhanced it.  Ammonium fertilizers were about
    five times more effective than nitrate fertilizers.  This suggests
    that nitrification as a source of NOx is more important than
    denitrification.  According to Slemr & Seiler (1984), an annual global
    flux of 10 million tonnes of nitrogen represents an upper limit to the
    release of NOx from soils.  Galbally et al. (1985) presented more
    detailed estimates for arid lands, and Table 4 provides a compilation
    of current literature used to develop the global budgets.  Soil is
    also a source of N2O and NH3 emissions.

         In the presence of low concentrations, plants can emit NH3,
    rather than absorb it.  This is especially true with scenescing and
    with highly fertilized plants (Grnhage et al., 1992; Holtan-Hartwig &
    Bockman 1994; Fangmeijer et al., 1994).  Release to the atmosphere of
    N2 and NO by plants has also been reported.  In some cases this was
    part of the response following exposure to nitrogen-containing
    pollutants, but other mechanisms are involved (Wellburn, 1990). NO and
    N2O are emitted in significant quantities by the soil.  The reason
    why the deposition velocity of NO is relatively low see (see Table 5)
    is partly due to the fact that the downward flux (and uptake by the
    canopy) is "mathematically" compensated by soil emissions.  In other
    words: a low deposition velocity does not always mean that the uptake
    by the vegetation is low.  In the case of N2O, soil emissions are
    mostly larger than deposition; this emission is the result of
    denitrification and is positively related to the nitrogen and water
    content and the temperature of the soil.  This is why the release of
    nitrogen from the ecosystem in the form of N2O is dependent on the
    ecosystem type, climate and land use (fertilization and water table
    height).  Skiba et al. (1992) estimated for the United Kingdom the NO
    and N2O emissions from agricultural land to be 2-6% of the nationwide
    NOx emissions and 16-64% of the N2O emissions, respectively.

        Table 4.  Global and North America natural emissions (average and range) of nitrogen oxides (NOx)
              from lightning, soils and oceans
                                                                                                          

                  Global               North America        Reference
                  (106 tonnes/year)    (106 tonnes/year)
                                                                                                          

    Lightning     8.6 (2.6-26)                              Borucki & Chameides (1984)
                  18                   1.7                  Albritton et al. (1984)
                  13 (7-26)            1 (0.3-2)            Kowalczyk & Bauer (1981); Placet et al. (1991)

    Soils         50 (as NO2)                               Lipschultz et al. (1981)
                  30 (as NO)                                Levine et al. (1984); Galbally & Roy (1978)
                  36                                        Slemr & Seiler (1984)
                                       2                    Placet et al. (1991)

    Oceans        0.35                                      Zafiriou & McFarland (1981); Logan (1983)
                                                                                                          
    
    Table 5.  Deposition velocity of nitrogen-containing gases and
              aerosols
                                                                        

                 Deposition velocity   Reference
                 (mm/second)
                                                                        

    NO2          0.1-10                Grennfelt et al. (1983);
                                       Anonymous (1991)

    NO           0.2-1                 Prinz (1982)

    NH3          12 (-5 - +40)         Grnhage et al. (1992);
                                       Sutton et al. (1993);
                                       Fangmeijer et al. (1994);
                                       Holtan-Hartwig & Bockman (1994)

    NH4+         1.4 (0.03-15)         Fangmeijer et al. (1994)
                                                                        

         Estimates of global emissions of N2O and ammonia are summarized
    in Table 6.


    Table 6.  Annual global estimates (average and range) of N2O and
              NH3 emissions to the troposphere (106 tonnes
              of nitrogen)
                                                                        

    Source             N2O          NH3      Reference
                                                                        

    Soils              10 (2-20)    15       Dawson (1977);
                                             Boettger et al. (1978)

    Ocean              26 (12-38)            Hahn (1981)

    Biomass burning    2            2-8      Crutzen et al. (1979);
                                             Crutzen (1983)

    Fossil fuels       1.6          0.2      Weiss & Craig (1976);
                                             Boettger et al. (1978)

    Fertilizer         0.1          3        Boettger et al. (1978);
                                             Crutzen et al. (1979);
                                             Crutzen (1983);
                                             Stedman & Shetter (1983)

    Domestic animals                22       Soederlund & Svensson (1976)
                                             Boettger et al. (1978);
                                             Crutzen et al. (1979);
                                             Crutzen (1983);
                                             Stedman & Shetter (1983)
                                                                        

    3.2.1.5  Oceans

         There have been few measurements of NOx, N2O or NH3 fluxes
    over the ocean, and current literature suggests that the sea is a
    negligible source of NO.  Zafiriou & McFarland (1981) observed a
    supersaturation of seawater with regard to NO in regions of relatively
    high concentrations of nitrite, owing to upwelling conditions. The

    excess NO must, in this case, arise from photochemical decomposition
    of nitrite by sunlight.  Logan (1983) estimated a local source
    strength of 1.3  1012 molecules/m2 per second under these
    conditions.  Linear extrapolation results in an annual global flux
    estimate of 350 000 tonnes of nitrogen.

    3.2.2  Removal from the ambient environment

         Wet precipitation and dry deposition provide two of the major
    mechanisms for removal of NOx from the atmosphere.  The addition to
    the plant soil ecosystem of nitrate (and ammonium) by rainwater
    constitutes an important source of fixed nitrogen to the terrestrial
    biosphere, and until 1930 practically all studies of nitrate in
    rainwater were concerned with the input of fixed nitrogen into
    agricultural soils.  Eriksson (1952) and Boettger et al. (1978) have
    compiled many of the available data.  Despite the wealth of
    information, it remains difficult to derive a global average for the
    deposition of nitrate, because of an uneven global coverage of the
    data, unfavourably short measurement periods at many locations, and
    inadequate collection and handling techniques for rainwater samples. 
    In addition, the concentration of nitrate in rainwater has increased
    in those parts of the world where the utilization of fossil fuels has
    led to a rise in the emissions of NOx, i.e. primarily western Europe
    and the USA.

         Dry deposition is important as a sink for those gases that are
    readily absorbed by materials covering the earth surface.  In the
    budget of NOx, the gases affected most by dry deposition are NO2 and
    HNO3.  The deposition velocity of NO is too small and the
    concentration of peroxyacetyl nitrates is not high enough for a
    significant contribution.

         According to Grennfelt et al. (1983) and Wellburn (1990), NO3-
    and HNO3 have a higher deposition velocity then NH3, but this was
    not quantified. HNO2 is assumed to have a deposition velocity equal
    to SO2: 1-30 mm/second (Table 5).

         There are several other nitrogen-containing air pollutants with
    relatively high deposition velocities.  These generally add only small
    amounts to the total nitrogen deposition, because most of the time
    their ambient concentrations are relatively low.

         Atmospheric nitrogen deposition can significantly change the
    chemical composition of the soil. In the rooting zone these changes
    have an impact on vegetation. The changes in deeper soil layers
    are particularly relevant if groundwater is used as a source of
    drinking-water. Groundwater under fertilized agricultural land can be
    heavily polluted with nitrate (and aluminium), but this is beyond the
    scope of this chapter. Due to atmospheric nitrogen deposition, the

    groundwater under forests and other non-fertilized vegetation can
    become polluted with nitrate.  For instance, in 20% of the forested
    area of the Netherlands, the nitrate concentration in phreatic
    groundwater is higher than 50 mg/litre (the EC drinking-water
    standard); in 37% it is higher than 25 mg/litre (Boumans & Beltman,
    1991).  The average annual nitrogen deposition in the Netherlands is
    45 kg/ha; approximately 10 kg/ha is from dry deposition of NOx.  The
    nitrate concentration in groundwater is strongly related to the soil
    type.  With the same atmospheric deposition, the nitrate concentration
    increases as follows: peaty soils < moderately drained sandy soils
    < well-drained rich sandy soils (Boumans, 1994).  A distinct relation
    also exists concerning the age of the trees: tree stands in Wales
    showed nitrate leaching (measured in the stream water draining the
    catchments), but only with stands older than 30 years. Younger trees
    used the nitrogen as nutrient, but the nitrogen demand of the older
    trees was lower.  The annual nitrogen deposition in that region was
    estimated to be 20 kg/ha (Emmett et al., 1993).

    3.2.3  Summary of global budgets for nitrogen oxides

         The principal routes to the production of NOx are combustion
    processes, nitrification and denitrification in soils, and lightning
    discharges.  The major removal mechanism is oxidation to HNO3,
    followed by wet and dry deposition.  In developing Table 7, the dry
    deposition velocities for NO2 over bare soil, grass and agricultural
    crops were assumed to fall in the range of 3 to 8 mm/second.  However,
    over water the velocities are significantly smaller, so that losses of
    NO2 by deposition onto the ocean surface can be ignored.  The
    absorption of nitric acid by soil, grass and water is rapid, and dry
    deposition correspondingly important, but the global flux is difficult
    to estimate because information on HNO3 mixing ratios is still
    sparse.  Logan (1983) adopted NO mixing ratios of 50 pptv over the
    oceans and 100 pptv over the continents.  The mixing ratios assumed
    for NO2 were 100 and 400 pptv, respectively.  Allowance was made for
    higher mixing ratios in industrialized areas affected by pollution. 
    Logan (1983) included the deposition of particulate nitrate over the
    oceans, using a settling velocity of 3 mm/second.  This process
    contributes 2 million tonnes nitrogen/year to a total dry deposition
    rate of 12 to 22 million tonnes nitrogen/year.

         Efforts by Boettger et al. (1978), Ehhalt & Drummond (1982),
    Galbally et al. (1985) and Warneck (1988) to quantify the sources and
    sinks have led to an improved understanding of the global budget of
    NOx, in which the flux of NOx into the troposphere and the rate of
    nitrate deposition are approximately balanced.  Ehhalt & Drummond
    (1982) relied on the detailed evaluation of data by Boettger et al.
    (1978).  Their analysis emphasized measurements from the period 1950
    to 1977, and they prepared a world map for nitrate deposition rates, 

    which were then integrated along 5 latitude belts.  Logan (1983)
    considered recent network data from North America and Europe; Galloway
    et al. (1982) reported measurements of nitrate in precipitation at
    remote locations in Alaska, South America, Australia and the Indian
    Ocean.  Both estimates gave wet nitrate deposition rates in the range
    of 2 to 14 million tonnes nitrogen/year for the marine environment and
    8 to 30 million tonnes nitrogen/year on the continents.  An earlier
    appraisal by Soederlund & Svensson (1976) led to rather similar
    values, i.e. 5 to 16 and 13 to 30 million tonnes nitrogen/year,
    respectively, although it was primarily based on Eriksson's (1952)
    compilation of data from the period 1880 to 1930.

    Table 7.  Global budget (average and range) of nitrogen oxides
              in the troposphere (from US EPA, 1993)a
                                                                        

    Type of source or sink                       Global flux
                                         (106 tonnes nitrogen/year)
                                                                        

                                            Ehhalt &       Logan (1983)
                                         Drummond (1982)
                                                                        

    Production

     Fossil-fuel combustion              13.5 (8.2-18.5)  21 (14-28)
     Biomass burning                     11.2 (5.6-16.4)  12 (4-24)
     Release from soils                  5.5 (1-10)       8 (4-16)
     Lightning discharges                5.0 (2-8)        8 (2-20)
     NH3 oxidation                       3.1 (1.2-4.9)    uncertain (1-10)
     Ocean surface (biologic)            -                < 1
     High-flying aircraft                0.3 (0.2-0.4)    -
     Stratosphere                        0.6 (0.3-0.9)    approx. 0.5

    Total production                     39 (19-59)       50 (25-99)

    Losses

     Wet deposition of NO3-, land        17 (10-24)       19 (8-30)
     Wet deposition of NO3-, oceans      8 (2-14)         8 (4-12)
     Wet deposition, combined            24 (15-33)       27 (12-42)
     Dry deposition of NOx               -                16 (12-22)

    Total loss                           24 (15-40)       43 (24-64)
                                                                        

    a  Derived from estimates according to Ehhalt & Drummond (1982)
       and Logan (1983)

         On continents, one should also consider the interception of
    aerosol particulates by high growing vegetation.  The interception of
    nitrate is expected to be particularly effective.  Hoefken &
    Gravenhorst (1982) studied the enrichment of nitrate in rainwater
    collected underneath forest canopies compared to that collected in
    open areas outside forests.  The effect is caused by the wash-off of
    dry-deposited material from foliage.  Hoefken & Gravenhorst (1982)
    found that, in a beech forest, nitrate was enhanced by a factor of
    1.4, whereas in a spruce forest enhancement by a factor of 4.1
    occurred.  Unfortunately, they were unable to differentiate between
    contributions of particulate nitrate versus gaseous nitrate to the
    total dry deposition.

         If losses of NO2 and HNO3 by dry deposition are included in the
    total budget of NOx, one obtains a reasonable balance between the
    sources and sinks, as Table 7 shows.  Ehhalt & Drummond (1982) noted
    that an appreciable part of their dry deposition is already included
    in their wet deposition rates, because rain gauges frequently are left
    open continuously, so that the collection of nitrate occurs during
    both wet and dry periods.  For NO2, they estimated a dry deposition
    rate of 7 million tonnes nitrogen/year.  Because of the uncertainty,
    they chose to include it in the error bounds and not in the mean value
    of total NOx-derived nitrogen deposition.  Clearly, the total budget
    of NOx is far from being well defined.  Moreover, in view of the
    relatively short residence times of chemical species involved in the
    NOx cycle, it is questionable whether a global budget gives an
    adequate description of the tropospheric behaviour of NOx and its
    reaction products.  Supplemental regional budgets could be more
    appropriate.

    3.3  Ambient concentrations of nitrogen oxides

         Because cities usually have an aggregation of emissions sources
    ambient concentrations of NO and NO2 tend to be greatest in cities. 
    High concentrations of NO are common in street canyons, owing to motor
    vehicle emissions.  In rural areas the emissions may have spent
    considerable time in the atmosphere and have undergone reactions to
    produce significant concentrations of other species, such as HNO3 and
    PAN.

    3.3.1  International comparison studies of NOx concentrations

         Data for monthly average concentrations of NOx collected by the
    World Meteorological Organization at five background locations in
    Europe for the period 1983 to 1985 are summarized in Fig. 3 (WMO,
    1988, 1989).  Fig. 4 presents published monthly averages of NO2 in
    1987 for 12 stations in a cooperative network under the Organisation
    for Economic Co-operation and Development (OECD) (Grennfelt et al.,
    1989).  These two figures show that concentrations of both NOx and
    NO2 tend to be higher during winter months. 

    FIGURE 4

    FIGURE 5

         Measurements of NO2 in several countries during the late 1970s
    and early 1980s are summarized in "Assessment of Urban Air Quality"
    (WHO, 1988).  The trends in composite annual averages for urban NO2
    monitoring stations in five countries are portrayed in Fig. 5 for the
    period 1975 to 1985.  The trend in the Canadian data appears to have
    been downward, but essentially stable trends were evident for data
    from the other countries.  Annual averages in the 1980-1984 period for
    42 cities around the world are summarized in the same report (WHO,
    1988).  During that period, only one city, Sao Paulo, reported an
    annual average greater than 0.053 ppm (100 g/m3).

         Short-term peak values (1-h or 30-min maxima, or 98th or 95th
    percentile values) have been reported for 18 cities during the
    1980-1984 period (WHO, 1988).  Ten of these cities (Amsterdam,
    Brussels, Hamilton, Hong Kong, Jerusalem, Montreal, Munich, Rotterdam,
    Tel Aviv and Toronto) reported values above the WHO 1-h guideline
    level of 400 g/m3 (0.21 ppm) for at least one year during that
    5-year period.  For eleven cities in the WHO report, both the annual
    average and a "1-hour" peak statistic were reported for the 1980-1984
    period.  Fig. 6 compares these two statistics.  It shows that three
    cities, Amsterdam, Jerusalem and Tel Aviv, reported an average peak
    value above the WHO 1-hour guideline value of 400 g/m3 (0.21 ppm). 
    It should be kept in mind that the peak-value statistic is more
    susceptible to undetected spurious measurements than is the annual
    average.  Data from the remaining eight cities place them in the
    quadrant below the target levels for both the annual average and the
    1-hour peak.  A similar situation is seen in the majority of cities in
    the USA and is discussed in the next section.

         More recent data on NO2 trends in the world's largest cities
    have been reported by WHO/UNEP (1992) in the monograph "Urban Air
    Pollution in Megacities of the World".  Such trends for six selected
    cities from various regions of the world are illustrated in Fig. 7, a
    composite of figures extracted directly from the WHO/UNEP (1992)
    report.  In general, the overall trends appeared to be relatively
    stable for most of the cities (and/or specific neighbourhoods). 
    However, there were a few exceptions, e.g., an apparent decrease in
    the late 1980s for Bombay and an apparent increase during the same
    period for some areas of Moscow.  There are substantial differences in
    the concentrations reported for different cities.

         Table 8 summarizes emissions of nitrogen oxides and ambient
    monitoring data from the WHO/UNEP (1992) report for the years
    indicated.  Included are estimates for total emissions and percentages
    attributed to mobile sources, primarily private motor vehicles and
    public land transport systems.  However, the quality and type of
    information contained in the report is mixed, reflecting a variety of
    monitoring methods and reporting policies in different countries. 
    Ambient data in some cities was reported as NOx, and in others as
    NO2; reporting periods varied from one hour to one year.

    FIGURE 6

    FIGURE 7

    FIGURE 7a

    FIGURE 7b

    FIGURE 7c

        Table 8.  Estimated mobile and stationary source emissions of nitrogen oxides in 
              megacities (from: WHO/UNEP, 1992)a
                                                                                                    

    City                Total emissions of       Mobile source       Ambient concentration
                        nitrogen oxides          contribution        (g/m3)
                        (tonnes/year)            (%)
                                                                                                    

    Bangkok             60 000 (1990)            30                  max 1 h NOx (as NO2)
                                                                     270 at one site; < 320 at
                                                                     three stations (1987)

    Beijing             na

    Bombay              56 000 (1990)            52                  NO2 70-85 (annual 98th
                                                                     percentile, 1990)

    Buenos Aires        27 000 (1989)            48                  na

    Cairo               24 700 (1989)            23                  NOx 380-1400 (1979,
                                                                     monthly means; single
                                                                     study)

    Calcutta            36 550 (1990)            29

    Delhi               73 000 (1990)            20                  NO2 500 (1990, 8 h)
                                                 (mostly diesel)

    Jakarta             20 500 (1989)            75                  NOx 28 (1990, annual mean)

    Karachi             50 000 (1989)            38                  38-544 (12-13 June 1988;
                                                                     single study)
                                                                                                    

    Table 8.  (Con't)
                                                                                                    

    City                Total emissions of       Mobile source       Ambient concentration
                        nitrogen oxides          contribution        (g/m3)
                        (tonnes/year)            (%)
                                                                                                    

    London              79 000 (1983)            75 (1984)           NO2 max 1 h 867; > 600
                                                                     for 8 h; > 205 for 72 h
                                                                     (episode 12-15 Dec. 1991);
                                                                     98th percentile > 135;
                                                                     50th percentile > 50 (1989);
                                                                     NO recorded but not
                                                                     reported

    Los Angeles         440 000 (1987)           76                  NO2 max 1 h 526; > 400
                                                                     at 8 out of 24 stations (1990)

    Manila              119 000 (1990 -          90                  na
                        dubious accuracy)

    Mexico City         177 300 (1991)           75                  NO2 hourly maxima
                                                                     301-714 (1986-91)

    Moscow              210 000 (1990)           19                  NO2 max daily means
                                                                     100-150

    New York            120 000 New York         na                  NO2 1 h max 402; daily
                        City; 513 000 New                            max 160; annual mean 87
                        York metropolitan                            (1990)
                        area (1985) 

    Rio de Janeiro      63 000 (1978)            92                  na

    Sao Paulo           245 000 (1988)           82                  NO2 max 1 h
                                                                     600-1500 (1988)
                                                                                                    

    Table 8.  (Con't)
                                                                                                    

    City                Total emissions of       Mobile source       Ambient concentration
                        nitrogen oxides          contribution        (g/m3)
                        (tonnes/year)            (%)
                                                                                                    

    Seoul               270 000 (1990)           78                  NO2 annual means only

    Shanghai            127 000 (1983);          na                  NOx annual mean 50;
                        1991 emissions                               indoor level 90
                        assumed 50%
                        higher, i.e.
                        approx. 190 000

    Tokyo               52 700 (1985)            67% from motor      daily mean 98th percentile
                                                 vehicles; 5% from   > 115 tolerable level at
                                                 ship and aircraft   25% of stations
                                                                                                    

    a  na = not available
    
         As shown in Table 8, of importance for air quality management is
    the large contribution of NOx from motor vehicles reported for some
    cities and the continuing growth in this contribution.  For example,
    emissions from vehicles in Bombay (about 29 000 tonnes per year in
    1990) are expected to increase by an additional 14 600 tonnes/year by
    the year 2000 (WHO/UNEP, 1992).

         Estimates for Jakarta attribute some three-quarters of NOx
    emissions to motor vehicles, which is comparable with London, Los
    Angeles and Mexico City.  Data from Manila indicate that some 90% of
    NOx originates from motor vehicles.

    3.3.2  Example case studies of NOx and NO2 concentrations

         Data from a range of countries and locations are given in Table 9
    (Agra, India) and Tables 10 and 11 (various cities in China).


    Table 9.  Concentrations of NO2 measured in the vicinity of the
              Taj Mahal, Agra Indiaa
                                                                        

              Year           Mean monthly concentration range (g/m3)
                                                                        

              1987                        5.5 to 41.9
              1988                        6.3 to 33.1
              1989                        4.2 to 15.2
                                                                        

    a  Highest concentrations tend to occur in winter
       Personal communication from R.R. Khan, Ministry of Environment and
       Forests, New Delhi, India (1994)

         In urban areas in the USA, hourly patterns at fixed-site ambient
    air monitors often follow a bimodal pattern of morning and evening
    peaks, related to motor vehicular traffic patterns. Sites affected by
    large stationary sources of NO2 (or NO that reacts to produce NO2)
    are often characterized by short episodes at relatively high
    concentrations, as the plume moves to downwind areas.

         Since 1980, the annual average level among NO2-reporting
    stations in the USA has been below 0.03 ppm, with no significant
    trend evident.  This is exemplified in Fig. 8 (US EPA, 1991) by
    annual averages for the period 1980 to 1989 for 60 metropolitan
    areas subdivided into three population categories: 16 areas with a
    population of 250 000 to 500 000, 14 with 500 000 to one million, and

        Table 10.  Annual average NOX concentration (g/m3) in China from 1981 to 1990a
                                                                                                                      

        Year      Cities all over China               Southern cities                    Northern cities
                                                                                                                      

                  Concentration    Annual             Concentration       Annual         Concentration       Annual
                  range            average            range               average        range               average
                                                                                                                      

        1981      10-90            50                 10-80               40             20-90               60
        1982      10-110           45                 10-90               40             30-110              50
        1983      9-94             46                 9-79                36             29-94               55
        1984      10-95            42                 13-75               37             10-95               46
        1985      13-49            50                 13-84               41             22-49               59
        1986      14-108           48                 14-98               41             18-108              55
        1987      17-199           56                 17-60               43             30-199              69
        1988      9-110            45                 9-110               42             8-120               48
        1989      10-140           47                 10-133              43             12-140              51
        1990      7-130            43                 12-71               38             7-130               47
                                                                                                                      

    a  General Environmental Monitoring Station of China (1991)

    Table 11.  Statistical data for the percentiles of ambient annual average NOx concentrations (g/m3) for Chinese cities (1986-1990)a
                                                                                                                               

    Year      Number      Minimum   Percentile                            Maximum    Arithmetic              Geometric
              of cities   value                                           value                                                
                                     5    10   25   50   75   90   95                Average   Standard      Average  Standard
                                                                                               deviation              deviation
                                                                                                                               

    1986      71           14       17    20   30   43   60   81   88       108        48         22          43         488

    1987      71           13       16    21   33   46   60   74   80       105        48         20          44         478

    1988      73           8        11    18   30   43   58   67   84       120        45         22          40         547

    1989      63           10       14    19   30   44   58   64   87       140        47         26          41         546

    1990      59           7        13    17   27   38   51   71   86       130        43         23          37         554
                                                                                                                               

    a  General Environmental Monitoring Station of China (1991)
        FIGURE 8

    30 with over one million.  No group exhibited a time trend, but the
    areas with more than one million people clearly reported levels higher
    than the smaller metropolitan areas.  For 103 Metropolitan Statistical
    Areas (MSA) reporting a valid year's data for at least one station in
    1988 and/or 1989, peak annual averages ranged from 0.007 to 0.061 ppm
    (Fig. 9). The only recently measured concentrations exceeding the USA
    annual average standard (0.053 ppm) have occurred at stations in
    southern California.

         The seasonal patterns at stations in California are usually quite
    marked and reach their highest levels through the autumn and winter
    months.  Stations elsewhere in the USA usually have less prominent
    seasonal patterns and may peak in the winter or summer, or may contain
    little discernable variation (Fig. 10) (US EPA, 1991).

         One-hour NO2 values at stations in the USA can exceed 0.2 ppm,
    but in 1988 only 16 stations (12 of which are in California) reported
    an apparently credible second high 1-h value above 0.2 ppm (Fig. 11). 
    Because at least 98% of 1-h values at most stations are below 0.1 ppm,
    these values above 0.2 ppm are quite rare excursions whose validity
    should be verified (US EPA, 1991).

    3.4  Occurrence of nitrogen oxides indoors

         This section summarizes emissions of NOx from sources that
    affect indoor air quality and are commonly found in residential
    environments. There are several reasons for considering these
    emissions.  Firstly, examining emissions from several types of sources
    and source categories can help identify the relative impact of each
    source on indoor air quality and thus its influence on human exposure. 
    Secondly, such information is needed to understand the fundamental
    physical and chemical processes influencing emissions.  This
    understanding can be used to help develop strategies for reducing
    emissions.  Finally, studying emissions from indoor sources can
    provide source strength input data needed for indoor air quality
    modelling.  Knowledge of indoor concentrations is an important
    component in estimating the total exposure of individuals to nitrogen
    oxides.

         An important factor for indoor air quality is how (or if) the
    combustion products from appliances are vented outside the building. 
    It should be noted that several common types of vented appliances
    usually emit NOx to the outdoors; examples include gas-fired
    furnaces, water heaters and clothes dryers, as well as stoves and
    furnaces using wood, coal and other fuels.  Under some circumstances
    even these vented emissions may filter back inside and contribute to
    elevated NOx levels indoors.  For example, Hollowell et al. (1977)
    reported high NO and NO2 concentrations in a house where a vented
    forced-air gas-fired heating system was used.  Elevated concentrations
    may also be a problem with malfunctioning vented appliances.  Other

    FIGURE 9

    FIGURE 10a

    FIGURE 10b

    FIGURE 11

    data (e.g., Fortmann et al., 1984), however, suggest that fugitive
    emissions of NOx from vented appliances are small.  The importance of
    unvented appliances to indoor NOx levels is well documented; this
    section focuses on emissions from such appliances.

    3.4.1  Indoor sources

    3.4.1.1  Gas-fuelled cooking stoves

         Several research programmes have investigated NOx emissions
    from stoves fuelled with natural and liquid petroleum gas (Himmel &
    DeWerth, 1974; Cote et al., 1974; Massachusetts Institute of
    Technology, 1976; Yamanaka et al., 1979; Traynor et al., 1982b; Cole
    et al., 1983; Caceres et al., 1983; Fortmann et al., 1984;
    Moschandreas et al., 1985; Cole & Zawacki, 1985; Tikalsky et al.,
    1987; Borrazzo et al., 1987a).  Most of these studies have included
    investigations of several other pollutants, including CO, aldehydes
    and unburned hydrocarbons.  Table 12 lists average emission factors
    for range-top burners and for oven and broiler burners operated at
    maximum heat input rate.  Data are shown for both well-adjusted blue
    flames and for poorly adjusted yellow flames.  Each of the averages is
    based on the total number of stoves tested for that category, using
    data from the above studies.  For top burners with blue flames, a
    total of 27 values are represented; for yellow flames, there are 23
    values (24 for NOx).  Averages for the oven and broiler burners
    represent 20 blue flame and 16 yellow flame values.  Values are
    generally very similar for emissions from these two types of burners
    on the same stove.  Overall, the results show that well-adjusted blue
    flames emit more NO but less NO2 than poorly adjusted yellow flames. 
    Emission factors from range-top burners are comparable to those from
    oven and broiler burners.

    Table 12.  Average emission factors for nitric oxide (NO),
               nitrogen dioxide (NO2) and nitrogen oxides (NOx)
               from burners on gas stoves
                                                                           

                        Flame     Factor for    Factor for     Factor for
                        type      NO (g/kJ)    NO2 (g/kJ)    NOx (g/kJ)
                                                                           

    Top burners         blue      20.0  4.5    10.2  3.1     41.0  8.2
    Top burners         yellow    16.9  4.5    15.0  4.8     42.0  9.1
    Ovens and broilers  blue      21.9  6.3    7.23  3.01    40.9  8.6
    Ovens and broilers  yellow    19.8  9.6    11.4  5.7     39.0  10.8
                                                                           

    3.4.1.2  Unvented gas space heaters and water heaters

         The findings of several investigators (Thrasher & DeWerth, 1979;
    Traynor et al., 1983a, 1984b; Zawacki et al., 1986) are summarized in
    Table 13.  The most significant result is the markedly lower emissions
    from heaters equipped with catalytic burners, radiant ceramic tile
    burners and improved-design steel burners (radiant and Bunsen),
    compared to emissions from simpler convection designs using
    conventional cast-iron Bunsen burners.  Equipping convective heaters
    with radiant tiles does not make much difference to emission levels,
    nor does the choice of natural gas or liquid petroleum gas fuel. 
    Other studies by Billick et al. (1984), Zawacki et al. (1984) and
    Moschandreas et al. (1985) produced similar results.

    3.4.1.3  Kerosene space heaters

         The data presented in Table 14 show that emission factors of NO
    and NO2 for radiant kerosene heaters are generally much smaller than
    those for convective kerosene heaters.  Emissions of NO from two-stage
    heaters are only slightly greater than those from radiant heaters,
    whereas emissions of NO2 are the lowest of the three heater types. 
    Most of the emissions from radiant heaters are in the form of NO2;
    for convective heaters that are two-stage heaters, the emissions of NO
    and NO2 are of comparable magnitude.  There are insufficient data
    to evaluate changes in emissions as kerosene heaters age.  Other
    products, including particles, present in these emissions may also be
    of concern for their possible health effects.

    3.4.1.4  Wood stoves

         A number of studies have examined pollutant emissions from wood
    stoves.  Some of these studies have developed emission factors based
    on concentrations in the flue gases; such information would be useful
    for assessing the contribution of wood stove emissions to ambient air
    quality.  Very little information is available, however, on fugitive
    emissions from wood stoves into the indoor living space.

         In a detailed literature survey, Smith (1987) reported that
    emissions of pollutants from wood stoves are highly variable,
    depending on the type of wood used, stove design, the way the stove is
    used and other factors.  He reported emission factors for NOx and
    other pollutants for wood stoves used in developing countries.  Many
    of these stoves are unvented, which results in excessive indoor
    concentrations as the combustion products are exhausted into the room. 
    The major health concerns for wood fires without chimneys arise from
    pollutants other than NO2, such as particulate matter.

        Table 13.  Summary of studies with unvented convective (C) and infrared (I) space heaters
                                                                                                                         

    Type of         Number    Heat input     NO emission     NO2 emission     NOx emission    Reference
    heater                    (kJ/min)       (g/kJ)         (g/kJ)          (g/kJ)
                                                                                                                         

    Convective      5         86-661         24-47           2.2-7.3          39-77           Thrasher & DeWerth (1979)

    Convective      8         188-830        9.5-22          9.5-20           34-47           Traynor et al. (1983a)

    Infrared        5         245-352        0.1-1           4.1-6.2          4.9-6.2         Traynor et al. (1984b)
    Convective      4         335-626        17.8-28.7       10-18.3          40.1-57.5

    Infrared        5         264-334        0.005-1.7       1.6-4.8          2.7-5.7         Zawacki et al. (1986)
    Convective      5         176-703        5.3-44.4        7.6-23.3         27.1-76.4
                                                                                                                         

    Table 14.  Average emission factors for nitric oxide (NO), nitrogen dioxide (NO2) and nitrogen oxides (NOx) from kerosene heaters
                                                                                                                                           

    Type of heater           Heat input rate     Emission factor       Emission factor       Emission factor      Reference
                             (kJ/min)            for NO (g/kJ)        for NO2 (g/kJ)       for NOx (g/kJ)
                                                                                                                                           

    Radiant, new             144                 0.45  0.05           4.4  0.2             5.1  0.2            Leaderer (1982)
    Radiant, new             113                 0.08  0.05           5.0  0.2             5.1  0.2
    Radiant, new             84.4                0                     5.9  0.3             5.9  0.3

    Convective, new          158                 17  0.3              7.0  0.4             33  0.6
    Convective, new          97.9                12  0.6              15  0.3              33  1.0
    Convective, new          37.3                11  0.9              17  1.0              34  1.7

    Radiant, new             137                 1.3  0.7             4.6  0.8             6.6  1.3            Traynor et al. (1983b)

    Radiant, 1 year old      111                 2.1                   5.1                   8.3

    Convective, new          131                 25  0.7              13  0.8              51  1.3

    Convective, 5 years old  94.8                11  0.1              32  2.8              49  2.8

    Radiant                  110-200             -                     -                     13  1.8             Yamanaka et al. (1979)

    Convective               110-200             -                     -                     70  6.8
                                                                                                                                           
             Traynor et al. (1984a) have studied wood stoves (three airtight
    and one non-airtight) used in a house.  For each experiment, airborne
    concentrations of several pollutants were measured inside and outside
    the house during operation of one of the stoves.  The results showed
    that all indoor and outdoor concentrations of NO and NO2 were
    below 0.02 ppm.  Moreover, indoor air concentrations of some other
    pollutants were high during use of the non-airtight stove.  The
    airtight stoves had little influence on indoor concentrations of any
    pollutants.  In another study, Traynor et al. (1982a) found elevated
    airborne concentrations of NO and NO2 in three occupied houses during
    operation of wood stoves and a wood furnace.  The concentrations were
    highly variable.

         Because of the limited data, it is difficult to reach
    quantitative conclusions regarding the importance of wood stoves. 
    However, the limited information available suggests that wood stoves
    are not a major contributor to indoor nitrogen oxide exposures.  This
    is consistent with the small NO emission rates expected from the low
    temperature combustion processes characteristic of wood stoves.

    3.4.1.5  Tobacco products

         A number of studies have compared concentrations of NOx and
    other pollutants in houses with smokers and houses without smokers. 
    In general, these studies have shown that concentrations are somewhat
    greater in the homes of smokers.

         A few studies have reported emissions of NOx from cigarettes
    while sampling both sidestream and mainstream smoke together. 
    Woods (1983) reported 0.079 mg NOx/cigarette, while Moschandreas
    et al. (1985) listed emissions of 2.78 mg/cigarette for NO and
    0.73 mg/cigarette for NO2.  The National Research Council (1986)
    reported total NOx emissions of 100 to 600 g/cigarette for
    mainstream smoke, with values 4 to 10 times greater for sidestream
    smoke.  According to the report, virtually all of the emitted NOx is
    in the form of NO; once emitted, the NO is gradually oxidized to NO2. 
    Thus environments containing cigarette smoke may have higher
    concentrations of both NO and NO2 than environments without such
    smoke.  The NO2 concentration on trains travelling between Changchun
    and Harbin, China, was found to be related to the amount of cigarette
    smoking, which was greater on daytime trains than on night-time ones. 
    On a one-way daytime train the average NO2 concentration was 54 ppb
    (range, 37-84 ppb), whereas on a two-way night-time train it was
    40.6 ppb (range, 30-59 ppb) (Du et al., 1992).

    3.4.2  Removal of nitrogen oxides from indoor environments

         A number of field studies of NO2 levels in residences have
    reported that NO2 is removed more rapidly than can be accounted for
    by infiltration alone (Wade et al., 1975; Macriss & Elkins, 1977;

    Oezkaynak et al., 1982; Traynor et al., 1982a; Ryan et al.,
    1983; Leaderer et al., 1986).  Indoors, NO2 is removed by
    infiltration/ventilation and by interior surfaces and furnishings. 
    The removal of NO2 by interior surfaces and furnishings and reactions
    occurring in air is often referred to as the reactive decay rate of
    NO2, and it can be a significant factor in the actual NO2 levels
    measured in residences.  Failure to account for the reactive decay
    rate can lead to a serious underestimation of emission rate
    measurements in chamber and test house studies and a serious
    overestimation of indoor concentrations when using emission rates to
    model indoor levels.  The NO2 reactive decay rate is typically
    determined by subtracting the decay of NO2, after a source is shut
    off, from that of a relatively non-reactive gas (e.g., CO, CO2, SF6,
    NO), which can be related to ventilation rates, expressed in room
    air changes per hour.  The measured reactive decay rates in the
    above-mentioned field studies ranged from 0.1 to 1.6 air change
    times/hour.  All studies noted that the reactive decay of NO2 is as
    important and in some cases more important than infiltration in
    removing NO2 indoors.  Leaderer et al. (1986) monitored NO2, NO, CO
    and CO2 continuously in seven houses over periods ranging from 2 to
    8 days.  They reported that the NO2 decay rate was always greater
    than that due to infiltration alone and was highly variable among
    houses and among time periods within a house.

         In an effort to identify the factors that control the NO2
    reactive decay rate, a number of small chamber (Miyazaki, 1984; Spicer
    et al., 1986), large chamber (Moschandreas et al., 1985; Leaderer et
    al., 1986) and test house studies (Yamanaka, 1984; Borrazzo et al.,
    1987b; Fortmann et al., 1987) have been conducted.  The most extensive
    small chamber work was reported by Spicer et al. (1986), where 35
    residential materials were screened for NO2 reactivity in a 1.64-m3
    chamber and a limited number of the materials were tested for the
    impact of relative humidity on the reactivity rate.  Fig. 12 shows the
    relative rates of NO2 removal for the materials screened.  The figure
    indicates that many of the materials used for building construction
    and furnishings are significant sinks for NO2 and that their removal
    rate is highly variable.  Many of the materials were found to reduce a
    significant proportion of the removed NO2 to NO.  In no cases was
    NO2 re-emitted, although some materials emitted NO.  The authors
    noted that the materials that removed NO2 most rapidly fall in two
    categories: (1) porous mineral materials of high surface area; and (2)
    cellulosic material derived from plant matter.  Higher relative
    humidities were found to enhance the removal rate for some materials
    (e.g., wool carpet), reduce the removal rate for some (e.g., cement
    block), and have little effect on others (e.g., wallboard).  In a
    series of small (0.69 m3) chamber studies (Miyazaki, 1984) reactive
    decay rates for NO2 were found to vary as a function of material type
    and to increase with increasing surface area of the material, degree
    of stirring in the chamber, temperature and relative humidity.  A
    saturation effect was noted on some of the carpets tested.

    FIGURE 12

         In a series of large chamber studies (34-m3 chamber), Leaderer
    et al. (1986) evaluated the reactive decay rate of NO2 as a function
    of material type, surface area of material, relative humidity and air
    mixing.  The reactive decay rate was found to vary as a function of
    material surface roughness and surface area.  Carpeting was found to
    be most effective in removing NO2, whereas painted wallboard was
    least effective.  Increases in relative humidity were associated with
    increases in removal rates for all materials tested, but the slope was
    a shallow one.  Of particular interest is the finding in this study
    that the degree of air mixing and turbulence was a dominant variable
    in determining the reactive decay rate for NO2.  Moschandreas et al.
    (1985) evaluated six materials in a 14.5-m3 chamber and found
    variations in decay rates according to material types and a positive
    impact of relative humidity on NO2 decay rates in an empty chamber.

         Yamanaka (1984), in assessing NO2 reactive decay rates in a
    Japanese living room, found the decay to consist of both homogeneous
    and heterogeneous processes.  The rates were found to vary as a
    function of surface property and sharply as a function of relative
    humidity.  NO production during the decay was noted.  In a test house
    study, Fortmann et al. (1987) noted that the NO2 decay rate tends to
    decrease as the concentration increases.  It is not clear whether this
    is due to surface saturation or second-order kinetics.  This study
    also noted a sharp increase in NO levels during the NO2 decay,
    indicating NO production as a result of the NO2 decay. In a test
    house study conducted over a 7-month period, Borrazzo et al. (1987b)
    found that reaction rates for NO2 in the test house were sensitive to
    the location in the house where they were measured.  This indicates
    that reaction losses during transport of NO2 from room to room in a
    house may be important.

         Reactive decay of NO2 associated with interior surface materials
    and furnishings is an important mechanism for removing NO2 from the
    air within homes. Reactive decay rates for NO2 vary as a function of
    the type and surface area of the material.  The impact of relative
    humidity on the decay rate is unclear, with some studies showing a
    pronounced impact (Yamanaka, 1984), while others show only moderate or
    little impact (e.g., Spicer et al., 1986; Leaderer et al., 1986).  The
    degree of air mixing or turbulence can have an important effect on the
    reactive decay rate.  A by-product of NO2 removal by materials may be
    NO production, and a saturation effect may occur for some materials. 
    Reactive decay of NO2 in residences is highly variable between
    residences, within rooms in a residence, and on a temporal basis
    within a residence.  The large number of variables controlling the
    reactive decay rate make it very difficult to assess in large field
    studies through questionnaire or integrated air sampling.

    3.5  Indoor concentrations of nitrogen oxides

         Indoor concentrations of NO2 are a function of outdoor
    concentrations, indoor sources (source type, condition of source,
    source use, etc.), infiltration/ventilation, air mixing within and
    between rooms, reactive decay by interior surfaces, and air cleaning
    or source venting.

    3.5.1  Homes without indoor combustion sources

         Typical studies in homes without indoor sources of NO2,
    summarized in Table 15, have reported concentrations lower than
    outdoor levels due to removal from the air of NOx by the building
    envelope and interior surfaces. Thus indoor/outdoor concentration
    ratios are consistently less than unity.  These homes provide some
    degree of protection from outdoor concentrations.  Indoor/outdoor
    ratios vary considerably according to the season of the year, the
    lowest ratios occurring in the winter and highest occurring during the
    summer.  Although urban concentrations are often highest in winter,
    this pattern in the indoor/outdoor ratio, attributed to seasonal
    differences in infiltration rates, NO2 reactivity rates, the
    penetration factor and outdoor concentrations, can result in higher
    indoor concentrations in summer than in winter.  The indoor-to-outdoor
    ratio for these homes does not appear to depend on geographical area,
    housing type or outdoor concentration.   Results of monitoring in
    Portage, Wisconsin, USA, show that the presence of a gas stove
    contributes dramatically to the indoor NO2 levels.  Table 16, taken
    from the report of Quackenboss et al. (1986) and based on data
    collected in 1981 and 1982, clearly shows that gas stoves increase not
    only indoor concentrations but also the personal exposure of children.

    3.5.2  Homes with combustion appliances

         It is estimated that gas (natural gas and liquid propane) is used
    for cooking, heating water or drying clothes in about 45% of all homes
    in the USA (US Bureau of the Census, 1982) and in nearly 100% of homes
    in some other countries (e.g., the Netherlands).  Gas appliances
    (gas cooker/oven, water heater, etc.) are the major indoor source
    category for indoor residential NO2 by virtue of the number of homes
    with such sources.  NO2 concentrations in homes with gas appliances
    are higher than those without such appliances.  Within this category,
    the gas cooker/oven and unvented heaters are by far the major
    contributors.  Cookers and ovens are especially important sources when
    used inappropriately as a supplementary room heater.  Average indoor
    concentrations (based on a 1- to 2-week measurement period) in excess
    of 100 g/m3 have been measured in some homes with gas cookers
    (Table 17).  Homes where gas cookers with pilot lights are used have

    higher NO2 levels than homes that have gas cookers without pilot
    lights.  Average NO2 concentrations in homes with gas cookers/ovens
    exhibit a spatial gradient within and between rooms. Kitchen
    concentrations of NO2 are higher than other rooms and a steep
    vertical concentration gradient in the kitchen has been observed in
    some homes, concentrations being highest nearest the ceiling.  Average
    NO2 concentrations are highest during the winter months and lowest
    during the summer months. This seasonal temporal gradient is
    attributed to differences in infiltration, appliance use, NO2
    reactivity rates and indoors and outdoor concentrations.  The impact
    of gas appliance use on indoor NO2 levels may be superimposed upon
    the background level resulting from outdoor concentrations.  Only very
    limited data exist on short-term average (3 h or less) indoor
    concentrations of NO2 associated with gas appliance use.  These data
    suggest that short-term average concentrations of NO2 are several
    times the longer-term average concentrations measured.

         A wide variety of fuel types can be used for cooking and heating
    in different localities. These can produce various effects on indoor
    air quality.  As an example, Table 18 gives data for indoor NOx
    concentrations measured at Lanzhou City, China, where coal and
    liquified gas were used in apartments and houses (Duan et al., 1992).

        Table 15.  Average outdoor concentrations of nitrogen dioxide (NO2) and average indoor/outdoor ratios in homes without gas appliances or
               unvented space heatersa
                                                                                                                                              

    Location             Housing         Averaging  Seasons         Number   Average NO2     Indoor/outdoor ratios    Reference
                         typeb           time                       of       outdoor                              
                                                                    homes    concentration
                                                                             (g/m3)         Kitchen      Bedroom
                                                                                                                                              

    Southern California  Mixed           7 days     Summer          70       71.9            0.80         0.75        Southern California
                                                    Spring          100      43.5            0.72         0.60        Gas Company (1986)
                                                    Winter          69       91.2            0.56         0.47

    New Haven, CT        Single family   14 days    Winter          60       13.2            0.56         0.55        Leaderer et al. (1986)
                         unattached

    Albuquerque, NM      Mixed           14 days    Winter 1        60       14.1            -            0.50        Marbury et al. (1988)
                                                    Winter 2        56       19.6            -            0.32

    California           Mobile homes    7 days     Summer          46       25.9            0.61         0.54        Petreas et al. (1988)
                                                    Winter          23       44.6            0.27         0.26

    Portage, WI          Mixed           7 days     Summer          47       15.2            0.91         0.72        Quackenboss et al. (1986)
                                                    Winter          47       17.2            0.65         0.45

    Tucson, AZ           Mixed           14 days    Summer          56       19.9            0.86         0.76        Quackenboss et al. (1986)
                                                    Spring/Autumn   41       25.6            0.71         0.55
                                                    Winter          23       36.8            0.64         0.52

    Boston, MA           Mixed           14 days    Summer          117      31.7            0.76         0.75        Ryan et al. (1988)
                                                    Autumn          117      37.8            0.43         0.40
                                                    Winter/Spring   124      33.5            0.53         0.47
                                                                                                                                              

    Table 15.  (Con't)
                                                                                                                                              

    Location             Housing         Averaging  Seasons         Number   Average NO2     Indoor/outdoor ratios    Reference
                         typeb           time                       of       outdoor                              
                                                                    homes    concentration
                                                                             (g/m3)         Kitchen      Bedroom
                                                                                                                                              

    Northern Central     Single family   5 days     Winter          9        53.8                                     Koontz et al. (1986)
    Texas                unattached

    Suffolk County,      Single family   7 days     Winter          49       35.5            0.47         -           Research Triangle
    NY                   unattached                                                                                   Institute (1990)

    Onondago County,     Single family   7 days     Winter          66       21.7            0.70         -
    NY                   unattached      

    Portage, WI          Single family   7 days     Average over    25       12.8            0.65         0.51        Spengler et al. (1983)
                         unattached                 all seasons

    Watertown, MA        Not given       3-4 days   November        18       37.0            0.65         0.51        Clausing et al. (1984)
                                                    December        10       46.0            0.39         0.30

    Middlesbrough, UK    Not given       7 days     Winter          87       35.0            0.97         0.75        Goldstein et al. (1979)

    Middlesbrough, UK    Not given       7 days     Winter          15       34.7            -            0.75        Melia et al. (1982a,b)
                                                                                                                                              

    a  Data from field studies of private residences in the USA and United Kingdom
    b  "Mixed" indicates a single family in an attached or unattached dwelling, condominium or apartment
            Table 16.  Nitrogen dioxide concentrations (ppm) according to season and 
               stove type in Portage, Wisconsin, USAa
                                                                                      

    Season       Stove          Indoor              Outdoor             Personal
                                                                                      
                             Mean      SD        Mean      SD        Mean      SD
                                                                                      

    Summer       Gas         0.016     0.006     0.006     0.003     0.014     0.004
                 Electric    0.007     0.003     0.008     0.003     0.009     0.003

    Winter       Gas         0.027     0.013     0.008     0.003     0.023     0.009
                 Electric    0.005     0.003     0.009     0.003     0.008     0.003
                                                                                      

    a  From: Quackenboss et al. (1986); SD = standard deviation
            Table 17.  Indoor and outdoor concentrations of nitrogen dioxide (NO2) in homes with gas appliances, and the calculated average
               contribution of those appliances to indoor residential NO2 levels
                                                                                                                                               

    Location    Housing   Averaging  Type of     Season    No. of  Average measured NO2              Indoor NO2 due to source       Reference
                typea     time       appliance             homes   (g/m3)                           (g/m3)
                          (days)                                                                                                    
                                                                   Outdoors Kitchen Bedroom  Other   Kitchen Bedroom Other Commentb
                                                                                                                                               

    USA

    Southern    Mixed      7         Oven/range,  Summer   147     75.3     91.6    68.4     -       31      12      -     1,2      Southern
    California                        pilot      Spring   202     49.2     79.2    51.3     -       35      22      -     1,2      California
                                                  Winter   141     104      101.5   69       -       48      20      -     1,2      Gas Company
                                                                                                                                   (1986)
                                     Oven/range,  Winter   98      107      113     76       -       53      26      -     1,2
                                     pilot

                                     Oven/range,  Winter   38      97       74      53       -       20      7       -     1,2
                                     no pilot

                                     Water heater Winter   21      92       59      50       -       11      11      -     1,2,3
                                     in home

                                     Wall furnace Winter   90      121      161     113      -       49      36      -     1,4

                                     Floor        Summer   42      119      177     126      -       66      52      -     1,4
                                     furnace
                                                                                                                                               

    Table 17.  (Con't)
                                                                                                                                               

    Location      Housing  Averaging  Type of     Season   No. of  Average measured NO2              Indoor NO2 due to source       Reference
                  typea    time       appliance            homes   (g/m3)                           (g/m3)
                           (days)                                                                                                   
                                                                   Outdoors Kitchen Bedroom  Other   Kitchen Bedroom Other Commentb
                                                                                                                                               

    New Haven,    Single      14      Oven/range,  Winter    42    14.8     44.7    27.6     30.4    36      20      22    1,5      Leaderer
    CT            family,              pilot                                                                                       et al.
                  unattached                                                                                                        (1986)

    Albuquerque,  Mixed       14      Oven/range,  Winter    82    19.1     -       33.1     41.9    -       24      31    1,5,6    Marbury et
    NM                                 pilot      Winter    75    20.3     -       30.9     39.3    -       24      32             al. (1988)

    California    Mobile      7       Oven/range,  Summer    265   21.1     43.1    30.2     -       30      19      -              Petreas et
                  homes                pilot      Winter    231   42.1     53.7    37.5     -       42      27      -     1,7      al. (1988)

    Portage,      Mixed       7       Oven/range,  Summer    36    11.5     38.9    21.1     29.6    29      13      20             Quackenboss
    WI                                 pilot      Winter    34    15.4     69.6    31.2     50.7    60      15      42    1,8      et al.
                                                                                                                                    (1986)

    Tucson,       Mixed       14      Oven/range,  Summer    13    23.1     39.1    26.3     30.7    19      8       11             Quackenboss
    AZ                                 pilot      Spring/   11    36.3     45.8    31.9     42.4    20      12      17             et al.
                                                   Autumn                                                                           (1986)
                                                   Winter    10    45.2     60.6    43.4     50.7    32      20      25    1,9

    Boston,       Mixed       14      Oven/range,  Summer    301   41.6     65.9    45.6     50.9    33      15      19             Ryan et al.
    MA                                 pilot      Autumn    277   43.7     74.3    47.5     52.8    56      30      34             (1988)
                                                   Winter/   298   40.5     73.5    48.6     55.1    52      30      34    1,9
                                                   Spring
                                                                                                                                               

    Table 17.  (Con't)
                                                                                                                                               

    Location      Housing  Averaging  Type of     Season   No. of  Average measured NO2              Indoor NO2 due to source       Reference
                  typea    time       appliance            homes   (g/m3)                           (g/m3)
                           (days)                                                                                                   
                                                                   Outdoors Kitchen Bedroom  Other   Kitchen Bedroom Other Commentb
                                                                                                                                               

    Central       Single      5       Oven/range,  Winter    22    34.6     -       -        54.1    -       -       37    1,10     Koontz et
    Texas         family,              pilot                                                                                       al. (1986)
                  unattached

    Suffolk Co.,  Single      7       Oven/range,  Winter    42    37.6     77.5    -        52.4    60      -       37             Research
    NY            family,              pilot                                                                                       Triangle
                  unattached                                                                                                        Institute
                                                                                                                                    (1990)

    Onondago      Single      7       Oven/range,  Winter    56    30.6     62.6    0        50      41      -       27    1,9
    Co., NY       family,              pilot
                  unattached

    New York,     Apartments  2       Oven/range   Summer    14    109      122     98       106     30      6       13             Goldstein
    NY                                             Autumn 1  15    61       96      65       71      53      22      18             et al. 
                                                   Autumn 2  9     73       108     66       76      45      15      25             (1985)
                                       pilot      Winter 1  8     100      121     76       95      61      16      35
                                                   Winter 2  18    75       126     63       82      81      18      37    9,11,12
                                                   Spring    13    95       121     82       99      55      16      33

    Portage, WI   Single      7       Natural gas  All       36    15.8     65.5    36.7     -       55      29      -              Spengler et
                  family,             Oven/range,  seasons                                                                          al. (1983)
                  unattached          no pilot

                                      Liquified    All       76    11.6     65.6    37.6     -       58      31      -     1,13
                                      petroleum    seasons
                                      gas
                                      Oven/range,
                                      no pilot
                                                                                                                                               

    Table 17.  (Con't)
                                                                                                                                               

    Location      Housing  Averaging  Type of     Season   No. of  Average measured NO2              Indoor NO2 due to source       Reference
                  typea    time       appliance            homes   (g/m3)                           (g/m3)
                           (days)                                                                                                   
                                                                   Outdoors Kitchen Bedroom  Other   Kitchen Bedroom Other Commentb
                                                                                                                                               

    Watertown,    Not given   3-4     Gas cooking  Novemb.   60    37       74      45       51      50      26      33    1,9,14   Clausing et
    MA                                             Decemb.   51    46       86      46       60      68      32      44             al. (1984)

    Netherlands

    Arnet         Not given   7       Gas cooking  Autumn/   294   35       118     -        97      97      -       37             Noy et al.
    Enschede                          no pilot     Winter                                                                           (1984)
                                      Water
                                      heaters

    Ede           Not given   7       Gas cooking  Autumn/   173   44       113     43       54      89      17      28             Noy et al.
                                      no pilot     Winter                                                                           (1984)
                                      Water
                                      heaters

    Vlagttwedde   Rural area  7       Gas cooking  Autumn/   162   28       107     24       51      90      7       34
                                      no pilot     Winter                                      Water
                                      heaters

    Rotterdam I,  Inner city  7       Gas cooking  Autumn/   228   45       144     51       80      117     24      53
                                      no pilot     Winter
                                      Water
                                      heaters

    Rotterdam II, Inner city  7       Gas cooking  Autumn/   102   45       143     64       73      117     37      46    9,17
                                      no pilot     Winter                                       Water
                                      heaters
                                                                                                                                               

    Table 17.  (Con't)
                                                                                                                                               

    Location      Housing  Averaging  Type of     Season   No. of  Average measured NO2              Indoor NO2 due to source       Reference
                  typea    time       appliance            homes   (g/m3)                           (g/m3)
                           (days)                                                                                                   
                                                                   Outdoors Kitchen Bedroom  Other   Kitchen Bedroom Other Commentb
                                                                                                                                               

    United Kingdom

    Middlesbrough Not given   7       Gas cooking  Winter    428   35       213     58       -       179     24      -     1,15     Goldstein 
                                      no pilot                                                                                      et al. 
                                                                                                                                    (1979)

    Middlesbrough Not given   7       Gas cooking  Winter    183   34.7     -       60       82.7    -       39      61    1,16     Melia et 
                                                                                                                                    al.
                                                                                                                                    (1982a,b)
                                                                                                                                              

    a  "Mixed" indicates a single family in an attached or unattached dwelling, condominium or apartment
    b  1.   Background correction determined by multiplying: (a) the indoor/outdoor ratio for homes in the study with no indoor NO2 sources
            for a given season; by (b) the outdoor NO2 concentration measured for the home with sources; and subtracting the product from
            the indoor level measured in the house.
       2.   Homes containing forced air gas furnace.  These homes are thought not to contribute significantly to indoor levels for this
            sample.
       3.   Homes with electric cooker/oven, forced air gas furnace, and gas water heater in home.  Comparison is made with electric
            cooker/oven, forced air gas furnace, and gas water heater located outside home.
       4.   Homes have gas cooker/oven with source contribution calculated after correction of a gas cooker/oven.  Values are background
            corrected with gas stove.
       5.   Living room or activity room.
       6.   Sampling was done over two different periods for the same houses within the same winter period.
       7.   Outdoor values were obtained from five locations, housing type, mobile home.
       8.   Other location in home; bedroom refers to average of levels in one or more bedrooms in house.
       9.   Other location in the main living room.
       10.  Other location is point nearest centre of home.

    Table 17  (Con't)

       11.  48-h samples over 30 consecutive days.
       12.  Indoor/outdoor (I/O) ratio is assessed to be 0.6, 0.7, and 0.85 for the Winter, Spring/Autumn and Summer periods,
            respectively, for all locations, because no control home (no gas appliances) mean measurements were available.  Using these
            I/O ratios, the impact of sources was calculated as footnote 1.
       13.  Each home was sampled six times over a 1-year period.
       14.  Outdoor levels are average for homes with or without gas appliances.
       15.  Outdoor levels were recorded at 75 locations in the general sampling area and were not home-specific.  Bedroom levels were
            obtained for 107 of the 428 homes.
       16.  Outdoor levels were recorded at 82 locations in the general sampling areas and were not home-specific.  Outdoor levels were
            recorded at the beginning and end of the study.
       17.  Indoor/outdoor (I/O) ratio is assumed to be 0.6 for all locations, because no control home (no gas appliances) measurements
            were available.  Using I/O ratio of 0.6, the impact of sources was calculated as in footnote 1.
        Table 18.  Indoor concentration of NOx in Lanzhou city, Chinaa
                                                                        

    Type of residence                                 Average NOx
                                                 concentration (mg/m3)
                                                                        

                                                 Winter         Summer
                                                                        

    Apartment building with central              0.141          0.059
    heating, liquified gas for cooking

    Apartment building without central           0.136          0.059
    heating, coal for cooking and heating

    One-storey house, coal for cooking           0.106          0.046
    and heating
                                                                        

    a  From: Duan et al. (1992)

    3.5.3  Homes with combustion space heaters

         Unvented kerosene and gas space heaters are important sources of
    NO and NO2 in homes, both because of the NO and NO2 production rates
    of the heaters and the long periods of time that they are in use.  The
    concentrations of NO emitted are usually several times higher than
    those of NO2. However, in the literature, indoor air concentrations
    of NO are frequently not reported.

         Field studies indicate that average residential concentrations
    (1- or 2-week average levels) exhibit a wide variation, depending
    primarily on the amount of heater use and the type of heater
    (Table 19).  Under similar operating conditions, unvented gas space
    heaters appear to be associated with higher indoor NO2 concentrations
    than kerosene heaters.  Average concentrations in homes using unvented
    kerosene heaters have been found to be well in excess of 100 g/m3. 
    In one study (Leaderer et al., 1986), calculations of NO2
    concentrations in residences during kerosene heater use (in homes
    without gas appliances) indicate that approximately 50% of the homes
    have NO2 concentrations above 100 g/m3 and 8% above 480 g/m3. A
    peak NO2 concentration of 847 g/m3 was measured over a 1-h period
    in a home during use of a kerosene heater.

    Table 19.  Two-week average nitrogen dioxide (NO2) levels for homes
               in New Haven, Connecticut, USA, during winter, 1983a
                                                                    

    Source category;                   NO2 (g/m3)
      location                                                      

                             n      Mean        SDb   % above
                                                        100 g/m3
                                                                    

    No kerosene heater
     or gas stove
       Outdoors              144     13.2       5.3       0
       House average         145      7.4       4.2       0
       Kitchen               147      7.6       3.7       0
       Living room           146      7.3       3.4       0
       Bedroom               145      7.3       8.6       0

    One kerosene heater,
     no gas stove
       Outdoors              95      12.9       4.6       0
       House average         95      36.8      32.8       2.1
       Kitchen               96      39.1      35.5       4.2
       Living room           96      38.5      36.6       5.2
       Bedroom               95      31.9      30.8       5.3

    No kerosene heater,
     one gas stove
       Outdoors              42      14.8       4.2       0
       House average         42      34.3      26.2       4.8
       Kitchen               42      44.7      31.4       4.8
       Living room           42      30.4      24.8       4.8
       Bedroom               42      27.8      25.1       4.8

    One kerosene heater,
     one gas stove
       Outdoors              18      14.5       5.2       0
       House average         18      66.8      43.9      16.7
       Kitchen               18      74.5      52.0      22.2
       Living room           18      57.4      38.6      11.1
       Bedroom               18      68.5      56.5      16.7

    Two kerosene heaters,
     no gas stove
       Outdoors              13      16.5       9.4       0
       House average         13      69.5      38.0      23.0
       Kitchen               13      73.0      31.7      23.0
       Living room           13      73.6      44.3      38.5
       Bedroom               13      67.8      44.9      23.1
                                                                    

    Table 19.  (Con't)
                                                                    

    Source category;                   NO2 (g/m3)
      location                                                      

                             n      Mean        SDb   % above
                                                        100 g/m3
                                                                    

    Two kerosene heaters,
     one gas stove
       Outdoors              3       22.1       6.2       0
       House average         3       85.8      24.4      33.3
       Kitchen               3       94.0      22.7      66.6
       Living room           3       77.6      38.4      33.3
       Bedroom               3       85.8      19.5      33.3

                                                                    

    a  From: Leaderer et al. (1986); repeat monitoring data (n = 19)
         are included
    b  SD = standard deviation

         A large field study (Koontz et al., 1986) of indoor NO2
    concentrations in Texas homes using unvented gas space heaters (most
    also had gas cookers) found that approximately 70% of the homes had
    average concentrations in excess of 100 g/m3 and 20% had average
    concentrations in excess of 480 g/m3.  This study found that the
    indoor/outdoor temperature difference was the best indicator of
    average indoor NO2 levels during the colder winter periods when
    heating demands are greatest.

         Only limited data have so far been published on short-term peak
    indoor concentrations for homes with unvented gas space heaters, and
    no data are available on spatial variations or concentrations solely
    during the hours of heater operation.

         No spatial gradient of NO2 was found in homes with unvented
    kerosene space heaters, contrary to the strong spatial gradient noted
    for homes with gas appliances.  This is probably due to the strong
    convective heat output and the long operating hours of the heaters,
    which result in rapid mixing within the homes.

         Ferrari et al. (1988) conducted a study of air quality in
    homes with unvented space heaters in Sydney, Australia, over
    two winters.  NO2 concentrations were measured by both continuous
    (chemiluminescence with O3 method) and passive monitors (badges and

    Palmes tubes).  Concentrations of NO2 exceeded 0.10 ppm (average
    concentration) in 85% of homes tested, and 0.16 ppm in 44% of homes.
    More than 10% of homes had average NO2 concentrations exceeding
    0.32 ppm, and the maximum recorded was greater than 0.5 ppm.  Unvented
    gas space heaters are common in Sydney, and average use is about 3 h
    per night during the winter.  As a result, an estimated 0.5 million
    residents are exposed to NO2 concentrations exceeding 0.16 ppm for
    several hours per night during the colder months of the year.

         Improper use of gas appliances (e.g., using a gas oven or
    stove to heat a living space) and improperly operating gas appliances
    or vented heating systems (e.g., out-of-repair gas cooker or
    improper operation of a gas wall or floor furnace) can be important
    contributors to indoor NO2 concentrations, but few data are available
    to assess the magnitude of that contribution.  Little or no field data
    exist that would allow for an assessment of the contributions of wood-
    or coal-burning stoves or fireplaces to indoor NO2 concentrations,
    but such a contribution would be expected to be small.  Cigarette
    smoking is expected to add relatively small amounts of NO2 to homes
    (see also Tables 15 and 18).

         In developing countries, biomass fuels (e.g., wood, biogas,
    animal dung, etc.) are much more widely used for home heating and/or
    cooking than in developed countries, these fuels often being burnt in
    open hearth fires or poorly vented appliances within indoor spaces of
    residential dwellings (WHO, 1992).  As noted by Sims & Kjellstrm
    (1991), a very conservatively estimated 400 million people are
    affected by biomass smoke problems worldwide, mostly in rural areas of
    developing countries.  A disproportionate number of women and young
    children are exposed, owing to the greater numbers of hours typically
    spent by them indoors and their involvement in cooking and other
    household chores.  Increased NOx concentrations, as well as greater
    concentrations of carbon monoxide, suspended particulate matter (SPM)
    and volatile organic compounds (VOCs) are normally found in biomass
    smoke (Chen et al., 1990).  Reviews of field studies in rural areas of
    developing countries indicate that exposure levels to biomass smoke
    components are usually rather high.  Indoor concentrations for NO2,
    for example, were found to fall within the range of 0.1 to 0.3 mg/m3
    in India, Nepal, Nigeria, Kenya, Guatemala and Papua New Guinea, as
    reported in reviews by WHO (1984) and Smith (1986, 1987).  Similarly,
    Hong (1991) reported NO2 concentrations in the range of 0.01 to
    0.22 mg/m3 resulting from indoor combustion of biogas in homes in
    Chengdu, Szechuan Province, China.  Hong (1991) also reported NOx
    concentrations in the range of 0.02 to 0.16 mg/m3 in other houses in
    Gansu Province, China, where dried cow dung was used as a fuel.  The
    above NO2 indoor air concentrations from biomass smoke should be
    compared with the WHO Air Quality Guidelines recommendation of
    0.15 mg/m3 for daily exposures to NO2 (WHO, 1987).

    3.5.4  Indoor nitrous acid concentrations

         Recent studies have demonstrated that substantial concentrations
    of HNO2 can be present inside residential buildings, especially when
    unvented combustion sources are used.  HNO2 is formed by the reaction
    of NO2 with water on surfaces and the reaction is promoted by high
    humidity.  HNO2 may also be produced by other mechanisms, and this is
    the subject of active research.  Brauer et al. (1993) found that HNO2
    can represent over 10% of the concentrations usually reported as NO2.

    3.5.5  Predictive models for indoor NO2 concentration

         Efforts to model indoor NO2 levels have employed two distinct
    approaches: physical/chemical and empirical/statistical models.

         The physical/chemical modelling approach has been used by
    numerous investigators in chamber, test house and small field studies
    (involving a small number of homes) to estimate emission rates of NO2
    from combustion sources (e.g., Traynor et al., 1982a; Leaderer, 1982;
    Moschandreas et al., 1984), to estimate reactive decay rates (e.g.,
    Oezkaynak et al., 1982; Yamanaka, 1984; Leaderer et al., 1986; Spicer
    et al., 1986; Borrazzo et al., 1987a), to estimate the impact of
    ventilation and mixing on the spatial and temporal distribution of
    NO2 (e.g., Oezkaynak et al., 1982; Traynor et al., 1982b; Borrazzo
    et al., 1987a), and to evaluate the applicability of emission
    rates determined under controlled conditions in estimating indoor
    concentrations of NO2 (e.g., Traynor et al., 1982b; Borrazzo et al.,
    1987a).  More recently, studies have reported the use of distributions
    of the input variables to the mass balance equation (emission rates,
    source use, decay rates, ventilation rates, etc.), determined from the
    published literature, to estimate distributions of indoor NO2 levels
    for specific sources and combinations of sources (Traynor et al.,
    1987; Hemphill et al., 1987).

         Prediction of indoor concentrations or concentration
    distributions of NO2 in homes with combustion sources using
    physical/chemical (mass-balance) models requires accurate information
    for input parameters (e.g., emission rates).  Although data are
    available for some of the input parameters under controlled
    experimental conditions, there are very limited data available
    concerning either the variability of such input parameters in actual
    homes or the factors that control variability (e.g., variability of
    emission or decay rates).  Obtaining field measurements or estimates
    of the inputs in large numbers of homes would be expensive and
    time-consuming.  Such modelling efforts do, however, help to identify
    the potential range of indoor NO2 concentrations, factors that may
    result in high levels, and the potential effectiveness of mitigation
    efforts.

         Empirical/statistical models have been developed from large field
    studies that have measured NO2 concentrations in residences and
    associated outdoor levels for time periods of a week or more.  These
    have typically used questionnaires to obtain information on sources in
    the residences, source use, building characteristics (house volume,
    number of rooms, etc.), building use, and meteorological conditions.
    In some cases, additional measurements, including temperature have
    been recorded.  Several investigators have attempted to fit simple
    regression models to their field study databases in an effort to
    determine whether the variations in NO2 levels seen among houses can
    be explained by variations in questionnaire responses.  The goal has
    been to see how well questionnaire information or easily available
    information (meteorological data) can predict indoor NO2 levels.  In
    most cases a linear model has been used, but several investigators
    have used log transformations of variables.  These employ
    questionnaire responses and measured physical data (house volume,
    etc.) as independent variables and have met with moderate success. 
    Linear regression models, with the exception of the Petreas et al.
    (1988) model, explain from 40 to 70% of the variations in residential
    NO2 levels and typically have large standard errors associated with
    their estimates.  Although log transformations of variables have
    always produced a higher percentage of explained variation due to the
    skewed distribution of the original variables, interpretation of the
    coefficients in a nonlinear model can require special attention.

         Regression models developed from field studies employing
    questionnaires to explain variations in indoor levels of NO2 have met
    with only moderate success.

         Better information, through additional measurements and better
    questionnaire design, is needed on a range of factors if the
    statistical/empirical models are to be used to estimate indoor
    concentrations of NO2 in homes without measurements.  Factors include
    source type and condition, source use, contaminant removal
    (infiltration and reactive decay) and between and within room mixing.

    3.6  Human exposure

         To assess the health impact of exposure to nitrogen oxides, it is
    essential to conduct an accurate exposure assessment.  Such data are
    of paramount importance for the definition of dose-effect and
    dose-response relationships.  In fact, the risk to human health is not
    simply determined by indoor and outdoor concentrations of nitrogen
    oxides, but rather by the personal exposure of every individual.  The
    integrated exposure is the sum of the individual exposures to oxides
    of nitrogen over all possible time intervals for all settings or
    environments.  It requires, thus, the consideration of long-term

    average concentration level, variations and short-term exposures, as
    well as the activity patterns and personal and home characteristics of
    individuals (Berglund et al., 1994).

         Exposure is a function of concentration and time.  People spend
    various periods in different types of micro-environments with various
    concentration levels.  On average, people spend about 90% of their
    time indoors (at home, work, school, etc.), about 5% in transit
    (Chapin, 1974), and 7% (range 3-12%) near smokers (Quackenboss et al.,
    1982).  These values vary with the season, day of the week, age,
    occupation and other factors but it is decidedly important to predict
    indoor pollutant levels when total exposure is being estimated.

         Adequate exposure assessment for NO2 is particularly critical in
    conducting and evaluating epidemiological studies.  Failure to measure
    or estimate exposures adequately and address the uncertainty in the
    measured exposures can lead to misclassification errors (Shy et al.,
    1978; Gladen & Rogan, 1979; Oezkaynak et al., 1986; Willett, 1989;
    Dosemeci et al., 1990; Lebret, 1990).  Early studies comparing the
    incidence of respiratory illness in children living in homes with and
    without gas stoves used a simple categorical variable of NO2
    exposure; the presence or absence of a gas cooker.  Such a dichotomous
    grouping can result in a severe non-differential misclassification
    error in assigning exposure categories.  This type of error is likely
    to underestimate the true relationship and could possibly result in a
    null finding.

         In assessing human exposure to NO2 (and other oxides of
    nitrogen), averaging times chosen should account for the type of
    effect to be expected.  With regard to NO2, the principal biological
    responses include (a) relatively transient effects on respiratory
    function associated with acute, short-term (< 1 h) exposures, and (b)
    the likelihood of increased risk for respiratory disease in children
    associated with frequently repeated short-term peak exposures and/or
    lower level long-term exposures.

         Indirect and direct methods for personal exposure assessment are
    available.  Indirect methods combine measures of concentrations at
    fixed sites in various types of micro-environments with information
    on where people have spent their time (time-activity patterns). 
    Time-weighted average (TWA) exposure models have been developed to
    estimate total personal exposure (Fugas, 1975; Duan, 1982; Duan,
    1991).  The NO2 exposure levels predicted from TWA exposure models
    have been shown to correlate closely with the exposure levels obtained
    by direct measurements of personal exposure (Nitta & Maeda, 1982;
    Quackenboss et al., 1986; Sega & Fugas, 1991).  However, the large
    variation in NO2 concentrations (distribution) within each type of

    micro-environment (because of variability in, for example, stove use,
    emission rates, ventilation frequencies, and the day-to-day and
    person-to-person variations in the use of time) decreases the accuracy
    of the predicted exposure and increases the risk for misclassification
    of the exposure.

         Direct measurements of concentrations in the breathing zone
    of a person using personal passive exposure monitors provide
    time-integrated measurements of exposure for a certain period across
    the various micro-environments where a person spends time.  It is
    important to collect exposure data over time intervals consistent with
    the expected effects.  Effects from long-term, low-level exposure may
    be different from effects from short periods of high concentration
    (intermittent peak exposure).  Intermittent peak exposure, which
    occurs during cooking on a gas stove, may be significant to total
    exposure and adverse health effects.  If effects from peak exposure
    are to be considered in the exposure assessment, the sampling time
    must be short enough to detect these peak exposures. Such a short
    sampling time is possible with the more sensitive passive samplers and
    with conventional air monitors, such as chemiluminescence NOx
    monitors.  However, direct methods of measuring personal exposure
    are relatively costly and time-consuming.  Within-person and
    between-person variability, both in personal exposure and personal
    use of time, can be large.

         Hence a sufficient number of personal exposure measurements must
    be collected for each person (repeated measurements), and a sufficient
    number of individuals must be sampled before the measurements can be
    considered to be representative.  Personal daily exposures have been
    shown to vary between individuals on the same day by a factor of up to
    about 15 in the urban area of Stockholm and between days for the same
    individual by a factor of up to 10 (Berglund et al., 1993).

         A comparison of personal NO2 exposures, as measured by Palmes
    diffusion tubes, and NO2 exposures measured in residences had a
    correlation of 0.94 for a subsample of 23 individuals (Leaderer et
    al., 1986).  Results of this comparison are depicted in Fig. 13 and
    show an excellent correlation between average household exposure and
    measured personal exposure.

         It is important to note that indoor concentrations are strong
    predictors of personal exposure.  In the case of homes with gas or
    electric stoves, personal exposure has been shown to be closely
    related to the household indoor average concentrations (Quackenboss
    et al., 1986; Harlos et al., 1987a).

    FIGURE 13

         Results of monitoring in Portage, Wisconsin, verify that the
    presence of a gas stove contributes dramatically to personal NO2
    exposure levels. Table 16, derived from the reports of Quackenboss et
    al. (1986) and based on data collected in 1981 and 1982, clearly shows
    that gas stoves increase not only indoor concentrations but also the
    personal exposure of children.

         On the other hand, outdoor concentrations, even if measured
    outside each residence, have been found to be relatively poor
    predictors of personal exposure (Quackenboss et al., 1986; Leaderer et
    al., 1986).  The association between personal exposure and outdoor
    levels of NO2 is weakest during the winter for both gas and electric
    stove groups.

         The only route of NO2 exposure is inhalation.  The dose is
    dependent on the inhalation volume and thus on physical activity, age,
    etc.  Lung absorption of NO2 is about 80-90% during rest and over 90%
    during physical activity (WHO, 1987).

         Efforts have been made to find a sufficient biological marker for
    NO2 exposure and dose.  Increased urinary excretion of collagen and
    elastin (pulmonary connective tissue) breakdown products (including
    hydroxyproline, hydroxylysine and desmosine) has been suggested as a
    marker of diffuse pulmonary injury related to inhaled NO2.  A
    significant relationship between urinary hydroxyproline excretion and
    daily NO2 exposure was found among housewives in Japan, but the
    hydroxyproline excretion fell within the normal distribution for
    healthy people (Yanagisawa et al., 1986).  The majority of the
    housewives were exposed to active or passive cigarette smoke, and this
    exposure was independently related to the excretion of hydroxyproline. 
    Other investigators have not been able to substantiate the
    relationship between urinary hydroxyproline excretion and NO2
    exposure (Muelenaer et al., 1987; Adgate et al., 1992).

         Measurements of the NO-haem protein complex in bronchoalveolar
    lavage (Maples et al., 1991) and of 3-nitrotyrosine in urine (Oshima
    et al., 1990) have been suggested as biological markers for NO2
    exposure.  The work in progress to find a suitable biological marker
    for NO2 exposure at levels found in the general environment is
    promising; however, no metabolite has yet proved to be sensitive or
    specific enough.

         Personal exposure to air pollutants can be assessed by direct or
    indirect measures.  Direct measures include biomarkers and use of
    personal monitors.  No validated biomarkers for exposure are presently
    available for NO2.

         Studies using passive monitors to measure NO2 exposures lasting
    one day to one week have been conducted in the USA (Dockery et al.,
    1981; Clausing et al., 1986; Leaderer et al., 1986; Quackenboss et
    al., 1986; Harlos et al., 1987; Schwab et al., 1990), in the
    Netherlands (Hoek et al., 1984), in Japan (Nitta & Maeda, 1982;
    Yanagisawa et al., 1984), and in Hong Kong (Koo et al., 1990).  These
    studies generally indicate that outdoor levels of NO2, although
    related to both personal levels and indoor concentrations, are poor
    predictors of personal exposures for most populations.  Average indoor
    air residential concentrations (e.g., whole-house average or bedroom
    level) tend to be the best predictor of personal exposure, typically
    explaining 50 to 80% of the variation in personal exposures.

         Indirect measures of personal exposure to NO2 employ various
    degrees of micro-environmental monitoring and questionnaires to
    estimate an individual's or population's total exposure.  One such
    model (Billick et al., 1991), developed from an extensive monitoring
    and questionnaire database, can estimate population exposure
    distributions from easily obtained data on outdoor NO2 concentrations,
    housing characteristics and time-activity patterns.  This model is
    proposed for use in evaluating the impact of various NO2 mitigation
    measures.  The model is promising, but has not yet been validated nor
    has associated uncertainty been characterized.

    3.7  Exposure of plants and ecosystems

         The sensitivity of plants to nitrogen oxides is determined both
    by their genetic characteristics and by environmental conditions.

         The relation between exposure and uptake by plants depends on
    aerodynamic and stomatal resistance and thus increases with increasing
    light intensity, wind velocity and air humidity.  After uptake, the
    response of a plant depends on its metabolic activity, and thus
    increases with poorer nutritional supply and lower temperature.

         Moreover, the sensitivity of plants depends on the general air
    pollution situation.  Emission of SO2 is often combined with NOx,
    and these compounds act synergistically.  Therefore, the impact of
    NOx may be higher in regions with elevated SO2 concentrations.  NOx
    forms part of photochemical smog.  Although ozone is the most
    phytotoxic, the contribution of NOx to adverse effects on plants is
    not negligible.

         For vegetation and ecosystems the impact of NOx is through its
    contribution to total nitrogen disposition rather than its direct
    toxicity.  Thus, other nitrogen-containing pollutants have to be taken
    into consideration.

         The dependencies of sensitivity, as summarized above, mean that
    wide variation exists in the vulnerability of different regions of the
    world.

    4.  EFFECTS OF ATMOSPHERIC NITROGEN COMPOUNDS (PARTICULARLY NITROGEN
        OXIDES) ON PLANTS

         Effects of nitrogen on ecosystems are caused through deposition
    onto soil and foliar uptake of nitrogen in various forms. Total
    effects are often difficult to separate into component effects.  This
    section, therefore, covers nitrogen inputs in all forms to ecosystems. 
    Much of the research focuses on European ecosystems, where the
    majority of the research has been conducted.  Here NHy deposition
    either dominates or is a major constituent of total nitrogen input. 
    However, this is not true for other parts of the world.  All effects
    of atmospheric nitrogen input, in whatever form, are included as
    indicators of more globally relevant effects on ecosystems but the
    reader should bear in mind local conditions of nitrogen input when
    assessing likely local consequences.

         NOx, as used in this chapter, refers to the total nitrogen
    measured by chemiluminescence detectors; this is NO2 following
    conversion to NO, and NO itself. Other nitrogen oxides may interfere
    to some extent in this method.

         Elemental nitrogen (N2) forms 80% of the atmosphere of the
    earth.  This is equivalent to about 75  106 kg above each hectare of
    the earth's surface. In unpolluted conditions a small fraction
    (1-15 kg nitrogen per ha per year) is converted by nitrogen-fixing
    microorganisms to biologically more active forms of nitrogen: NH4+
    and NO3-. The natural deposition of nitrogen-containing atmospheric
    compounds other than N2 is much less.  The soil contains 5 times more
    nitrogen than the atmosphere, but weathering of rock is a negligible
    source of biologically active nitrogen.  By denitrification (reduction
    of NO3- to N2 and to a lesser extent N2O, NO and NH3), 1-30 kg
    nitrogen per ha per year is recycled from the earth to the atmosphere.

         Human activities, both industrial and agricultural, have greatly
    increased the amount of biologically active nitrogen compounds,
    thereby disturbing the natural nitrogen cycle.  Various forms of
    nitrogen pollute the air, mainly NO, NO2 and NH3 as dry deposition
    and NO3- and NH4+ as wet deposition.  Another contribution
    is from occult deposition (fog and clouds).  There are many more
    nitrogen-containing air pollutants (e.g., N2O5, PAN, N2O, amines)
    but these have not been considered in this chapter, either because
    their contribution to the total nitrogen deposition is considered to
    be small or because their concentrations are probably far below the
    effect thresholds.

         Transformations of nitrogen, as it moves from the atmosphere,
    through ecosystems and back to the atmosphere, form the nitrogen
    cycle.  This is illustrated, together with anthropogenic sources of
    nitrogen, in Fig. 14.  The component processes affected by chronic
    nitrogen deposition are indicated in Fig. 15.

    FIGURE 14

    FIGURE 15

         Nitrogen-containing air pollutants can affect vegetation
    indirectly, via chemical reactions in the atmosphere, or directly
    after being deposited on vegetation, soil or water surfaces.  The
    indirect pathway is largely neglected in this chapter, although it
    includes very relevant processes, and should be taken into account
    when evaluating the entire impact of nitrogen-containing air
    pollutants: NO and NO2 are precursors for tropospheric ozone (O3),
    which acts both as a phytotoxin and a greenhouse gas.  The effects of
    O3 will be discussed in a forthcoming Environmental Health Criteria
    monograph.  N2O contributes to the depletion of stratospheric O3,
    resulting in increasing ultraviolet radiation.  This and other aspects
    of global climate change will be evaluated in a WHO/WMO/UNEP document
    entitled "Climate and Health: potential impacts of climate change". 
    The direct impact of airborne nitrogen is due to toxic effects,
    eutrophication and soil acidification.  One effect of soil
    acidification is that aluminum enters into solution, hence increasing
    its bioavailability.  This result in root damage.  Aluminum toxicity
    will be discussed in a further Environmental Health Criteria
    monograph.

         Most biodiversity is found in (semi-)natural ecosystems, both
    aquatic and terrestrial.  Nitrogen is the limiting nutrient for plant
    growth in many (semi-)natural ecosystems.  Most of the plant species
    from these (semi-)natural habitats are adapted to nutrient-poor
    conditions, and can only compete successfully in soils with low
    nitrogen levels (Chapin, 1980; Tamm, 1991). Ellenberg (1988b) surveyed
    the nitrogen requirements of 1805 plant species from Germany and
    concluded that 50% can compete successfully only in habitats that are
    deficient in nitrogen.  Furthermore, of the plants threatened by
    increased nitrogen deposition, 75-80% are indicator species for
    low-nitrogen habitats.  When stratified by ecosystem type, it is also
    clear that the trend of rare species occurring with greater frequency
    in nitrogen-poor habitats is a common phenomenon across many
    ecosystems (Fig. 16 and Fig. 17).  Plant species threatened by high
    nitrogen deposition are common across many ecosystem types (Ellenberg,
    1988b).  The critical loads for nitrogen depend on (i) the type of
    ecosystem; (ii) the land use and management in the past and present;
    and (iii) the abiotic conditions (especially those which influence the
    nitrification potential and immobilization rate in the soil).  The
    impact of increased nitrogen deposition upon biological systems can be
    the result of direct uptake by the foliage or uptake via the soil. 
    The most relevant effects at the level of individual plants are injury
    to the tissue, changes in biomass production and increased
    susceptibility to secondary stress factors.  At the vegetation level,
    this results in changes in competitive relationships between species
    and loss of biodiversity.

    FIGURE 16

    FIGURE 17

         Effects on individual plants are discussed in section 4.1.  The
    following natural ecosystems are treated in detail in section 4.2:
    freshwater ecosystems (shallow soft-water bodies, lakes and streams)
    and terrestrial ecosystems (wetlands and bogs, species-rich
    grasslands, heathlands and forests).  Estuarine and marine systems
    are also considered.

         Air quality guidelines refer to thresholds for adverse effects. 
    Two different types of effect thresholds exist: critical levels and
    critical loads.

         The critical level is defined as:

         the concentration in the atmosphere above which direct adverse
         effects on receptors, such as plants, ecosystems or materials,
         may occur according to present knowledge.

         The critical load is defined as:

         a quantitative estimate of an exposure (deposition) to one or
         more pollutants below which significant harmful effects on
         specified sensitive elements of the environment do not occur
         according to present knowledge.

         Generally, critical levels for nitrogen-containing air pollutants
    are expressed in terms of exposure (g/m3 and exposure duration),
    while critical loads are expressed in terms of deposition (kg nitrogen
    per ha per year).  Both critical level and load are intended to be
    set so as to protect vegetation, and can be converted into each
    other knowing the deposition velocity.  Thus, it might seem to be
    superfluous to assess both critical levels and loads.  However, with
    the currently accepted approach, critical levels and loads are more or
    less complementary: critical levels focus on effect thresholds for
    short-term exposure (1 year or less), while critical loads focus on
    safe deposition quantities for long-term exposure (10-100 years):
    critical levels are not aimed to protect plants completely against
    adverse effects.  No-observed-effect concentrations (NOECs) are
    usually lower than critical levels.  For instance, a critical level
    can be set at 5% yield reduction. Thus, owing simply to differences in
    definition, the critical level is generally higher than the critical
    load (Fig. 18b).

         In current practice there are other differences between critical
    levels and loads: critical levels give details on individual compounds
    and focus on responses on plant level, while critical loads cover all
    nitrogen-containing compounds and focus on the vegetation or ecosystem
    level.  In other words: critical loads focus on functioning of the
    ecosystem, while critical levels focus on protection of the relatively
    sensitive plant species.

         In the critical level concept, the different nitrogen-containing
    compounds are evaluated separately, because of their differences in
    phytotoxic properties, even when their load in terms of kg nitrogen
    per ha per year is the same (Ashenden et al., 1993).  Another
    difference between critical level and critical load is that critical
    level considers the possibility of more- or less-than-additive effects
    (Wellburn, 1990), while in the critical load concept additivity of
    nitrogen-containing or acidifying compounds is presumed.  Moreover,
    nitrogen-containing air pollutants have their impact not only because
    of their contribution to the nitrogen supply.  Sometimes other effects
    seem to dominate.  For instance, although occult deposition is
    generally small in terms of nitrogen deposition, it may be of great
    significance because of its ability to affect plant surfaces.

         It was concluded for these reasons that both critical levels and
    loads are necessary within the scope of air quality guidelines for
    nitrogen-containing compounds.

         Assessing effect thresholds is relatively simple in the case of
    toxic compounds with an exposure/response relationship which follows
    the usual sigmoid curve: the lowest exposure level that results in a
    response that is significantly different from the control treatment is
    the effect threshold.  However, this approach is essentially invalid
    for exposure of nitrogen-limited vegetation to nitrogen-containing air
    pollutants.  Nitrogen is a macro-nutrient and so each addition of
    nitrogen can result in a physiological response: growth stimulation
    gradually increases with higher exposure levels and changes in growth
    inhibition at higher levels (Fig. 18a).  Moreover, depending on the
    definition of adverse effects, the status of the vegetation may not be
    optimal at background levels (Fig. 18b).  These features complicate
    the assessment of effect thresholds for nitrogen-containing compounds. 
    Nevertheless, in this chapter effect thresholds are presented,
    according to current practice.

    4.1  Properties of NOx and NHy

         In this section general information is initially presented on
    uptake, detoxification, metabolism and growth aspects.  In the
    following subsections the data determining the critical levels for
    individual compounds and mixtures are discussed. The relevance of this
    information and possibilities for generalization are discussed in
    sections 4.1.8 and 4.1.9, where the critical levels are estimated. 
    Deposition on and emission from soils and vegetation is discussed in
    chapter 3.

    FIGURE 18

    4.1.1  Adsorption and uptake

         The impact of a pollutant on plants is determined by its
    adsorption, rate of uptake (flux) and the reaction of the plants. 
    Foliar uptake is probably dominant for NO, NO2 (Wellburn, 1990) and
    NH3 (Prez-Soba & Van der Eerden, 1993), while the pathway via soil
    and roots is the major route for nitrogen-containing pollutants in wet
    deposition.

         The flux of the compounds from the atmosphere into the plant is a
    complicated process, which is highly dependent on the properties of
    both plant and compound and on environmental conditions.  This is why
    deposition velocities proved to be highly variable (chapter 3).

         The flux from the atmosphere to the leaf surface (and soil)
    depends on the aerodynamic and boundary layer resistances, which
    are determined by meteorological conditions and plant and leaf
    architecture.  The flux from the leaf surface to the final site of
    reaction in the cell is determined by stomatal, cuticular and
    mesophyll resistance.  The reaction of the plant to the nitrogen that
    arrives at the target side is dependent on the intrinsic properties of
    the plant and on its nutritional status, and again on environmental
    conditions.

         The flux of atmospheric nitrogen through the soil is conditioned
    by properties of soil and vegetation and by meteorological conditions. 
    The chemical composition of soil water, the rate of nitrification
    (NH4+ -> NO3-), the preference of the plant for either NH4+ or
    NO3-, the root architecture and the metabolic activity of the plants
    play major roles in this uptake (Schulze et al., 1989).

         Adsorption on the outer surface of leaves certainly takes
    place.  Exposure to relatively high concentrations of gaseous NH3
    (180 g/m3) or NH4+ in rainwater (5 mmol/litre) damages the
    crystalline structure of the epicuticular wax layer of the needles of
     Pseudotsuga menziesii (Van der Eerden & Prez-Soba, 1992).  NO2
    (Fowler et al., 1980) and NH4+ and NO3- in wet and occult
    deposition can disturb leaf surfaces in several ways (Jacobson, 1991). 
    The quantitative relevance of this effect for the field situations has
    not yet been shown in detail.

         Uptake of NH3 and NOx is driven by the concentration gradient
    between atmosphere and mesophyll.  It is generally directly determined
    by stomatal conductance and thus depends on factors influencing
    stomatal aperture.  Although in higher plants uptake through the
    stomata strongly dominates, there are indications that penetration
    through the cuticle is not completely negligible.  This has been
    demonstrated for NO and NO2 (Wellburn, 1990).  While stomata greatly
    influence the foliar uptake of aerial nitrogen compounds, many of
    these compounds subsequently alter stomatal aperture and the extent of

    further uptake.  The nitrogen status of plants is also known to affect
    stomatal behaviour towards other environmental conditions such as
    drought (Ghashghaie & Saugier, 1989).

         The flux of NH3 into a plant appeared to be linearly related to
    the atmospheric concentration (Van der Eerden et al., 1991), there
    being no mesophyll resistance (Van Hove et al., 1989).  This relation
    can become less then linear with high concentrations, e.g., some
    hundreds of g/m3 (Wollenheber & Raven, 1993).  Mesophyll resistance
    is, however, probably more significant for NO and NO2 (Capron et al.,
    1994).

         There is increasing evidence that foliar uptake of nitrogen
    reduces the uptake of nitrogen by the roots (Srivastava & Ormrod,
    1986; Prez-Soba & van der Eerden, 1993), although the driving
    mechanism is not yet clear.

         In the presence of low concentrations plants can emit NH3,
    rather than absorb it (chapter 3).  NO and N2O are emitted in
    significant quantities by the soil (chapter 3).

         Rain, clouds, fog and aerosols always contain significant amounts
    of ions including NH4+ and NO3-.  In the past, foliar uptake of
    nitrogen from wet deposition was considered to be negligible, but
    recent research using 15N and throughfall analysis shows that this
    path can contribute a high proportion of the total plant uptake (see
    Pearson & Stewart, 1993, and section 2.4).  In general, cations (e.g.,
    NH4+) are more easily taken up through the cuticle than anions
    (e.g., NO3-).  A substantial foliar uptake of NH4+ from rainwater
    has been measured in several tree species (Garten & Hanson, 1989). 
    Lower plants, such as bryophytes and lichens do not have stomata and a
    waxy waterproof cuticle, and thus the potential for direct uptake of
    pollutants in the form of wet or dry deposition is greater.  Epiphytic
    lichens are active absorbers of both NH4+ and NO3- (Reiners &
    Olson, 1984).  Uptake and exchange of ions through the leaf surface is
    a relatively slow process, and thus is only relevant if the surface
    remains wet for long periods.

    4.1.2  Toxicity, detoxification and assimilation

         One would expect a positive relationship between the solubility
    of a compound and its biological impact.  NO is only slightly soluble
    in water, but the presence of other substances can alter its
    solubility.  NO2 has a higher solubility, while that of NH3 is much
    higher.

         Much information exists on mechanisms of toxicity, although it is
    sometimes confusing.  NO2, NO, HNO2 and HNO3 can be incorporated
    into nitrogen metabolism using the pathway: NO3- -> NO2- ->

    (NH3 <--> NH4+) <--> glutamate -> glutamine -> other amino
    acids, amides, proteins, polyamines, etc.  The enzymes involved
    include nitrate reductase (NR), nitrite reductase (NiR) and glutamine
    synthetase (GS).  Glutamate dehydrogenase (GDH) plays a role in the
    internal cycling of NH4+.

         After exposure to NO2, nitrate can accumulate for some weeks;
    accumulation of nitrite is rarely reported, although it is certainly
    an intermediate.  Nitrite levels can be elevated for some hours due to
    the fact that NR activity is induced faster than that of NiR.  In many
    cases storage of excess nitrogen has been found to be in the form of
    arginine (Van Dijk & Roelofs, 1988), which could last months or
    longer.

         NO2-, NH3 and NH4+ are highly phytotoxic, and could well be
    the cause of adverse effects of nitrogen-containing air pollutants. 
    Wellburn (1990) suggested that the free radical *N=O plays a role in
    the phytotoxicity of NOx.

         High concentrations can cause visible injury via lipid breakdown
    and cellular plasmolysis.  At lower concentrations inhibition of lipid
    biosynthesis may dominate, rather than damage of existing lipids
    (Wellburn, 1990).

         Raven (1988) assumed that the adverse effects of nitrogen-
    containing compounds are due to their interference with cellular
    acid/base regulation.  They can influence cellular pH both before
    and after assimilation.  Assimilation of most air pollutants,
    including NH3, has been shown to result in production of protons
    (Wollenheber & Raven, 1993).  Assimilation of nitrate and a high
    buffer capacity can prevent the plant from being damaged by this
    acidification (Pearson & Stewart, 1993).  If these adverse effects can
    effectively be counteracted, assimilation of nitrogen-containing
    compounds will result in growth stimulation.

         Synergistic effects have been found in nearly all studies
    concerning SO2 and NO2 (Wellburn et al., 1981).  Inhibition of NiR
    by SO2, resulting in the inability of the plant to detoxify nitrite,
    might be the cause of this interaction.

    4.1.3  Physiology and growth aspects

         When climatic conditions and nutrient supply allow biomass
    production, both NOx and NHy result in growth stimulation at low
    concentrations and growth reduction at higher concentrations. 
    However, the exposure level at which growth stimulation turns into
    growth inhibition is much lower for NOx than for NHy (see Fig. 18a).

         Foliar uptake of NH3 generally results in an increase in
    photosynthesis and dark respiration, and in the amount of RUBISCO
    (ribulose 1,5-biphosphate carboxylase oxygenase) and chlorophyll. 
    Some authors have shown that stomatal conductance increases and the
    stomata remain open in the dark, resulting in enhanced transpiration
    and drought sensitivity (Van der Eerden & Prez-Soba, 1992).  Most
    experiments with NO and NO2 have been conducted with relatively high
    concentration levels (> 200 g/m3).  These experiments show
    inhibition of photosynthesis by both NO and NO2, possibly additively
    (Capron & Mansfield, 1976).  Inhibition by NO may be stronger than
    that of NO2 (Saxe, 1986).  The threshold for this response is well
    below the threshold for visible injury (Wellburn, 1990) and
    transpiration (Saxe, 1986).  With lower (nearer to ambient) NOx
    concentrations, stimulation of photosynthesis may well occur.  Both
    NOx and NHy generally cause an increase in shoot/root ratio.  The
    specific root length and the amount of mycorrhizal infection can be
    reduced by both compounds. However, these alterations in root
    properties resemble general responses to increased nitrogen nutrient
    supply.

    4.1.4  Interactions with climatic conditions

         Evidence suggests that exposure of vegetation to NH3 and to
    mixtures of NO2 and SO2 can influence the subsequent response to
    drought and frost stress.  There is also evidence that environmental
    conditions can affect the response to NOx and to NH3.

         The foliar uptake of nitrogenous compounds in the form of wet and
    occult deposition is largely via the cuticle.  Uptake and exchange of
    ions through the leaf surface is a relatively slow process, and thus
    is especially relevant if the surface remains wet for longer periods,
    e.g., in regions where exposure to mist and clouds is frequent.

         The solubility of most gases, including NO, NO2 and NH3, rises
    as temperature falls, while the metabolic activity of plants and thus
    their detoxification capacity is lower.  On the other hand, stomatal
    conductivity and thus the influx of gases generally falls as
    temperature falls.

         Guderian (1988) proposed a lower critical level in winter than
    for the whole year, in acknowledgement of several results that
    indicate greater toxicity of NO2 during winter conditions.  For
    example, exposure of  Poa pratensis in outdoor chambers to 120 g/m3
    inhibited growth during winter but had little effect or even
    stimulated growth in summer and autumn (Whitmore & Freer-Smith, 1982). 
    Mortensen (1986) found that low light and non-injurious low
    temperature conditions can enhance the toxicity of NOx.  Capron et
    al. (1991) found that the depression relative to the control of net
    photosynthesis by 1250 g NO/m3 plus 575 g NO2/m3 at 10C was
    three times, and at 5C was almost five times, that recorded at 20C.

    An interaction between NOx and temperature may also occur at lower
    realistic concentrations.  This is suggested by the observation of
    nitrite accumulation at low temperatures during fumigation of
     Picea rubra with 38 g NO2/m3 plus 54 g SO2/m3 (Wolfenden et
    al., 1991).  This temperature effect may play a role in combination
    with elevated concentrations of CO2 as well: the stimulating effect
    of CO2 on net photosynthesis was inhibited by NOx to a larger extent
    when applied at lower temperature (Capron et al., 1994).  Observation
    of NH3 injury to plants also indicates that this is greatest in
    winter (Van der Eerden, 1982).

         In contrast with the view that NOx (and NH3) injury is greater
    at low temperatures, Srivastava et al. (1975) found that inhibition by
    NOx of photosynthesis was greatest under optimal temperature and high
    light conditions, when stomatal conductance to the gas would be
    highest.

         The exposure of plants to NOx and NH3 may reduce their ability
    to withstand drought stress, owing to loss of control of transpiration
    by stomata and to an increase in the shoot/root ratio (Lucas, 1990;
    Atkinson et al., 1991; Fangmeijer et al., 1994).

    4.1.5  Interactions with the habitat

         Whether the atmospheric input of nitrogen has a positive or
    negative impact depends on the plant species and habitat.  Based on
    experimental evidence, Pearson & Stewart (1993) hypothesized that
    species which are part of a climax vegetation on nutrient-poor acidic
    soils are often relatively sensitive to NOx and NHy.  Morgan et al.
    (1992) found that NOx disrupted the NR activity to a greater extent
    in calcifuge than calcicole moss species.  Ombrotrophic mires and
    other strongly nitrogen-limited systems may be especially prone to
    detrimental effects from input of nitrogen-containing air pollutants.

         The assimilation of low concentrations of NO2 and the
    incorporation into amino acids by NR (Morgan et al., 1992; Table 20)
    are indicators that this pollutant can contribute to the nitrogen
    budget of plants (Prez-Soba et al., 1994).  The contribution of NOx
    to the nitrogen supply increases as root-available nitrogen is lowered
    (Okano & Totsuka, 1986; Rowland et al., 1987).  Srivastava & Ormrod
    (1986) observed reduced ability to respond to a supply of nitrate to
    the roots when  Hordeum vulgare was fumigated with NO2.  Similarly,
    Prez-Soba & Van der Eerden (1993) found reduced uptake of NH4+ from
    the soil when  Pinus sylvestris was fumigated with NH3.  Although
    there is much evidence that nitrogen-containing air pollutants play a
    role in the nitrogen demand and nitrogen metabolism of the plant,
    Ashenden et al. (1993) found no obvious relationship between
    sensitivity to NO2 and nitrogen preference, as indicated by Ellenberg
    (1985).

    4.1.6  Increasing pest incidence

         Any change in chemical composition of plants due to the uptake of
    nitrogenous air pollutants could alter the behaviour of pests and
    pathogens.  Evidence indicates that, in general, NOx and NHy
    increase the growth rate of herbivorous insects (Dohmen et al., 1984;
    Flckiger & Braun, 1986; Houlden et al., 1990; Van der Eerden et al.,
    1991).  This may also apply to fungal pathogens (van Dijk et al.,
    1992).

    4.1.7  Conclusions for various atmospheric nitrogen species and
           mixtures

    4.1.7.1  NO2

         In Table 20 the lowest effective exposure levels for NO2 are
    given.  Three different types of effects are considered:

    *    (bio)chemical: e.g., enzyme activity, consumption quality
    *    physiological: e.g., CO2 assimilation, stomatal conductivity
    *    growth aspects: e.g., biomass, reproduction, stress sensitivity

         Four exposure durations are used in this table.  These are
    (including an indication of the exposure durations and the margins):

    *    short term (hours): < 8 h
    *    air pollution episodes (days): 8 h to 2 weeks
    *    growing season or winter season (months): 2 weeks to 6 months
    *    long term (years): > 6 months

         To avoid the information being too selective, in each cell in
    this table a species is used only once.  For each cell the three
    lowest effective concentrations and exposure durations are given;
    species and references are mentioned in footnotes.  Exposure levels
    far higher than current levels measured in the field situation have
    not been considered.

         The fact that not all cells in Table 20 are filled with
    information is because many of the experiments have been conducted
    with unrealistically high concentrations. The majority of observations
    mentioned in the table are on crops; several of these show growth
    stimulation.  Most of the responses on a biochemical level deal with
    enhanced NR activity, which shows that the plants are capable of
    assimilating the NO2.  A general effect threshold as derived from
    Table 20 would be substantially higher if enhanced NR and biomass
    production of crops were not assumed to be an adverse effect. 
    However, growth stimulation is often considered an adverse effect in
    most types of natural vegetation.  Moreover, Pearson & Stewart (1993)

    assumed detoxification of NHy and NOx to be a potentially adverse
    effect, because it contributes to cellular acidification, which can
    not always be counteracted.

    4.1.7.2  NO

         In Table 21 the lowest effective exposure levels for NO are
    given.

         Most research into the effects of nitric oxide has been based on
    glasshouse crops, particularly the tomato  (Lycopersicon esculentum).
    Virtually all experiments deal with photosynthesis or enzymatic
    reactions and a few growth aspects were measured.  The existing data
    are rather difficult to interpret since controlled fumigation with NO
    inevitably results in some oxidation to NO2.  Thus atmospheres will
    contain a mixture of the oxides.  There is growing interest in the
    distinct properties and effects of NO and NO2, and the mechanisms of
    their cellular action probably differ (Wellburn, 1990).  The exchange
    properties of NO and NO2 over vegetation (personal communication by
    D. Fowler to the IPCS) and single plants (Saxe, 1986) appear quite
    different.  Their effects are also contrasting, and there is clearly
    some dispute over which oxide is the most toxic.  Earlier studies of
    the inhibition of photosynthesis found NO to act more rapidly than
    NO2 (at several ppm) but to cause less overall depression of the
    photosynthetic rate (Hill & Bennet, 1970).  More recent photosynthetic
    studies by Saxe (1986), using similar concentrations, found NO to be
    considerably more toxic than NO2.  There is very little information
    on contrasting effects of the two oxides at low concentrations, but
    this also adds weight to the suggestion that NO is biologically more
    toxic.  In her studies of NR in bryophytes, Morgan et al. (1992)
    discovered that exposure to NO initially inhibited NR while NO2
    induced activity.  At present, however, there is insufficient
    knowledge across a range of species to establish separate critical
    levels for NO and NO2, and studies using a wider variety of
    vegetation are urgently required.

    4.1.7.3  NH3

         The lowest effective exposure levels for NH3 are given in Table
    22.

         The toxicity of NH3 during very short exposure periods has been
    tested for the purpose of evaluating accidental releases during
    transport or industrial processes.  The estimated critical level for
    10 min is (100 ppm) (personal communication by Lee & Davison to the
    IPCS).  This type of exposure is out of the context of this monograph. 


        Table 20.  Lowest exposure levels (in g/m3) and durations at which NO2
               caused significant effectsa
                                                                                      

                     (Bio)chemical     Physiological            Growth aspects
                                                                                      

    Long term                                                   200 (130); 104 h/week;
                                                                7 monthsr
                                                                120-500; 9.5 monthss
                                                                122; 37 weekst

    Growing season   50; 39 daysb      120; 22 daysj            10-43; 130 daysu
    or winter        125; 140 daysc    190 (65); 105 h          55-75; 62 daysv
                     940; 19 daysd     in 15 daysk              150-190 (28-33);
                                                                120 h in 40 daysw

    Air pollution    140; 1 daye       375 (165); 35 h in       375; 2 weeksx
    episodes         160; 7 daysf      5 daysl  190; 3 daysm    100 (25);
                     65; 1 dayg        375 (165); 35 h          20 h in 5 daysy
                                       in 5 daysn

    Short term       7500, 6 hh        940; 1 ho                2000-3000; 3.5 hz
                     7500; 4 hi        850; 7 hp
                                       1100; 1.5 hq
                                                                                      

    a  If the fumigation was not continuous an average value has been estimated
       and quoted in parentheses (calculated assuming 10 g/m3 during the periods
       in which the fumigation was switched off).
    b   Pinus sylvestris; changes in amino acid composition, with no physiological
       changes (Nsholm et al., 1991)
    c   Lolium perenne; increase in GDH activity (Wellburn et al., 1981)
    d   Lycopersicum esculentum; decrease in nitrate content of the leaves (Taylor
       & Eaton, 1966)
    e   Picea rubens, increase in NR activity (Norby et al., 1989)

    Table 20  (Con't)

    f   Pinus sylvestris, increase in NR activity (Wingsle et al., 1987)
    g  Several bryophyte species; increase in NR activity (Morgan et al., 1992)
    h   Zea mais; increase in NiR activity (Yoneyama et al., 1979)
    i   Vicia faba; change in amino acid composition (Ito et al., 1984)
    j   Betula sp; increased water loss (Neighbour et al., 1988)
    k   Phaseolus vulgaris; reversible increase in dark respiration (Sandhu & Gupta, 1989)
    l   Glycine max; increase in photosynthesis (Sabarathnam et al., 1988a,b)
    m   Phaseolus vulgaris; increase in transpiration (Ashenden, 1979)
    n   Glycine max; enhanced dark respiration (Sabarathnam et al., 1988b)
    o   Vicia faba; reversible structural damage on cellular level (Wellburn et al., 1972)
    p   Pisum sativum; emission of stress ethylene (Mehlhorn & Wellburn, 1987)
    q   Medicago sativa, Avena sativa; inhibition of photosynthesis (Hill & Bennet, 1970)
    r  Several grass species; reduction in shoot growth (Whitmore & Mansfield, 1983)
    s   Citrus sinensis; increased fruit drop (Thompson et al., 1970)
    t   Polytrichum formosum and 3 fern species; injury and changes in growth (Ashenden
       et al., 1990; Bell et al., 1992)
    u   Brassica napus and  Hordeum vulgare; growth stimulation (resp.: Adaros et al.,
       1991a,b)
    v   Phaseolus vulgaris; increase in total dry matter, not in yield (Bender et al.,
       1991)
    w   Raphanus sativus; growth stimulation (Runeckles & Palmer, 1987)
    x   Helianthus annuus; reduction in net assimilation rate (Okano et al., 1985b)
    y   Pinus strobus; slight needle necrosis in 2 of 8 clones (Yang et al., 1983)
    z   Nicotiana tabacum; leaf necrosis (Bush et al., 1962)
        
    Table 21.  Lowest exposure levels (in g/m3) at which NO caused
               significant effectsa
                                                                         

                      (Bio)chemical     Physiological     Growth aspects
                                                                         

    Growing season    44; 21 daysb                        625; 16 daysn
                      500; 28 daysc                       500;o

    Air pollution     375; 8 daysd      1250; 4 daysi     1250; 5 daysp
    episodes          44; 8-24 he       125; 20 hj
                      1875; 18 hf

    Short term        188; 7 hg         750; 1 hk
                      500; 3 hh         2500; 10 minl
                                        1875; 20 minm
                                                                         

    a  If the fumigation was not continuous an average value has been
       estimated and quoted in parentheses (calculated assuming 10 g/m3
       during the periods in which the fumigation was switched off).
    b  Four bryophyte species; inhibition of nitrate-induction of NR
       (Morgan et al., 1992)
    c   Lycopersicon esculentum; induction of NiR (Wellburn et al., 1980)
    d   Lactuca sativa; induction of NiR (Besford & Hand, 1989)
    e   Ctenidium molluscum (bryophyte); inhibition of NR (Morgan et al., 1992)
    f   Capsicum annum; reduction in NiR activity (Murray & Wellburn, 1980)
    g   Pisum sativum; increase in ethylene release (Mehlhorn & Wellburn, 1987)
    h   Lycopersicon esculentum; induction of NiR (Wellburn et al., 1980)
    i  Eight indoor ornamental species; inhibition of photosynthesis
       (Saxe, 1986)
    j   Lycopersicon esculentum; inhibition of photosynthesis (Capron &
       Mansfield, 1989)
    k   Avena sativa &  Medicago sativa; inhibition of photosynthesis (Hill &
       Bennet, 1970)
    l   Lactuca sativa; inhibition of photosynthesis (Capron, 1989)
    m   Lycopersicon esculentum; inhibition of photosynthesis (Mortensen, 1986)
    n   Lactuca sativa; reduction in plant mass (Capron et al., 1991)
    o   Lycopersicon esculentum; reduction in plant mass (Anderson &
       Mansfield, 1979)
    p   Lycopersicon esculentum; reduction in plant mass (Bruggink et al., 1988)

    Table 22.  Lowest exposure levels (in g/m3) at which NH3 caused 
               significant effectsa
                                                                        

                     (Bio)chemical    Physiological    Growth aspects
                                                                        

    Long term        50; 8 monthsb    53; 9 monthsh    25; 1 yeark
                                                       53; 8 monthsl
                                                       35; 16 monthsm

    Growing season   100; 6 weeksc    50; 6 weeksi     60; 2 monthsn
    or winter        60; 14 weeksd                     20; 90 dayso
                     180; 13 weekse                    30; 23 daysp

    Air pollution    2000; 24 hf      213; 5 daysj     120; 11 daysq
    episodes         213; 5 daysg                      1000; 2 weeksr
                                                       300; 3 dayss

    Short term                                         30 000; 1 ht
                                                       2000 2 hu
                                                       2000 6 hv
                                                                        

    a  If the fumigation was not continuous an average value has been
       estimated and quoted in parentheses (calculated assuming
       10 g/m3 during the periods in which the fumigation was
       switched off).
    b  Species of  Violion caninea alliance; imbalanced nutrient
       status (Dueck & Elderson, 1992)
    c   Deschampsia flexuosa; change in amino acid composition (Van
       der Eerden et al., 1990)
    d   Pinus sylvestris; increased GS activity (Prez-Soba et al.,
       1990)
    e   Pseudotsuga menziesii; imbalanced nutrient status (Van der
       Eerden et al., 1992)
    f   Lycopersicum esculentum; increase of NH4+ in the dark
       (Van der Eerden, 1982)
    g   Lolium perenne; 30% of N in the plant is derived from
       foliar uptake (Wollenheber & Raven, 1993)
    h   Pinus sylvestris; increased loss of water after two weeks
       of desiccation (Dueck et al., 1990)
    i   Populus sp.; increase in stomatal conductance in leaves;
       increase in mesophyll conductance and maximum photosynthetic
       rate at a slightly higher exposure level (Van Hove et al., 1989)
    j   Lolium perenne; significant impact acid/base regulation and
       nutrients status

    Table 22  (Con't)

    k   Pseudotsuga menziesii; erosion of wax layer (Thijse & Baas,
       1990; the authors have some doubts about the causality of this
       effect (personal communication)
    l   Calluna vulgaris; reduction in survival rate after winter
       (Dueck, 1990)
    m   Arnica montana; reduced survival after winter and flowering
       (Van der Eerden et al., 1991)
    n  Field exposure during winter; median concentration; severe
       injury of several conifer species (Van der Eerden, 1982)
    o   Viola canina, Agrostis capillaris; 50% growth stimulation
       of the shoot (not of the roots) (Van der Eerden et al., 1991)
    p   Racomitrium lanuginosum; chlorosis (Van der Eerden et al.,
       1991)
    q   Hypnum jutlandicum; chlorosis (Van der Eerden et al., 1991)
    r   Lepidium sativum; reduction in dry weight (Van Haut &
       Prinz, 1979)
    s  Several horticultural crops; leaf injury
    t  Various deciduous trees; leaf injury (Ewert, 1979)
    u   Brassica sp., Helianthus sp.; leaf injury (Benedict & Breen,
       1955)
    v   Rosa sp.; leaf injury rose (Garber, 1935)


    Several cells in Table 22 could not be filled; the majority of quoted
    effects are on biomass production and tissue injury.  It is clear that
    the data in this table are not random; nearly all of the information
    originating from one Dutch research group.  Only a few pollution
    climates were considered.  The results may be representative for
    mild oceanic climates, but probably not for cold climates with dark
    winters: toxicity of NH3 increases with lower temperature and lower
    light intensity.  The effects of NH3 need to be studied with more
    plant species and under more climatic conditions in order to obtain
    critical levels with sufficient potential for generalization.

    4.1.7.4  NH4+ and NO3- in wet and occult deposition 

         NH4+, NO3- and H+ make up about half of the ionic
    composition of rain, clouds, fog and aerosols.  The impact of the
    acidity of rain and clouds has received much attention in recent years
    (Jacobson, 1991). This is not the case with other compounds in wet
    deposition, although their relevance is recognized. At the same pH,
    Cape et al. (1991) found a much greater effect of sulfuric acid than
    of nitric acid, indicating that the impact of acid rain is not only
    through protons, but also through anions.

         There is an abundance of information on the effects of NH4+ in
    soil solution.  However, threshold concentrations for NH4+ in the
    soil (e.g. Schenk & Wehrman, 1979) can not be considered a critical
    level for rain water quality, because the type of exposure and
    response is completely different.

         Wet deposition containing NH4+ can reduce frost tolerance (Cape
    et al., 1990) and induce leaching of K+ and other cations (Roelofs
    et al., 1985).  It is not yet clear whether this type of ion exchange
    can have deleterious effects on its own in the field situation.

         Currently, too few quantitative data on the effects of nitrogen-
    containing wet and occult deposition are available for critical levels
    for this group of compounds to be derived.

    4.1.7.5  Mixtures

         A polluted atmosphere generally consists of a cocktail of
    compounds, but certain combinations are more frequent.  Because of
    their role in the formation of tropospheric O3, simultaneous
    co-occurrence of relatively high levels of O3 and NO are rarely
    observed, while sequential co-occurrences are much more frequent
    (Kosta-Rick & Manning, 1993).  If burning of fossil fuels results in
    emission of SO2, this is often combined with emission of NOx.

    a)  SO2 plus NO2

         Synergism has been found in nearly all studies concerning this
    combination, with only few exceptions (Kuppers & Klump 1988; Murray et
    al., 1992). Based on data presented by Whitmore (1985), for  Poa
     pratensis the effect threshold for combinations of SO2 and NO2, in
    equal concentrations when expressed in ppm, is in the range of 1.2-2.0
    ppm.days (Fig. 19).  This threshold applies to effects by combinations
    of SO2 and NO2; the effects of single exposures were not assessed in
    this study.  However, it is reasonable from other references to expect
    synergism, and thus to include this threshold in Table 23, in which
    combined effects are summarized.  Another threshold for combinations
    of SO2 and NO2  was defined by Van der Eerden & Duym (1988) (Fig.
    20; Table 23).

    b)  SO2 plus NH3

         Adsorption of either NH3 or SO2 on leaf surfaces is enhanced by
    the presence of the other compound (Van Hove et al., 1989). 
    Interactive physiological effects have been found as well (Dueck,
    1990; Dueck et al., 1990; Dueck & Elderson, 1992).  Currently, there
    is far too little information on this combination to quantify this
    interaction.

    FIGURE 19

    FIGURE 20

    Table 23.  Lowest exposure levels at which NO2 increases the
               effect of SO2, O3, or SO2 plus O3
                                                                        

                    (Bio)chemical    Physiological  Growth aspects
                                                                        

    Long term                                       150-190; 9 monthsf
                                                    220; 60 weeksg
                                                    19; 10-41 weeksh

    Growing season  55-75; 34 daysb  135; 28 daysd  30; 38 daysi
    or winter       135; 28 daysc                   10-43; 130 daysj
                                                    30; 43 daysk

    Air pollution                                   80; 2 weeksl
    episodes                                        75; 1 daym

    Short term                       153; 1 he      325; 1 hm
                                                    400; 1 hn
                                                                        

    a  If the fumigation was not continuous an average value has
       been estimated and quoted in parentheses (calculated
       assuming 10 g/m3 during the periods in which the
       fumigation was switched off).
    b   Phaseolus vulgaris; inhibition of parts of nitrogen
       metabolism, when exposed sequentially with O3
       (100-120 g/m3; 8 h/day)
    c   Lolium perenne; decrease in proline content during winter
       hardening when applied in combination with SO2 at
       188 g/m3 (Davison et al., 1987)
    d   Lolium perenne; less negative osmotic potential during
       winter hardening when applied in combination with SO2 at
       188 g/m3 (Davison et al., 1987)
    e   Phaseolus vulgaris; Inhibition of photosynthesis when in
       combination with SO2 (215 g/m3); without SO2
       inhibition at 760 g/m3 (Bennet et al., 1990)
    f  Several crops; growth stimulation by NO2 turns into a
       reduction in synergism with sequential treatment with O3
       (160-200 g/m3; 6 h/day) (Runeckles & Palmer, 1987)
    g  Six tree species; reduced plant growth in combination with
       SO2 (280 g/m3), both antagonism and synergism
       (Freer-Smith, 1984)
    h  10 grass species were tested in combination with SO2
       (27 g/m3). Three species showed growth stimulation.
       Reduced growth was found at higher concentrations.
       Interactions with acidic mist and with O3 were found
       (Ashenden et al., 1993).

    Table 23  (Con't)

    i   Poa pratensis; inhibition of biomass production; in
       combination with SO2 (42 g/m3) for 38 days; the longest
       exposure period used in the experiments. Calculated from
       data from Whitmore (1985), assuming synergism and a critical
       level for SO2 plus NO2 of 1.2 ppm.days (Whitmore,     
       1985).
    j   Brassica napus and  Hordeum vulgare; antagonism (and
       rarely synergism) with O3 (6-44 g/m3; 8 h/day) and       
       SO2 (9-33 g/m3, continuously): enhanced yield turns into
       reduction (Adaros et al., 1991a,b) 
    k   Plantago mayor; reduced shoot dry weight synergism with
       SO2 (60 g/m3) and O3 (60 g/m3, 8 h/day)
       (Mooi, 1984)
    l   Poa pratensis; inhibition of biomass production; in
       combination with SO2 (110 g/m3) for 2 weeks (the upper
       margin of the exposure period of this cell in the table; the
       shortest fumigation in this survey was 20 days. Calculated
       from data from Whitmore (1985), assuming synergism and a
       critical level for SO2 plus NO2 of 1.2 ppm.days
       (Whitmore, 1985).
    m  Critical level for NO2 assuming SO2 to be present at
       70 g/m3; considered to be a critical level for a 24-h mean
       (UNECE, 1994) (Van der Eerden & Duym, 1988)
    n   Lycopersicon esculentum; reduction in plant mass if in
       combination or preceded by O3 (160 g/m3; 1 h)
       (Goodyear & Ormrod, 1988).

    c)  NO plus NO2

         When activated charcoal has been used as filter material in NO2
    fumigation experiments, NO must have been present as well, because
    activated charcoal has virtually no capacity to absorb NO.  In those
    studies, responses must have been due to NO2 plus NO.  Although the
    toxicity of NO was often considered to be much less than that of NO2,
    currently the two compounds are assumed to be equally toxic and to
    act additively.  However, Wellburn (1990) and others have stated
    that NO is more toxic, and Saxe (1994) showed that the variation in
    sensitivity amongst species is different for the two compounds.  This
    supports the suggestion of Wellburn that the mechanism of toxicity is
    different.

         For the purpose of deriving critical levels, the assumption of
    additivity may result in an underestimation.  However, there are not
    enough data to quantify this.

    d)  Mixtures with O3

         The combination NH3 plus O3 has rarely been studied.  No
    statistically significant interactions have been found as yet, but in
    one study the threshold for leaf injury was higher in the presence
    of NH3 (Van der Eerden et al., 1994).  The combination NO2 plus O3
    has been studied more frequently, but the responses differed
    considerably between experiments and species.  Additivity or
    antagonism was found by  Runeckles & Palmer (1987), Adaros et al.
    (1991a,b), and Bender et al. (1991).  Synergism was reported by Ito et
    al. (1984), Runeckles & Palmer (1987) and Kosta-Rick & Manning (1993).

         The combination of SO2 plus O3 plus NO2 has also been studied. 
    Again the responses varied between plant species and experiment. 
    Antagonism, additivity and synergism have all been found (Kosta-Rick &
    Manning, 1993).

    e)  Mixtures with elevated CO2

         Generally, an increased supply of CO2  to crops results in an
    enhanced biomass production.  The responses of native species are more
    variable but are also frequently positive.  This growth stimulation is
    limited by a deficiency of other nutrients.  Nitrogen can be one such
    limiting factor, and for this reason a nitrogen fertilizer such as
    NHy and possibly low NOx concentrations could be hypothesized to
    have a more-than-additive relationship with CO2.  However, as yet
    there is no experimental evidence for this.  Van der Eerden
    et al. (1994) and Prez-Soba et al. (1994) found stimulation of
    photosynthesis and growth by both NH3 and CO2, but not by a
    combination of these two compounds.

         Effects of the combination of NOx and CO2 have not yet been
    studied within the scope of global climate change.  But some relevant
    information could be gained from the literature dealing with CO2
    enrichment in glasshouses.  When the flue gases of the heating system
    are used as a CO2 source, NOx (in which NO is dominant) becomes a
    major contaminant.  The fertilizing effect of elevated CO2 can
    largely disappear in the presence of NOx (Anderson & Mansfield, 1979;
    Saxe & Voight Christensen, 1984; Mortensen, 1985; Bruggink et al.,
    1988; Capron et al., 1994).

         The CO2, NH3 and NOx concentrations used in combination in
    these experiments were relatively high and therefore cannot be used
    in the critical level assessment.  More experiments with lower
    concentrations are required.

         Table 23 indicates, surprisingly, that the effective long-term
    exposures are generally higher than those of shorter duration. 
    However, long-term responses (more than half a year) have rarely been
    studied.  Therefore, the information on effects over growing season
    periods may be much more representative of long-term effects.

         A study included in a report by UNECE (1994) used 21 g SO2/m3
    and 11 g NO2/m3, over the entire growing season and found synergism
    in reducing biomass production of  Pisum sativum and  Spinacea
     oleracea.  Similar results were found for  Hordeum vulgare and
     Brassica oleracea, when fumigation was conducted for 120-190 days
    with 30-40 g SO2/m3 and 30-50 g NO2/m3.  This study cannot be
    used for the assessment of critical levels because it has not yet been
    published, but it indicates that lower levels of the two pollutants
    than those quoted in Table 23 can influence plant responses.

    4.1.8  Appraisal

         Table 24 shows the former air quality guidelines for NO2 and
    some other critical levels assessed in the same period.  Fig. 21
    summarizes the results given in Tables 20 to 23.  In this figure
    curves are drawn to estimate critical levels according to current
    practice, known as the "envelope" approach.  After having plotted all
    effective exposure levels in a graph of concentration and exposure
    time, a curve is drawn just below the lowest effective exposures. 
    Critical levels can be derived from this curve.  Fig. 21 shows that
    more experiments with exposure periods of 0.5 to 5 days are required
    to give a more solid basis for the estimation of critical levels of
    24 h.

    Table 24.  Critical levels for NO2

    Concentration       Exposure time       Reference
    (g/m3)
                                                                        

    95                  4 h                 WHO (1987)
    30a                 annual mean         WHO (1987)
    800                 1 h                 Guderian (1988)
    60                  growing season      Guderian (1988)
    40                  winter              Guderian (1988)
                                                                        


    a    SO2 and O3 not higher than 30 g/m3 and 60 g/m3, respectively

    FIGURE 21

         A second approach to arrive at critical levels is the statistical
    model of Kooijman (1987).  Based on the variation in sensitivity
    between species, critical levels are calculated taking into account
    the number of tested species and the level of uncertainty (Van der
    Eerden et al., 1991).  The second approach is better, but only part of
    the available data is suitable for this approach.

         Tables 20 to 23 show that some new relevant information has
    appeared.  Comparing the data of Table 20 with those of Table 21
    (Fig. 21a and 21b), it appears that NO2 has slightly higher effect
    thresholds than NO.  However, this probably reflects the separate
    attention paid to these compounds, rather than any difference in
    toxicity.  It is now obvious that the toxicity of NO cannot be
    ignored, and it should be included in the guidance values.  The
    consideration of NO and NO2 together (leading to a guidance value for
    NOx) seems the best way of evaluating the impact of NO.  However,
    future research should evaluate the specific phytotoxic properties of
    the individual compounds and their combinations.

         It is not yet possible to discriminate in the critical level
    assessment between separate types of vegetation, such as crops,
    plantation forests, natural forests and other natural vegetation.  A
    1-h average for NO2 of 800 g/m3 to prevent acute damage
    (Table 24) is probably too high.  A critical level for NOx of around
    300 g/m3 would be better.  A critical level of 95 g/m3 as a 4-h
    mean, as proposed in the previous WHO guidelines (WHO, 1987), is still
    realistic, but not very practical.  If critical levels for short
    periods (e.g., 1 or 8 h) should be defined, it is probably necessary
    to separate day- and night-time exposures.  A critical level for a
    24-h mean is more practical, as this is relevant for both peak
    concentrations of several hours and air pollution episodes of several
    days.

         For the growing season and winter half year, Guderian (1988)
    suggested critical levels of 60 and 40 g/m3, respectively.  From
    Table 20 it can be seen that the critical level of 60 g/m3 can cause
    substantial growth stimulation rather than reduction. Within the
    context of air quality guidelines, this type of response must be
    regarded as potentially adverse because, for instance, of its
    influence on competition within the natural vegetation.  From current
    knowledge it is evident that 60 g/m3 is too high to prevent growth
    stimulation. In addition, the critical level of 30 g/m3 for an
    annual mean, given in the 1987 WHO guidelines, will almost certainly
    not protect all plant species.  However, for crops, where growth
    stimulation is rarely an adverse effect, this could be acceptable if
    secondary effects are negligible.  The experimental basis for the WHO
    air quality guidelines of 1987 was relatively poor, but evidence is
    increasing that these are certainly not unrealistically low.  Not even

    all direct adverse effects are eliminated by these levels (Adaros et
    al., 1991a,b; Bender et al., 1991; Ashenden et al., 1993).  Thus, the
    updated guidelines/guidance values should be lower than the ones of
    1987.

         A long-term critical level for NO2 of 10 g/m3, especially to
    avoid eutrophication of nutrient-poor vegetation, was proposed by
    Guderian (1988) and Zierock et al. (1986).  The basis for this
    proposal was the work of Lee et al. (1985) and Press et al. (1986),
    who found reduced growth of  Sphagnum cuspidatum in regions with an
    annual mean concentration of 38 and 11 g/m3, respectively, compared
    to the growth in another region with 4 g/m3 after 140 days of
    exposure.  However, Lee et al. (1985) also showed that the poor growth
    of  Sphagnum was more closely related to the excessively high
    concentrations of nitrate and ammonium ions in bog water rather than
    to the concentration of NO2 alone.  Thus, this information could well
    be used to assess water quality guidelines, but is not very useful as
    a basis for air quality guidelines.

    4.1.8.1  Representativity of the data

         Critical levels for adverse effects of NH3 on plants were
    estimated using the model of Kooijman (Van der Eerden et al., 1991). 
    To protect 95% of the species at P < 0.05, a 24-h critical level of
    270 and an annual mean critical level of 8 g/m3 were estimated. 
    With the graphical approach the 24-h average was a little lower and
    the annual mean somewhat higher (13 and 200 g/m3, respectively;
    Fig. 21).

         On the basis of a review by Cape (1994), critical levels for H+
    and NH4+ were adopted for locations where ground-level cloud is
    present for more than 10% of the time and where calcium and magnesium
    concentrations in rain or cloud do not exceed H+ and NH4+
    concentrations (mainly high elevation areas in cold climate zones):
    300 mol NH4+/litre as an annual mean (UNECE, 1994).

         There remains considerable deficiency in the amount and scope of
    experimentally derived information on which to base air quality
    guidelines.  This results from the fact that most experiments have
    been performed under conditions that cannot directly be compared to
    outdoor circumstances.  In most experiments, only primary effects such
    as photosynthesis and biomass production were evaluated, and rarely
    secondary effects such as alteration of stress tolerance or
    competitive ability.  The plant species chosen in most experiments
    were crops, although evidence suggests that some native species are
    relatively more sensitive.  For instance, lower plants such as
    bryophytes and lichens are not protected by a waxy waterproof cuticle
    and do not have the potential to close stomata.  Furthermore, Pearson
    & Stewart (1993) suggested that plants species from nutrient-poor
    acidic soils are more sensitive.

         Further work, employing low concentrations of NHy and NOx
    (especially NO) in different climates, is urgently required.  It is
    not realistic to screen for all likely growth and physico-chemical
    effects in the majority of species in order to arrive at general
    effect thresholds.  Selections must be made on the basis of an
    understanding of differences in sensitivity between species.  However,
    an obvious mechanistic explanation for sensitivity differences is not
    yet available.  For instance, there appears to be no relationship
    between the sensitivity to NO2 and the nitrogen preference
    (Ellenberg, 1985; Ashenden et al., 1993).  Sensitivity classifications
    for some tens of species have been made for NO2 and NH3 (e.g. US
    EPA, 1978; Taylor et al., 1987), but it appears difficult to extend
    predicitions very far beyond those examined.  The hypotheses of Raven
    (1988) and Pearson & Stewart (1993) should be studied in more detail
    in laboratory experiments and field studies, as they could result in
    an efficient selection criterium for further screening.

         An attempt to determine the global situation regarding
    nitrogen-containing compounds is being made.  The assumption that all
    deposited nitrogen-containing compounds (which is part of the critical
    load concept) act additionally in their impact on vegetation is poorly
    based on experimental results and is probably not valid for the short
    term.

         Generalizations and simplifications have to be made to arrive at
    conclusions that are applicable in environmental policy making, but
    this should be done with great care.  Mechanistic simulation models
    can become a powerful tool for making general predictions on the basis
    of various air pollution experiments (Van de Geijn et al., 1993). 
    However, sufficient knowledge of biochemical and physiological
    mechanisms to incorporate the impact of air pollution on vegetation
    into these models is still lacking.  This applies especially to
    natural vegetation where stress sensitivity and competition are key
    factors.

         Many gaps in understanding the impact of nitrogen-containing air
    pollution on vegetation still exist, and this is a good reason to use
    a safety factor in determining critical levels and loads.  However,
    currently there is still no broadly accepted approach to quantify such
    a safety factor.

    4.1.9  General conclusions

         The sum of information on gaseous NH3 and on NH4+ in wet and
    occult deposition is still too limited to arrive at air quality
    guidelines, as they should have a broad applicability.  The critical
    levels for NH3 and NH4+ are probably only valid for temperate
    oceanic climatic zones (see sections 4.1.7.3, 4.1.7.4 and 4.1.8).

         In most studies with NO and NO2, no significant effects were
    found at levels below 100 g/m3, but several relevant exceptions
    exist.  NO2 altered the response to O3 generally with a
    less-than-additive interaction.  In combination with SO2, NO2 acted
    more-than-additively in most cases.  With CO2 and with NO, no
    interaction and thus additivity were generally found.  The lowest
    effective concentration levels of NO2 are about equal for NO2 alone
    and in combination with O3 or SO2, although, generally, at
    concentrations near to its effect threshold NO2 causes growth
    stimulation if it is the only pollutant, while in combination with
    SO2 and/or O3 it results in growth inhibition.

         To include the impact of NO, a critical level for NOx instead of
    one for NO2 is proposed, assuming that NO and NO2 act in an additive
    manner.  A strong case can be made for the provision of critical
    levels for short-term exposures, but currently there are insufficient
    data to provide these with sufficient confidence.  Current evidence
    exists for a critical level of around 75 g/m3 for NOx as a 24-h
    mean.

         The critical level for NOx (NO and NO2, added in ppb and
    expressed as NO2 in g/m3) is 30 g/m3 as an annual mean.  At
    concentrations slightly above this critical level, growth stimulation
    will be the dominant effect if the ambient conditions allow growth and
    NOx is the only pollutant.  This stimulation may be combined with a
    minor increase in sensitivity to biotic and abiotic stresses.  In
    cases where biomass production is a positive effect, e.g., in
    agriculture and plantation forests, the critical level can be higher. 
    Current knowledge is insufficient to arrive at critical levels for
    these systems.

         The critical level can be converted into deposition quantities. 
    With deposition velocities of 3 and 0.3 mm/second for NO2 and NO,
    respectively (see section 3.2.2 and Table 5), the annual deposition
    corresponding to a NOx concentration of 30 g/m3 is 4.8 kg/ha when
    half of the NOx is NO2 and 8.3 kg/ha when all is NO2.  This
    indicates that at a concentration near to its critical level the
    contribution of NOx to the nitrogen demand is negligible for
    fertilized crops but not for natural vegetation (see section 4.2).

    4.2  Effects on natural and semi-natural ecosystems

    4.2.1  Effects on freshwater and intertidal ecosystems

         In this section the effects of atmospheric nitrogen deposition
    on freshwater and intertidal ecosystems are evaluated.  The
    effects of increased emissions of nitrogen compounds with respect to
    eutrophication are examined in order to establish ecosystem guidelines

    for nitrogen deposition.  The ecological effects of nitrogen
    deposition are reviewed for (i) shallow softwater lakes and (ii) lakes
    and streams.

    4.2.1.1  Effects of nitrogen deposition on shallow softwater lakes

         In the lowlands of western Europe, soft water is often found on
    sandy soil which is poor in calcium carbonate or almost devoid of it. 
    The water is poorly buffered and the concentrations of calcium in the
    water layer are very low.  The water bodies are shallow and fully
    mixed, with periodically fluctuating water levels.  They are mainly
    fed by rain water and thus are oligotrophic. These softwater
    ecosystems are characterized by plant communities from the
    phytosociological alliance Littorellion (Schoof-van Pelt, 1973;
    Wittig, 1982; Roelofs, 1986; Vge, 1988; Arts, 1990).  The stands of
    these communities are characterized by the presence of rare and
    endangered isoetids, such as  Littorella uniflora, Lobelia dortmanna,
     Isoetes lacustris, I. echinospora, Echinodorus species,  Luronium
     natans and many other softwater macrophytes.  These softwater bodies
    are now almost all within nature reserves and have become very rare in
    western Europe.  A strong decline in amphibians has also been observed
    in these water bodies (Leuven et al., 1986).

         The effects of nitrogen pollutants on these softwater bodies have
    been intensively studied in the Netherlands both in field surveys and
    experimental studies.  Field observations on about 70 softwater bodies
    (with well-developed isoetid vegetation in the 1950s) showed that the
    water, in which these macrophytes were still abundant in the early
    1980s, was poorly buffered (alkalinity of 50-500 eq/litre), slightly
    acidic (pH=5-6) and very poor in nitrogen (Roelofs, 1983; Arts et al.,
    1990).  The softwater sites where these plant species had disappeared
    could be divided into two groups.  In 12 of the 53 softwater sites,
    eutrophication, resulting from nutrient-enriched water, seemed to be
    the cause of the decline.  In this group of non-acidified water
    bodies, plant species, such as  Myriophyllum alterniflorum, Lemna
     minor or  Riccia fluitans had become dominant.  High concentrations
    of phosphate and ammonium ions were measured in the sediment. In some
    of the larger water bodies no macrophytes were found, as a result of
    dense plankton bloom.  In the second group of lakes and pools (41 out
    of 53) another development had taken place: the isoetid species were
    replaced by dense stands of  Juncus bulbosus or aquatic mosses such
    as  Sphagnum cuspidatum or  Drepanocladus fluitans.  This clearly
    indicates acidification of the water in recent decades, probably
    caused by enhanced atmospheric deposition.  In the same field study it
    was shown that the nitrogen levels in the water were higher in
    ecosystems where the natural vegetation had disappeared, compared with
    ecosystems where the Littorellion stands were still present (Roelofs,
    1983).  This strongly suggests the detrimental effects of atmospheric
    nitrogen deposition in these softwater lakes.

         Several ecophysiological studies have revealed the importance of
    (i) inorganic carbon status of the water as a result of intermediate
    levels of alkalinity, and (ii) low nitrogen concentrations for the
    growth of the endangered isoetid macrophytes.  Furthermore, almost all
    of the typical softwater plants had a relatively low potential growth
    rate.  Increased acidity and higher concentrations of ammonium ion in
    the water clearly stimulated the development of  Juncus bulbosus and
    submerged mosses such as  Sphagnum and  Drepanocladus species
    (Roelofs et al., 1984; Den Hartog, 1986).  Cultivation experiments
    confirmed that the nitrogen species involved (ammonium or nitrate
    ions) differentially influenced the growth of the studied species of
    water plants.  Almost all of the characteristic softwater isoetids
    developed better when nitrate was added instead of ammonium, whereas
     Juncus bulbosus and aquatic mosses  (Sphagnum & Drepanocladus) were
    clearly stimulated by ammonium (Schuurkes et al., 1986).  The
    importance of ammonium for the growth of these aquatic mosses was also
    reported by Glime (1992).

         The effects of atmospheric deposition have been studied in
    small-scale softwater systems during a 2-year treatment with different
    artificial rainwaters.  Acidification, without airborne nitrogen input
    (using sulfuric acid), did not result in a mass growth of  Juncus
     bulbosus, and a diverse isoetid vegetation remained present. 
    However, after increasing the nitrogen concentration in the
    precipitation (as ammonium sulfate), similar changes to those seen in
    field conditions were observed, i.e. a dramatic increase in the
    dominance of  Juncus bulbosus, of submerged aquatic mosses and of
     Agrostic canina (Schuurkes et al., 1987).  It became obvious that
    the observed changes occurred because of the effects of ammonium
    sulfate deposition, leading to both eutrophication and acidification. 
    The increased levels of ammonium in the system directly stimulated the
    growth of plants such as  Juncus bulbosus, whereas the surplus
    ammonium would be nitrified in this water (pH > 4.0).  During this
    nitrification process, H+ ions are produced, which increases the
    acidity of the system.  The results of this study clearly demonstrated
    that the changes in composition of the vegetation had already occurred
    after a 2-year treatment with > 19 kg nitrogen per ha per year.  A
    reliable critical load for nitrogen deposition in these shallow
    softwater lakes is thus most likely to be below 19 kg nitrogen per ha
    per year and probably between 5 to 10 kg nitrogen per ha per year. 
    This value is supported by the observation that the greatest decline
    in the species composition of the Dutch Litorellion communities has
    coincided with nitrogen loads of around 10-13 kg nitrogen per ha per
    year (Arts, 1990).

    4.2.1.2  Effects of nitrogen deposition on lakes and streams

         There is ample evidence that an increase of acidic and
    acidifying compounds in atmospheric deposition had resulted in recent
    acidification of lakes and streams in geologically sensitive regions

    of Scandinavia, western Europe, Canada and the USA (Hultberg, 1988;
    Muniz, 1991).  This acidification is characterized by a decrease in pH
    and acid neutralizing capacity and by increases in concentrations of
    sulfate, aluminium, and sometimes nitrate and ammonium.  It has been
    shown since the 1970s, using various approaches (field surveys,
    laboratory studies, whole-lake experiments), that these changes have
    had major consequences for plant and animal species (macrofauna,
    fishes) and for the functioning of these aquatic ecosystems.

         The critical loads of acidity (from SOy and NOy) for aquatic
    ecosystems, based on steady-state water chemistry models, were
    published by the UN Economic Commission for Europe (UNECE) in 1988 and
    1992.  These models incorporate both sulfur and nitrogen acidity, and
    critical loads are calculated on the basis of: (i) base cation
    deposition; (ii) internal alkalinity production or base cation
    concentrations; and (iii) nitrate leaching from the water system.  The
    calculated critical loads are thus site-specific (sensitive areas or
    not) and also depend on the local hydrology and precipitation (for
    full details, see Hultberg (1988), Henriksen (1988) and Kmri et al.
    (1992)).  The critical loads of nitrogen acidity (kg nitrogen per ha
    per year) for the most sensitive lakes and streams are:

    Scandinavian        1.4-4.2        (Hultberg, 1988; Henriksen,
    waters                             1988; Kmri et al., 1992)

    Alpine lakes        3.5-6.1        (Marchetto et al., 1994)
 
    Humic moorland      3.5-4.5        (Schuurkes et al., 1987;
    pools                              van Dam & Buskens, 1993)

         In many areas with high water alkalinity and/or high base cation
    deposition, the values of the critical load for nitrogen acidity are
    much higher than those for sensitive freshwaters.  At present, the
    possible effects of nitrogen eutrophication by ammonia/ammonium or
    nitrate deposition are not incorporated in the establishment of
    critical loads for nitrogen, except for shallow softwater lakes (see
    section 4.2.1.1).  This is because primary production in almost all
    aquatic ecosystems is limited by phosphorus availability, and thus
    nitrogen enrichment has been considered unimportant in this respect
    (Moss, 1988). This certainly holds for those aquatic ecosystems
    considered above, where the critical load with respect to acidifying
    effects are certainly more relevant than the effects of nitrogen
    eutrophication. It is, however, to be expected that the following
    aquatic ecosystems are sensitive to nitrogen enrichment: (i) alpine
    lakes; (ii) water with low background nitrogen; and (iii) humic lakes
    (Kmri et al., 1992).  The effects of nitrogen eutrophication
    (including ammonia/ammonium) in these water bodies need further
    research and should be taken into account in future critical loads
    determinations for nitrogen.  At present it is not possible to present

    reliable critical loads for nitrogen eutrophication in these aquatic
    ecosystems.  An overview of critical loads for nitrogen in aquatic
    ecosystems is given in section 8.2.2.

    4.2.2  Effects on ombrotrophic bogs and wetlands

         In this section the effects of atmospheric nitrogen deposition in
    (semi-)natural wetlands are evaluated.  The effects of enhanced
    nitrogen inputs are considered for: (i) ombrotrophic (raised) bogs;
    (ii) fens; and (iii) intertidal fresh- and saltwater marshes.  A
    common feature of all these systems is the anaerobic nature of their
    waterlogged soils, characterized by low redox potential, high
    concentrations of toxic reduced substances and high rates of
    denitrification (Gambrell & Patrick, 1978; Schlesinger, 1991).

    4.2.2.1  Effects on ombrotrophic (raised) bogs

         Ombrotrophic ("rain-nourished") bogs, which receive all their
    nutrients from the atmosphere, are particularly sensitive to airborne
    nitrogen loads.  These bogs are systems of acidic wet areas and are
    very common in the boreal and temperate parts of Europe.  Because of
    the anaerobic conditions, decomposition rates are slow, favouring the
    development of peat.  In western Europe and high northern latitudes,
    typical plant species include bog-mosses ( Sphagnum species), sedges
     (Carex; Eriophorum) and heathers ( Andromeda, Calluna and  Erica). 
    Insectivorous plant species (e.g.,  Drosera) are especially
    characteristic of these bogs; they compensate for low nitrogen
    concentrations by trapping and digesting insects (Ellenberg, 1988b).

         Clear effects of nitrogen eutrophication have been observed in
    Dutch ombrotrophic bogs. The composition of the moss layer in the
    small remnants of the formerly large bog areas has markedly changed in
    recent decades as nitrogen loads have increased to 20-40 kg nitrogen
    per ha per year (especially as NH4+/NH3).  The most characteristic
    species  (Sphagnum) are replaced by the more nitrophilous mosses
    (Greven, 1992).  A national survey of Danish ombrotrophic bogs has
    shown a decline of the original bog vegetation together with an
    increase of more nitrogen-dependent species in areas with high ammonia
    deposition (> 25 kg ammonium nitrogen per ha per year (Aaby, 1990).

         The effects of atmospheric nitrogen deposition on ombrotrophic
    bogs have also been intensively studied in the United Kingdom (Lee et
    al., 1989; Lee & Studholme, 1992).  Many characteristic  Sphagnum
    species are now largely absent from affected ombrotrophic bog areas
    in the United Kingdom, such as the southern Pennines in England.
    Atmospheric nitrogen deposition has more than doubled in these areas
    to around 30 kg nitrogen per ha per year, compared with areas of
    healthy  Sphagnum growth.  In contrast to the situation in
    continental western Europe, most of the nitrogen deposition in the
    United Kingdom is of nitrogen oxides, although the importance of

    ammonia/ammonium deposition is also increasing in the United Kingdom
    (Fowler et al., 1980; Sutton et al., 1993).  Several studies on bogs
    in the United Kingdom have shown that increased supplies of nitrogen
    are rapidly absorbed and utilized by bog-mosses  (Sphagnum),
    reflecting the importance of nitrogen as a nutrient and its scarcity
    in unpolluted regions (Woodin et al., 1985; Woodin & Lee, 1987).  The
    high nitrogen loadings are, however, supraoptimal for the growth of
    many characteristic  Sphagnum species, as demonstrated by restricted
    development in growth experiments and transplantation studies between
    clean and polluted locations.  In areas with high nitrogen loads, such
    as the Pennines, the growth of  Sphagnum is in general less than in
    unpolluted areas (Lee & Studholme, 1992).  After transplantation of
     Sphagnum from an unpolluted site to a bog in the southern Pennines,
    a rapid increase in plant nitrogen content from around 12 to 20 mg/g
    dry weight was observed (Press et al., 1986).  A large increase in
    arginine in the shoots of these bog-mosses was also found after
    application of nitrogen.  In field experiments in northern and
    southern parts of Sweden, nitrogen was found to be the limiting factor
    for the growth of  Sphagnum.  However, other nutrients, especially
    phosphorus, may become secondarily limiting to plant growth when
    nitrogen inputs reach a threshold (Aerts et al., 1992).

         A further possible effect of the increased nitrogen content of
     Sphagnum is an increased decay rate of the peat, as nitrogen content
    strongly influences decomposition rates (Swift et al., 1979).  The
    decay rate of  Sphagnum peat in Swedish ombrotrophic bogs has been
    studied along a gradient of nitrogen deposition (Hogg et al., 1994). 
    The results of this short-term decay experiment indicated that the
    decomposition rate of  Sphagnum peat is more influenced by the
    phosphorus content of the material than by its nitrogen content,
    although some relation with nitrogen supply has been observed. 
    Further evidence is necessary to evaluate the long-term effects of
    enhanced nitrogen supply on the decay of peat.

         All these studies strongly indicate the detrimental effects of
    atmospheric nitrogen on the development of the bog-forming  Sphagnum
    species.  However, enhanced nitrogen deposition can influence the
    competitive relationships in nutrient-deficient vegetation such as
    bogs.  The effects of the supply of extra nitrogen on the population
    ecology of  Drosera rotundifolia has been recently studied in a
    4-year experiment in Swedish ombrotrophic bogs (Redbo-Torstensson,
    1994).  It was demonstrated that experimental applications of more
    than 10 kg nitrogen (as NH4NO3) per ha per year clearly affected the
    population of this insectivorous species: the establishment of new
    individuals and the survival of the plants significantly decreased in
    the vegetation treated with extra nitrogen.  This decrease in the
    total population density of the characteristic bog species  Drosera
    was not caused by toxic effects of nitrogen, but by enhanced

    competition for light with tall species such as  Eriophorum and
     Andromeda, which responded positively to the increased nitrogen
    inputs.

         On the basis of the United Kingdom and Scandinavian studies, it
    has become clear that increased nitrogen loads strongly affect
    ombrotrophic bog ecosystems, especially because of the high nitrogen
    retention capacity and closed nitrogen cycling.  The growth of
    bog-mosses is reduced, directly by nitrogen and indirectly by a
    changed competitive relationship between the prostrate dominants
    (e.g.  Eriophorum) and the subordinate plant species.  A reliable
    critical load for nitrogen in these ombrotrophic bogs is 5-10 kg
    nitrogen per ha per year, although additional long-term studies with
    enhanced nitrogen (both nitrogen oxides and ammonia/ammonium) are
    necessary to validate this figure.

    4.2.2.2  Effects on mesotrophic fens

         Fens are wetland ecosystems that are typical of alkaline to
    slightly acidic habitats in many countries.  The alkalinity is due to
    groundwater draining from surrounding rocks or sediments that are
    relatively rich in calcium carbonate.  Most of these fen ecosystems
    are characterized by rare and endangered plants species.  The effects
    of nitrogen enrichment upon mesotrophic fens have been intensively
    studied in the Netherlands (Verhoeven & Schmitz 1991; Koerselman &
    Verhoeven, 1992).  These fen ecosystems are characterised by many
     Carex species and are rich in forbs (e.g.,  Pedicularis palustris;
    orchids).  Most of these Dutch fen ecosystems are managed as hay
    meadows, with removal of the plant material further restricting
    nutrient levels, and are now nature reserves.

         A considerable increase of tall graminoids (grass or  Carex
    species), with a somewhat higher potential growth rate, was observed
    after experimentally adding nitrogen to three Dutch fen ecosystems
    (Vermeer, 1986; Verhoeven & Schmitz, 1991).  This increase caused a
    significant decrease in the diversity of subordinate plant species. 
    In one of the Dutch fen sites investigated, which had a long history
    of hay making, it has been shown that phosphorus deficiency was also a
    major factor in the productivity of the system, since there was a high
    output of phosphorus from the ecosystem with the hay (Verhoeven &
    Schmitz, 1991; Koerselman & Verhoeven, 1992).  Using the results of
    fertilization trials and nutrient budget studies in these fen
    ecosystems (Koerselman et al., 1990; Koerselman & Verhoeven, 1992),
    with their relatively closed nitrogen cycle, it seems reasonable to
    establish a critical load of 20-35 kg nitrogen per ha per year, based
    upon the output of the nitrogen from the fen system via normal
    management.  In some fen ecosystems, the critical nitrogen load based
    on the change in diversity may be substantially higher, because of the
    limitation of productivity by phosphorus (Egloff, 1987; Verhoeven &
    Schmitz, 1991).  In this situation, however, the risks of nitrogen

    losses to surface water or groundwater will increase because of
    phosphorus limitation, and this effect should be taken into account in
    critical load evaluation.  High rates of denitrification could also
    influence the establishment of critical loads for these fen
    ecosystems, and this aspect needs further investigation.

    4.2.2.3  Effects on fresh- and saltwater marshes

         In the wetland ecosystems discussed above, the nitrogen cycle is
    more closed than that of intertidal marshes.  The data on atmospheric
    nitrogen inputs to the nitrogen cycling in intertidal fresh- and
    saltwater marshes (with large prostrate graminoids as species of
     Spartina, Typha and  Carex) have been reviewed by Morris (1991). 
    It has become evident that nitrogen inputs to these marsh ecosystems
    via surface water (well above 100 kg nitrogen per ha per year) are
    much higher than the atmospheric loading.  In non-tidal freshwater
    marshes, it has been demonstrated in many studies that denitrification
    is very high with a very large output of nitrogen from the ecosystem
    (Morris, 1991).  Because of the combined effect of these processes,
    atmospheric nitrogen deposition is of only minor importance for these
    marshes, and it is not useful to establish a critical load for
    airborne nitrogen to these systems.  In his review Morris (1991)
    formulated a critical load for atmospheric nitrogen in wetland
    ecosystems of around 20 kg nitrogen per ha per year.  It is more
    appropriate to make a distinction for different types of wetlands, as
    shown above.  An overview of the critical loads for wetlands is given
    in chapter 8.

    4.2.3  Effects on species-rich grasslands

         Almost all of the research on the effects of atmospheric
    deposition on terrestrial vegetation has focused on ecosystems
    (e.g., forest, heathland and bogs) involving poorly buffered acidic
    soils.  Semi-natural grasslands with traditional agricultural use have
    also been an important part of the landscape in western and central
    Europe, and contain, or used to contain, many rare and endangered
    plant and animal species. A number of these grasslands have been set
    aside as nature reserves in several European countries (Ellenberg,
    1988b; Woodin & Farmer, 1993).  These semi-natural grasslands, which
    are of conservation interest, are generally nutrient-poor because of
    long agricultural use with low levels of manure and the removal
    of plant growth by grazing or hay making.  The vegetation is
    characterized by many low growing species and is of nutrient-poor soil
    status (Ellenberg, 1988b).  Although these grasslands are nowadays
    rare, the proportion of endangered plant and animal species in these
    communities is very high (Van Dijk, 1992).  Many experiments have
    shown that application of artificial fertilizer (nitrogen, phosphorus
    and potassium) changes these grasslands into tall, species-poor
    stands, dominated by a few highly productive crop grasses (Van Den
    Bergh, 1979; Willems, 1980; Van Hecke et al., 1981).  To maintain a

    large diversity of species, addition of fertilizer has to be avoided. 
    It is thus to be expected that these species-rich grasslands will be
    affected by increased atmospheric nitrogen input (Wellburn, 1988;
    Liljelund & Torstensson, 1988; Ellenberg, 1988b).

         Many semi-natural grassland types are present in western and
    central Europe.  Most of these grasslands belong to the so-called
    neutral grasslands (Molinio-Arrhenateretea; moist to moderately dry),
    to the dry calcareous grasslands (Festuca-Brometea) or to the acid
    grasslands on very poor soils (Nardetalia).  Detailed descriptions
    have been given by Ellenberg (1988b) and Van Dijk (1992).  To obtain
    critical loads for nitrogen for all these grasslands, it would be
    essential to study the effects of nitrogen eutrophication in a
    representative range within these communities.  Detailed data are,
    however, scarce.  Therefore, the results of an integrated research
    programme on nitrogen eutrophication in Dutch calcareous grasslands
    are used as a target study in this chapter to obtain (i) information
    on observed changes in this type of grassland caused by enhanced
    nitrogen input, and (ii) a reliable estimation of the critical load
    for nitrogen in these well-buffered non-acidic grasslands.  The
    results of this calcareous grassland study will be discussed in
    respect to other semi-natural grasslands.

    4.2.3.1  Effects of nitrogen on calcareous grasslands

         Calcareous grasslands are communities on limestone.  The subsoils
    consist of various kinds of limestone with high contents of calcium
    carbonate (> 90%), covered by shallow well-buffered rendzina soils
    (A/C-profiles; pH of the top soil is 7-8 with a calcium carbonate
    content of around 10%).  The depth of the soil varies between 10 and
    50 cm and the availability of nitrogen and phosphorus is low.  The
    grasslands are generally found on slopes with limestone in the subsoil
    and a deep groundwater table. Plant productivity is low, and the peak
    standing crop is in general between 150 and 400 g/m2.  The canopy of
    the vegetation is open and low (10-20 cm).  Calcareous grasslands are
    among the most species-rich plant communities in Europe and contain a
    large number of rare and endangered species.  The area of these
    semi-natural grasslands has decreased substantially in Europe during
    the second half of this century (Wolkinger & Plank, 1981; Ratcliffe,
    1984).  Some remnants have become nature reserves in several European
    countries.  To maintain the characteristic calcareous vegetation a
    specific management is needed to prevent their natural succession
    towards woodland (Wells, 1974; Dierschke, 1985).  The calcareous
    grasslands in the Netherlands are mown in autumn with removal of the
    hay (Bobbink & Willems, 1987).

    a)  Nitrogen enrichment and vegetation composition

         The effects of nitrogen enrichment in Dutch calcareous grasslands
    on vegetation composition have been investigated in two field

    experiments (Bobbink et al., 1988; Bobbink, 1991).  Either potassium 
    (100 kg per ha per year), phosphorus (30 kg per ha per year) or
    nitrogen (100 kg per ha per year), as well as a complete fertilization
    (nitrogen, phosphorus and potassium), were applied for 3 years to
    study the long-term effects on vegetation composition.  Nitrogen was
    given as ammonium nitrate and was applied to two nature reserves with
    opposite aspects (north and south).  Total above-ground biomass
    increased considerably, as expected, after three years of nitrogen,
    phosphorus and potassium fertilization.  In the experiments where
    the nutrients were applied individually, a moderate increase in
    above-ground dry weight was only seen with nitrogen addition: (about
    330 g/m2 compared with about 210 g/m2 in the untreated plots). The
    dry weight distribution of the species was significantly affected by
    nutrient treatments.  In the nitrogen-treated vegetation, the dry
    weight of the grass species  Brachypodium pinnatum was about 3 times
    higher than in the control plots.  Nitrogen application also resulted
    in a drastic reduction of the biomass of forb species (including
    several Dutch Red List species) and of the total number of species.
    The observed decrease in species diversity in the nitrogen-treated
    vegetation was not caused by nitrogen toxicity, but by the change in
    vertical structure of the grassland vegetation. The growth of
     Brachypodium was strongly stimulated and its overtopping leaves
    reduced the light within the vegetation.  It overshadowed the other
    characteristic species and growth of these species declined rapidly
    (Bobbink et al., 1988; Bobbink, 1991).  Stimulation of  Brachypodium
    growth and a substantial reduction in species diversity were observed
    following application of nitrogen to a 5-year permanent plot study
    using a factorial design (Willems et al., 1993).

         Many characteristic lichens and mosses have also disappeared in
    recent years from European calcareous grasslands (During & Willen,
    1986).  This has been caused partly by the indirect effects of extra
    nitrogen inputs, as shown experimentally by Van Tooren et al. (1990). 
    Data on the effects of nitrogen eutrophication on the species-rich
    fauna of calcareous grassland are not available.  However, it is very
    likely that the diversity of animals, especially of insects, will also
    be reduced when tall grasses are strongly dominating the vegetation,
    because of the decreasing abundance of many herbaceous flowering
    species which act as host or forage plants.

    b)  Nitrogen enrichment and nutrient storage in calcareous grasslands

         The seasonal distribution of nutrients after nitrogen
    fertilization in spring (120 kg nitrogen as ammonium nitrate) has been
    studied with the repeated harvest approach (Bobbink et al., 1989). 
    It resulted in a significantly increased peak standing crop of
     Brachypodium . This grass proves to have very efficient nitrogen
    uptake and very efficient withdrawal from its senescent shoots into
    its well-developed rhizome system.  Brachypodium can benefit from the
    extra nitrogen redistributed to the below-ground rhizomes by enhanced

    growth in the next spring.  The distribution of nitrogen has also been
    quantified in 3-year fertilization experiments.   Brachypodium
    greatly monopolized (> 75%) the nitrogen storage in both the
    above-ground and below-ground compartments of the vegetation with
    increasing nitrogen availability (Bobbink et al., 1988; Bobbink,
    1991).

         Nitrogen cycling and accumulation in calcareous grassland can be
    significantly influenced by two major outputs from the system:
    (i) leaching from the soil; and (ii) removal with management regimes. 
    Nitrogen losses by denitrification in dry calcareous grasslands are
    low (< 1 kg nitrogen per ha per year), owing to the well-drained soil
    conditions (Mosier et al., 1981).  Ammonium and nitrate leaching has
    been studied in Dutch calcareous grasslands by Van Dam et al. (1992). 
    Both the water fluxes and the solute fluxes at different soil depths
    have been measured over 2 years in untreated vegetation and in
    calcareous grassland vegetation sprayed at 2-weekly intervals with
    ammonium sulfate (50 kg nitrogen per ha per year).  The nitrogen
    leaching from the untreated vegetation was very low (0.7 kg nitrogen
    per ha per year) and amounted to only 2% of the total atmospheric
    deposition of nitrogen.  After the spraying with ammonium sulfate,
    nitrogen leaching increased significantly to 3.5 kg nitrogen per ha
    per year, although this figure was also a very small proportion (4%)
    of the nitrogen input in this vegetation (Van Dam et al., 1992).  It
    is thus evident that calcareous grassland ecosystems retain nitrogen
    almost completely in the system.  This is caused by a combination of
    enhanced plant uptake (Bobbink et al., 1988; Bobbink, 1991) and
    increased immobilization in the soil organic matter (Van Dam et al.,
    1992).

    4.2.3.2  Critical loads for nitrogen in calcareous grasslands

         The most important output of nitrogen from calcareous grassland
    is via exploitation or management.  The annual nitrogen removal in the
    hay varies slightly between years and sites, but in general between
    17 and 22 kg nitrogen per ha is removed from the system under normal
    management conditions in the Netherlands (Bobbink, 1991; Bobbink &
    Willems, 1991).  The ambient nitrogen deposition in Dutch calcareous
    grasslands, as determined by Van Dam (1990), is high (35-40 kg
    nitrogen per ha per year) and is nowadays the major nitrogen input to
    the system.  Legume species  (Leguminosae) also occur in calcareous
    vegetation, and form an additional nitrogen input owing to the
    nitrogen-fixing microorganisms in their root nodules (about 5 kg
    nitrogen per ha per year).

         The nitrogen mass balance of Dutch calcareous grasslands is
    summarized in Table 25. It is obvious that calcareous grasslands now
    significantly accumulate nitrogen (16-26 kg per ha per year) in the
    Netherlands.  A critical nitrogen load has been determined with a mass
    balance model, because of the lack of long-term addition experiments

    with low nitrogen loads.  Assuming a critical long-term immobilization
    rate for nitrogen of 0-6 kg nitrogen per ha per year, the critical
    nitrogen load can be derived by adding the nitrogen fluxes due to net
    uptake, denitrification and leaching, corrected for the nitrogen input
    by fixation. In this way, 15-25 kg nitrogen per ha per year is
    considered as nitrogen critical load for this ecosystem. Nitrogen
    cycling within the system (between plants and soil) is not used for
    this calculation.

    Table 25.  Nitrogen mass balance (kg nitrogen per ha per year)
               for dry calcareous grassland in the Netherlands
                                                                        
 
    Input                              Output
                                                                        

    Atmospheric deposition   35-40     Harvest             17-22
    Nitrogen fixation        5         Denitrification     1
                                       Soil leaching       1

    Total                    40-45     Total               19-24
                                                                        

         In calcareous grassland in England, addition of nitrogen
    stimulated the dominance of grasses in most cases (Smith et al., 1971;
    Jeffrey & Pigott, 1973).  In these studies, the application of
    50-100 kg nitrogen per ha per year resulted in a strong dominance of
    the grasses  Festuca rubra, F. ovina or  Agrostis stolonifera.
    However,  Brachypodium and  Bromus erectus, the most frequent
    species in calcareous grassland in continental Europe, were absent
    from these sites.  Following a survey of data from a number of
    conservation sites in southern England, Pitcairn et al. (1991)
    concluded that  Brachypodium had expanded in the United Kingdom
    during the last 100 years.  They considered that much of the early
    spread could be attributed to a decline in grazing pressure but that
    the more recent spread had, in some cases, taken place despite grazing
    or mowing, and could be related to nitrogen inputs.  However, a study
    of chalk grassland at Parsonage Downs (United Kingdom) showed no
    substantial change in species composition over the twenty years
    between 1970 and 1990, a period when nitrogen deposition is thought to
    have increased significantly (Wells et al., 1993).   Brachypodium was
    present in the sward but had not expanded as in the Dutch grasslands. 
    In a linked experimental study, applications of nitrogen to eight
    forbs and one grass  (Brachypodium) at levels of 20, 40 and 80 kg
    nitrogen per ha per year for two years did not result in
     Brachypodium becoming dominant.

         Apart from the Dutch studies, the effects of enhanced nitrogen
    inputs have been little investigated in continental European
    calcareous grasslands.  Some data from a recent fertilization

    experiment at the alvar grasslands, a thin-soiled vegetation over flat
    limestone, on the Swedish island land, suggest that the vegetation
    hardly responds to applications of nitrogen or phosphorus (Sykes & Van
    der Maarel, 1991; personal communication by Van der Maarel).  Only
    irrigation in combination with nutrients has caused an increase in
    grasses.  This is probably due to the low annual precipitation in this
    area (400-500 mm).

         Increased nitrogen availability is probably of major importance
    in many European calcareous grasslands. An increased availability of
    nitrogen is indicated by enhanced growth of some tall grasses,
    especially stress-tolerant species, which have a slightly higher
    potential growth rate and efficient nitrogen utilization.  It clearly
    depends on the original species composition, as to which of the
    grass species will increase following enhanced nitrogen inputs. 
    Furthermore, the difference between the Dutch and United Kingdom
    results may reflect differences in management; the impacts of grazing
    in the United Kingdom grasslands could offset any competitive
    advantage the grasses may have obtained from additional nitrogen
    inputs.  The critical load for nitrogen in these calcareous grasslands
    could be influenced by management; long-term studies involving
    additional nitrogen input with various management schemes are needed
    to quantify these aspects.

    4.2.3.3  Comparison with other semi-natural grasslands

         Productivity in grasslands is strongly influenced by nutrients,
    as shown in many agricultural studies (e.g. Chapin, 1980).  It is also
    well-known that large amounts of fertilizer (nitrogen, phosphorus and
    potassium) alter almost all grassland types into tall, species-poor
    swards dominated by a few highly productive crop grasses (e.g. Bakelaar
    & Odum, 1978; Van Den Bergh, 1979; Willems, 1980; Van Hecke et al.,
    1981).  Most of these species-rich grasslands are deficient in
    nitrogen or phosphorous, and thus characterized by plant species of
    nutrient-poor habitats.  It is thus likely that these grasslands are
    sensitive to nitrogen eutrophication from the atmosphere (Wellburn,
    1988; Ellenberg, 1988b).  Thus, it is also important to establish
    critical loads for nitrogen in the species-rich grasslands, although
    data from experiments with nitrogen application in these semi-natural
    grasslands are scarce.

         Increased nitrogen availability can also affect other
    semi-natural grasslands, although experimental evidence is quite
    scarce.  A classical study into the effects of nutrients on neutral
    grasslands is the Park Grass experiment at Rothamsted, England, which
    has been running since 1856 (Williams, 1978). After application of
    nitrogen as ammonium sulfate or sodium nitrate (48 kg nitrogen per ha
    per year), the vegetation became very poor in species and dominated by
    grasses such as  Holcus lanatus or  Agrostis sp.  The effects of
    nutrients in dry and wet dune grasslands (1% calcium carbonate) on

    sandy soils have been studied at Braunton Burrows (Devon, England) by
    Willis (1963).  Nutrients were applied over 2 years (6  40 kg
    nitrogen per ha per year) using a factorial design for nitrogen and
    phosphorus.  Nitrogen proved to be the most important nutrient in
    stimulating the growth of some grass species ( Festuca rubra and  Poa
     pratensis).  This enhanced growth reduced significantly the abundance
    of many small plants such as prostrate phanerogamic species, mosses
    and lichens (Willis, 1963).  In this coastal area with low nitrogen
    deposition (currently about 10 kg nitrogen per ha per year) the
    vegetation of dune grasslands is at present still species-rich,
    whereas in many Dutch dune grasslands with higher nitrogen loading
    (20-30 kg nitrogen per ha per year) certain grasses have increased and
    it has become a problem to maintain diversity.  Recent studies of the
    response of mesothrophic grasslands in the United Kingdom have shown
    that additions as small as 25 kg per ha per year can lead to changes
    in species diversity after several years of fertilizer additions and
    that changes take place more rapidly at higher rates of addition
    (Mountford et al., 1994).  This indicates that many of these
    semi-natural grasslands are also sensitive to nitrogen eutrophication
    and that the critical loads are likely to be of the same magnitude or
    slightly higher (20-30 kg nitrogen per ha per year) than in calcareous
    grasslands.

         Many other semi-natural grassland types occur, especially in the
    montane-subalpine regions, containing a large proportion of the
    biodiversity of the area.  However, data are too scarce to establish
    reliable load for these grasslands, although it may be expected that:
    (i) most of these grassland are sensitive to nitrogen; and (ii) the
    critical load for nitrogen is probably lower than for lowland
    (calcareous) grasslands.  The presented critical loads for
    species-rich grasslands are summarized in section 8.2.2.

    4.2.4  Effects on heathlands

         Various types of plant communities have been described as heath,
    but the term is applied here to plant communities where the dominant
    vegetation is small-leaved dwarf-shrubs forming a canopy of 1 m or
    less above soil surface.  Grasses and forbs may form discontinuous
    strata, and there is frequently a ground layer of mosses or lichens
    (Gimingham et al., 1979; De Smidt, 1979).  Dwarf-shrub heathlands
    occur in various parts of the world, especially in montane habitats,
    but are widespread in the atlantic and sub-atlantic parts of Europe. 
    In these parts of the European continent, natural heathland is limited
    to a narrow coastal zone.  Inland lowland heathlands are man-made
    (semi-natural), although they have existed for several centuries. 
    Lowland healths are widely dominated by the  Ericaceae, especially
     Calluna vulgaris in the dry heathlands and  Erica tetralix in the
    wet heathlands (Gimingham et al., 1979).  In these heaths, development
    towards woodland has been prevented by mowing, burning, sheep grazing
    and sod removal.

         Until the beginning of this century, the balance of nutrient
    input and output was in equilibrium in the lowland heathlands of
    western Europe (De Smidt, 1979; Gimingham & De Smidt, 1983).  The
    original land use implied a regular, periodic removal of nutrients
    from the ecosystems via grazing and sod removal (Heil & Aerts, 1993).
    Sod removal was practised less systematically in many Scandinavian and
    Scottish heathlands (Gimingham & De Smidt, 1983).  Here  Calluna has
    been renewed by burning at regular intervals, varying from 4-6 years
    in southern Sweden to 15-20 years in western Norway (Nilsson, 1978;
    Skogen, 1979).  The original land use of the lowland heathland ceased
    in the early 1900s and the area occupied by this community decreased
    markedly all over its distribution area (Gimingham, 1972; De Smidt,
    1979; Ellenberg, 1988b).  Dwarf-shrub heathlands may be divided into
    four categories according to broad differences in habitat: (1) dry
    heathlands; (2) wet heathlands; (3) montane and (4) arctic-alpine
    heathlands.

    4.2.4.1  Effects on inland dry heathlands

         During recent decades many lowland heathlands in western Europe
    have become dominated by grass species.  An evaluation, using aerial
    photographs, has shown that more than 35% of Dutch heathland has been
    altered into grassland (Van Kootwijk & Van der Voet, 1989).  In recent
    years, similar changes have been observed in SW Norway, which has the
    largest local emission of ammonia as well as the heaviest nitrogen
    input through long-range deposition in Norway (Anonymous, 1991).  It
    has been suggested that nitrogen eutrophication might be a significant
    factor in this transition to grasslands.  Field and laboratory
    experiments confirm the importance of nutrients, especially in the
    early phase of heathland development (Heil & Diemont, 1983; Roelofs
    1986; Heil & Bruggink, 1987; Aerts et al., 1990).  However,  Calluna
    can compete successfully with the grasses, even at high nitrogen
    loading, if its canopy remains closed (Aerts et al., 1990).  Apart
    from the changes in competitive interactions between  Calluna and the
    grasses, heather beetle plagues and nitrogen accumulation in the soil
    are important factors in the changing lowland heaths.  Furthermore,
    evidence is growing that frost sensitivity of the dominant
    dwarf-shrubs may also be affected by increasing nitrogen inputs.

         Heathland canopies have a strong filtering effect on air
    pollutants, a varying canopy structure being an important factor.  For
    sulfur and nitrogen it has been shown that bulk deposition accounts
    for only about 35-40% of total atmospheric input (Heil et al., 1987;
    Bobbink et al., 1992b).  Total atmospheric deposition of nitrogen is
    30-45 kg nitrogen per ha per year in the heathland sites in the
    eastern part of the Netherlands.  More than 70% of the total nitrogen
    input is deposited as ammonium or ammonia (Bobbink et al., 1992b;
    Bobbink & Heil, 1993).  Although data for nitrogen inputs in other

    European lowland heathlands are missing, it is very likely that in
    many European agricultural regions nitrogen deposition has increased
    in recent years (Asman, 1987; Buijsman et al., 1987).

         In  Calluna heathland, outbreaks of the chrysomelid heather
    beetle  (Lochmaea suturalis) occur frequently.  These beetles feed
    exclusively on the green parts of  Calluna.  The closed  Calluna
    canopy is opened over large areas and the interception of light by
     Calluna decreases strongly (Berdowski, 1987, 1993).  Thus the growth
    of the under-storey grasses ( Deschampsia or  Molinia) is enhanced
    significantly.  In general insect grazing is affected by the nutritive
    value of the plant material, and the nitrogen content is especially
    important in this respect (Crawley, 1983).  Experimental applications
    of nitrogen to heathland vegetation cause the concentrations of this
    element in the green parts of  Calluna to increase (Heil & Bruggink,
    1987; Bobbink & Heil, 1993).  It is, therefore, very likely that the
    frequency and intensity of heather beetle outbreaks are stimulated by
    increased atmospheric nitrogen input in Dutch heathland.  This
    hypothesis is supported by the observations of Blankwaardt (1977), who
    reported that from 1915 onwards heather beetle outbreaks were observed
    in the Netherlands with an interval of about 20 years, whereas in the
    last 15 years the outbreaks have occurred with a periodicity of less
    than 8 years.  It has also been observed that during a heather beetle
    outbreak  Calluna plants are more severely damaged in nitrogen-
    fertilized vegetation (Heil & Diemont, 1983). In a rearing experiment
    with larvae of the heather beetle, Brunsting & Heil (1985)
    demonstrated that the growth of the larvae was increased by higher
    nitrogen concentrations in the leaves of  Calluna.  Van der Eerden
    et al. (1990) studied the effects of ammonium sulfate on the growth of
    heather beetle after a outbreak of the beetle in vegetation
    artificially sprayed under a cover.  They found no significant effect
    of the treatments on total number or on biomass of the first stage
    larvae.  However, the development of subsequent larval stages was
    accelerated by the application of ammonium sulfate in the artificial
    rain: the percentage of third stage larvae increased by 20%, compared
    with larvae in the control treatment.  Furthermore, heather beetle
    larvae were put on  Calluna shoots taken from plants which had been
    fumigated with ammonia in open-top chambers (12 months; 4 to
    105 g/m3) (Van der Eerden et al., 1991).  After 7 days the larvae
    were counted and weighed.  Both the mass and the development rate of
    the larvae clearly increased with increasing concentrations of
    ammonia.  The heather beetle has recently been found in SW Norway and
    it is expanding its territory.  It is probably an important cause of
     Calluna death in this region (Hansen, 1991).  It can be concluded
    that nitrogen inputs influence outbreaks of heather beetle, although
    the exact relationship between both processes needs further research.

         In the past Dutch inland heathlands were grazed by flocks of
    sheep and sods were generally removed at intervals of 25-50 years
    (De Smidt, 1979).  Nowadays these heathlands are mostly managed by

    mechanical sod removal, which can restore the heathland vegetation if
    a seed bank of the original heathland species is still present
    (Bruggink, 1993).  The increase in organic matter and in the amounts
    of nitrogen in the system during secondary succession is well known
    (Begon et al., 1990). It was shown in the 1970s that during secondary
    heathland succession the above-ground and below-ground biomass and the
    amount of litter increase (Chapman et al., 1975; Gimingham et al.,
    1979).  It is likely that changes in nitrogen accumulation will have
    occurred due to the increase in atmospheric deposition.

         Berendse (1990) performed a detailed study on the accumulation of
    organic matter and of nitrogen during the secondary succession after
    sod removal in the Netherlands.  He found a large increase in plant
    biomass, soil organic matter and total nitrogen storage in the first
    20 to 30 years after sod removal.  Furthermore, it was demonstrated
    that nitrogen mineralization was low during the first 10 years (about
    10 kg nitrogen per ha per year), but increased considerably over the
    next 20 years to 50-110 kg nitrogen per ha per year.  Regression
    analysis of the total nitrogen storage versus the years after sod
    removal revealed an annual nitrogen increase in the system of about
    33 kg nitrogen per ha per year (probably somewhat lower in the early
    years and higher in later years) (Berendse, 1990).  These values are
    in good agreement with measured nitrogen deposition in Dutch
    heathlands in the late 1980s (Bobbink et al., 1992b).

         Thus, the organic matter in the soil increases rapidly after sod
    removal, which removes almost all of the soil organic matter. 
    However, this process is accelerated by the enhanced dry matter
    production and litter production of the dwarf shrubs caused by the
    extra nitrogen inputs.  Nitrogen accumulation in the system also
    increases. Hardly any nitrogen disappears from the system because
    nitrate leaching to deeper layers is only of minor importance in Dutch
    heathlands, as shown by De Boer (1989) and Van Der Maas (1990). 
    Nitrogen availability from atmospheric inputs, in addition to
    mineralization, is within a relatively short period (about 10 years)
    high enough to stimulate the transition of heathland to grassland,
    especially after the opening of the heather canopy by secondary
    causes.

         It has been demonstrated that frost sensitivity of some tree
    species increases with increasing concentrations of air pollutants
    (e.g. Aronsson, 1980; Dueck et al., 1991).  This increase in frost
    sensitivity is sometimes correlated with enhanced nitrogen
    concentrations in the foliage of the trees.  Long-term effects of air
    pollutants on the frost sensitivity of  Calluna and  Erica are to be
    expected because of (i) the evergreen growth form of these species and
    (ii) the increasing content of nitrogen in the leaves of  Calluna,
    associated with increased nitrogen deposition in the Netherlands and
    Norway (Heil & Bruggink, 1987; Hansen, 1991).  It has been suggested
    that damage to  Calluna shoots in the successive severe winters of
    the mid-1980s was at least partly caused by the increased frost

    sensitivity.  Investigations into the effects of air pollutants on the
    frost sensitivity of heathland species outside the Netherlands started
    in the early 1990s (Hansen, 1991; Uren, 1992).

         The effects of sulfur dioxide, ammonium sulfate and ammonia upon
    frost sensitivity in  Calluna have been studied by Van der Eerden
    et al. (1990).  After fumigation with sulfur dioxide (90 g/m3 for
    3 months), increased frost injury in  Calluna was only found at
    temperatures that seldom occur in the Netherlands (lower than -20C). 
    Fumigation with ammonia of  Calluna plants in open-top chambers over
    a 4-7 month period (100 g/m3) revealed that frost sensitivity was
    not affected in autumn (September or November), whereas in February,
    just before growth started, frost injury increased significantly at
    -12C (Van der Eerden et al., 1991).  These authors also studied the
    frost sensitivity of  Calluna vegetation sprayed with six different
    levels of ammonium sulfate (3-91 kg nitrogen per ha per year). The
    frost sensitivity increased slightly, although significantly, after
    5 months in vegetation treated with the highest level of ammonium
    sulfate (400 mol/litre; 91 kg nitrogen per ha per year), compared
    with the control treatments.  However, frost sensitivity of  Calluna
    decreased again two months later and no significant effects of the
    ammonium sulfate application upon frost hardiness were seen at that
    time. Thus, high levels of ammonia or ammonium sulfate seem to
    increase the frost sensitivity of  Calluna plants, although the
    significance of this phenomenon is still uncertain at ambient nitrogen
    inputs.  The relation between frost sensitivity and nitrogen input has
    not yet been sufficiently quantified to use it for a precise
    assessment of critical loads in this respect.

         It has been shown above that atmospheric nitrogen is the trigger
    for changes of lowland dry heathlands into grass swards in the
    Netherlands.  Lowland dry heathlands in the United Kingdom do not show
    consistent patterns over the past 10 to 40 years.  Pitcairn et al.
    (1991) assessed changes in abundance of  Calluna in three heaths in
    East Anglia (eastern England) over recent decades.  All three heaths
    showed a decline in  Calluna and an increase in grasses.  The authors
    concluded that increases in nitrogen deposition was at least partly
    responsible for the changes, but also noted that the management had
    changed.  A wider assessment of heathlands in SE England showed that
    in some cases  Calluna had declined and subsequently been invaded by
    grasses while other areas were still dominated by dwarf shrubs (Marrs,
    1993).  This clearly stresses the importance of management for the
    maintenance of dwarf shrubs in heathlands.  A simulation model, which
    integrates processes such as atmospheric nitrogen input, heather
    beetle outbreak, soil nitrogen accumulation, sod removal and
    competition between species, has been used to establish the critical
    loads of nitrogen deposition in lowland dry heathlands (Heil &
    Bobbink, 1993a,b).  The model has been calibrated with data from field
    and laboratory experiments in the Netherlands.  As an indicator of the
    effects of atmospheric nitrogen, the proportion and increase of

    grasses in the heathland system are used.  Atmospheric nitrogen
    deposition has varied between 5 and 75 kg nitrogen per ha per year in
    steps of 5-10 kg nitrogen during different simulations.  From these
    simulations, the value for the critical load of nitrogen for the
    changes from dwarf shrubs to grasses was 15-20 kg nitrogen per ha per
    year.

    4.2.4.2  Effects of nitrogen on inland wet heathlands

         The western European lowland heathlands of wet habitats are
    dominated by the dwarf shrub  Erica tetralix (Ellenberg, 1988b) and
    are generally richer in plant species than the dry heathlands.  In
    recent decades a drastic change in species composition of Dutch wet
    heathlands has been observed.  Nowadays, many wet heathlands that were
    originally dominated by  Erica have become monospecific stands of the
    grass  Molinia.  Together with  Erica almost all of the rare plant
    species have disappeared from the system.  It has been hypothesized
    that this change has been caused by atmospheric nitrogen
    eutrophication.

         Competition experiments using micro-ecosystems have clearly shown
    that  Molinia is a better competitor than  Erica at high nitrogen
    availability.  After 2 years of application of nitrogen (150 kg per ha
    per year), the relative competitive strength of  Molinia compared
    with  Erica doubled (Berendse & Aerts, 1984).  A 3-year field
    experiment with nitrogen application in Dutch lowland wet heathland
    (around 160 kg nitrogen per ha per year) also indicated that  Molinia
    is able to outdo  Erica at high nitrogen availability (Aerts &
    Berendse, 1988).  In contrast to the competitive relations between
     Calluna and the grasses,  Molinia can outdo  Erica without opening
    of the dwarf shrub canopy.  This difference is caused by the lower
    canopy of  Erica (25-35 cm), compared with  Calluna, and the tall
    growth form of  Molinia, which can overgrow and shade  Erica if
    enough nitrogen is available.  It is in this respect also important
    that heather beetle plagues do not occur in wet heathlands and that no
    frost damage has been observed in this community.

         It has been demonstrated that in many Dutch wet heathlands the
    accumulation of litter and humus has led to increased nitrogen
    mineralization (100-130 kg nitrogen per ha per year) (Berendse et al.,
    1987).  In the first 10 years after sod removal the annual nitrogen
    mineralization is very low, but afterwards it increases rapidly.  The
    leaching of accumulated nitrogen from wet heathlands is extremely low
    (Berendse, 1990).  The observed nitrogen availabilities are high
    enough to change  Erica -dominated wet heathlands into monostands of
     Molinia.

         Berendse (1988) developed a wet heathland model to simulate
    carbon and nitrogen dynamics during secondary succession.  He
    incorporated in this model the competitive relationships between

     Erica and  Molinia, the litter production from both species, soil
    nitrogen accumulation and mineralization, leaching, atmospheric
    nitrogen deposition and sheep grazing.  He simulated the development
    of lowland wet heathland after sod removal, because almost all of the
    Dutch communities are already strongly dominated by  Molinia and it
    is impossible to expect changes in this situation without drastic
    management.  Using the biomass of  Molinia with respect to  Erica as
    an indicator, his results suggested 17-22 kg nitrogen per ha per year
    as the critical load for the transition of lowland wet heathland into
    a grass-dominated sward (Berendse, 1988).  The decrease in endangered
    wet heathland forbs is partly caused by the overshading by  Molinia,
    but some species had already disappeared from wet heathlands before
    the increase of  Molinia started.  The critical load for this decline
    is probably lower than the given values and is discussed in section
    4.2.4.4.

    4.2.4.3  Effects of nitrogen on arctic and alpine heathlands

         Semi-natural  Calluna heathlands are found in the lowlands along
    the Norwegian coast to 68N and show distinct plant gradients in the
    south-north direction, from coast to inland and from lowland to upland
    areas (Fremstad et al., 1991).  In central parts of western Norway the
    plant composition changes at an altitude of about 400 m, above which
    alpine species occur regularly in the heaths.  At this altitude
    oceanic upland  Calluna and  Erica heaths merge into alpine heaths,
    which are naturally occurring, non-anthropogenic communities.  Some
    oligotrophic alpine heaths also contain  Calluna, but most heaths in
    Fennoscandia and in European parts of Russia are dominated by other
    ericoid species ( Vaccinium spp.,  Empetrum nigrum s. lat.,
     Arctostaphylos spp.,  Loiseleuria procumbens, Phyllodoce caerulea,
     Betula nana, Juniperus communis and  Salix spp.).  Many heath types
    have a more or less continuous layer of mosses and lichens. Related
    heaths are found in alpine regions in the British Isles, in Iceland,
    in southernmost Greenland, in northern Russia, and on siliceous rocks
    in the Alps (Grabherr, 1979; Elvebakk, 1985; Ellenberg, 1988b).

         Alpine and arctic habitats have many ecological characteristics
    in common, although the climatic conditions are more severe in the
    arctic regions than in most alpine regions.  The growing season is
    short (3-3.5 months in the low arctic zone), air and soil temperatures
    are low, winds are frequent and strong, and the distribution of plant
    communities depends on the distribution of snow during winter and
    spring.  Most alpine and all arctic zones are influenced by frost
    activity or solifluction, except for soils in the low alpine and
    hemiarctic zones, where podzolic soils are found.  Decomposition of
    organic matter and nutrient cycling are slow, and a large amount of
    the nitrogen input is stored in the soil in forms which can not be
    used by plants (Chapin, 1980).  The low nutrient availability limits

    primary production.  Most species are adapted to a strict nitrogen
    economy and their nitrogen indicator values are generally low
    (Ellenberg, 1979).

         Barsdate & Alexander (1975) investigated the nitrogen balance of
    an arctic area in Alaska. The most important sources of nitrogen were
    nitrogen fixation (75%) and ammonia in precipitation (22%).  Most of
    the nitrogen input is retained in living biomass, and very little is
    leached from the soil.  Denitrification is also low, partly due to
    nutrient deficiency.  Nitrogen metabolism as such does not seem to be
    inhibited by low soil temperatures (Haag, 1974).  Nitrogen fixation in
    arctic habitats has been studied in bacteria, soil algae, lichens and
    legume species  (Leguminosae) (Novichkova-Ivanova, 1971).  Blue-green
    algae (cyanobacteria) are especially important in this respect, either
    as free-living species, species associated with mosses or phycobionts
    in lichens (e.g.  Peltigera, Nephroma and  Stereocaulon).  The rate
    of nitrogen fixation depends on temperature and moisture, and thus
    varies through the year (Alexander & Schnell, 1973).

         It is to be expected that arctic and alpine communities are
    sensitive to increased atmospheric nitrogen input, because nitrogen
    retention is very efficient, although primary production is also
    strongly regulated by factors other than nitrogen (temperature,
    moisture) (Tamm, 1991).  The effects of increased nitrogen
    availability on alpine/tundra vegetation have been studied in several
    fertilizer experiments.  In most experiments full nitrogen, phosphorus
    and potassium fertilizer was used, although sometimes nitrogen was
    applied separately.  The following effects of nitrogen addition have
    been observed:

    *    In alpine and arctic vegetation, nitrogen is quickly absorbed by
         phanerogamic species and incorporated into their tissues.  The
         increase in nitrogen contents differs for graminoids, deciduous
         and evergreen species (Summers, 1978; Shaver & Chapin, 1980;
         Lechowicz & Shaver, 1982; Karlsson, 1987).

    *    Phanerogamic plant species respond to nitrogen application in
         different ways: increased growth and biomass, enhanced number of
         tillers, more flowers and changes in phenology (Henry et al.,
         1986).

    *    Some phanerogamic plant species are damaged or even killed at
         high doses of nitrogen fertilizer (Henry et al., 1986).

    *    Changes in species cover and composition are likely when nitrogen
         is applied for a longer period of time (5-10 years).

         All these studies concentrated on effects on phanerogamic plant
    species; little information is available on the effects of nitrogen on
    cryptogams.  Many authors, however, stress that nitrogen fixation
    probably will decrease when atmospheric deposition increases in arctic

    and alpine ecosystems.  In forest studies it has already been shown
    that  Cladonia spp. and some mosses are very sensitive to nitrogen
    addition.  The suggested critical load for arctic and alpine heaths
    (5-15 kg nitrogen per ha per year) is lower than that for lowland
    heathland (15-20 kg nitrogen per ha per year).

    4.2.4.4  Effects on herbs of matgrass swards

         In recent decades, in addition to the transition from
    dwarf-shrub-dominated to grass-dominated heathlands, a reduced species
    diversity in these ecosystems has been observed.  Species of the
    acidic Nardetalia grasslands and related dry and wet heathlands seem
    to be especially sensitive.  Many of these herbaceous species (e.g.,
     Arnica montana, Antennaria dioica, Dactylorhiza maculata, Gentiana
     pneumonanthe, Genista pilosa, Genista tinctoria, Lycopodium inundatum,
     Narthecium ossifragum, Pedicularis sylvatica, Polygala serpyllifolia
    and  Thymus serpyllum) are declining or have even become locally extinct
    in the Netherlands.  The distribution of these species is related to
    small-scale, spatial variability of the heathland soils.  It has been
    suggested that atmospheric deposition has caused such changes (Van Dam
    et al., 1986).  Dwarf shrubs as well as grass species are nowadays
    dominant in the former habitats of these endangered species.

         Enhanced nitrogen fluxes into nutrient-poor heathland soil
    leads to an increased nitrogen availability in the soil.  However,
    most of the deposited nitrogen in western Europe originates from
    ammonia/ammonium deposition and may also cause acidification as a
    result of nitrification.  Whether eutrophication or acidification or a
    combination of both processes is important depends on pH, buffer
    capacity and nitrification rates of the soil.  Roelofs et al. (1985)
    found that, in dwarf-shrub-dominated heathland soils, nitrification is
    inhibited at pH 4.0-4.2 and that ammonium accumulates while nitrate
    decreases to almost zero at these or lower pH values.  Furthermore,
    nitrification has been observed in the soils from the habitats of the
    endangered species, owing to its somewhat higher pH and higher buffer
    capacity.  In soils within the pH rage of 4.1-5.9, the acidity
    produced is buffered by cation exchange processes (Ulrich, 1983). The
    pH will drop when calcium is depleted, and this may cause the decline
    of those species that are generally found on soils with somewhat
    higher pH.  To study the pH effects on root growth and survival rate,
    hydroculture experiments have been conducted over 4-week periods with
    several of the endangered species ( Arnica, Antennaria, Viola,
     Hieracium pilosella and  Gentiana) and with the dominant species
    ( Molinia and  Deschampsia) (Van Dobben, 1991).  The dominant
    species indeed have a lower pH optimum (3.5 and 4.0, respectively)
    than the endangered species (4.2-6.0).  However, the endangered
    species could survive very low pH without visible injuries during this
    short experimental period.

         The pH decrease may indirectly result in an increased leaching of
    base cations, increased aluminium mobilization and thus enhanced
    aluminium/calcium (Al/Ca) ratios of the soil (Van Breemen et al.,
    1982).  Furthermore, the reduction of the soil pH may inhibit
    nitrification and result in ammonium accumulation and consequently
    increased NH4/NO3 ratios.  In a recent field study the
    characteristics of the soil of several of these threatened heathland
    species have been compared with the soil characteristics of the
    dominant species ( Calluna vulgaris, Erica tetralix and  Molinia
     caerulea) (Houdijk et al., 1993). Generally the endangered species
    grow on soil with higher pH, lower nitrogen content, and lower Al/Ca
    ratios than the dominant species.  The NH4+/NO3 ratios were higher
    in the dwarf-shrub-dominated soils than in the soil of the endangered
    species.  Fennema (1990, 1992) has demonstrated that soil from
    locations where  Arnica is still present had a higher pH and lower
    Al/Ca ratio than soil of former  Arnica stands.  However, he found no
    differences in total soil nitrogen or NH4/NO3 ratios.  Both these
    studies indicate that high Al/Ca ratios or even increased NH4/NO3
    ratios are associated with the decline of these species.  However, no
    significant effects of Al and Al/Ca on growth rates have been observed
    in hydroculture experiments in which the effects of Al and Al/Ca
    ratios on root growth and survival rate were studied (Van Dobben,
    1991).  Comparable experiments of Pegtel (1987) with  Arnica and
     Deschampsia and Kroeze et al. (1989) with  Antennaria, Viola,
     Filago minima, and  Deschampsia showed similar results.  However,
    results of a hydroculture experiment with  Arnica showed that this
    species is very sensitive to enhanced Al/Ca ratios at intermediate or
    low nutrient levels (De Graaf, 1994).  Pot experiments have indicated
    that increased NH4/NO3 ratios lead to decreased health of  Thymus.
    Hydroculture experiments with this plant species confirmed that
    increased NH4/NO3 ratios affected the cation uptake (Houdijk, 1993). 
    In a pot experiment  Thymus, planted on acid heathland soil and on
    artificially buffered heathland soil, was sprayed with 0, 15 and
    150 kg nitrogen (as ammonium) per ha per year during 6 months (Houdijk
    et al., 1993).  In this relatively short period, a deposition of 15 kg
    nitrogen (as ammonium) per ha per year on the acid soil did not lead
    to ammonium accumulation in the soil.  As a result of nitrification,
    soil pH decreased faster than in the absence of ammonium deposition. 
    At the highest deposition (150 kg nitrogen (as ammonium) per ha per
    year), nitrification rates in the acid heathland soils were too low to
    prevent ammonium accumulation, and increased NH4/NO3 ratios probably
    caused the decline of  Thymus.  Only in the artificially buffered
    soils with higher pH were nitrification rates high enough to balance
    ammonium and nitrate.   Thymus plants on these soils were healthy
    despite very high total nitrogen contents.

         At present, however, there is too little information available on
    these rare heathland and acidic grassland species to formulate a
    critical load for nitrogen.  The observation that these heathland

    species generally disappear before dwarf shrubs are replaced by
    grasses leads to the assumption that their critical load is lower than
    the critical load for the transition to grasses (< 15-20 kg nitrogen
    per ha per year) and probably between 10 and 15 kg nitrogen per ha per
    year.  An overview of the critical loads in heathlands is given in
    section 8.2.2.

    4.2.5  Effects of nitrogen deposition on forests

    4.2.5.1  Effects on forest tree species

         The growth of the vast majority of the forest tree species in the
    Northern hemisphere was until recently limited by nitrogen.  In
    forestry, nitrogen fertilizers were used to increase wood production
    (Tamm, 1991).  An increase in the supply of an essential nutrient,
    including nitrogen, will stimulate tree growth; the initial impact of
    enhanced nitrogen deposition will, therefore, be a fertilizer effect. 
    However, continued high inputs of nitrogen produces negative effects
    on tree growth (Chapin, 1980).  Until the mid-1980s, almost all of the
    research on forest decline focused on acidification, but it has now
    become evident that enhanced nitrogen deposition may also be important
    in recent forest decline.

         The effects of high atmospheric nitrogen input are very complex
    (Wellburn, 1988; Pitelka & Raynal, 1989; Heij et la., 1991; Pearson &
    Stewart, 1993).  Chronic nitrogen deposition may result in nitrogen
    saturation, when enhanced nitrogen inputs no longer stimulate tree
    growth, but start to disrupt ecosystem structure and function, and
    increased amounts of nitrogen are lost from the ecosystem in leachate
    (Agren, 1983; Aber et al., 1989; Tamm, 1991).  The nitrogen input at
    which saturation occurs depends on a number of factors including the
    amount of deposition, vegetation type and age (see chapter 3), soil
    type and management history.  The following indirect processes,
    besides the direct effect of gaseous pollutants on the shoots, are
    important:

    *     Soil acidification, due to nitrification of ammonium.  This
         process leads to accelerating leaching of base cations and, in
         poorly buffered soils, to increased dissolution of aluminium,
         which can damage fine roots development and mycorrhizas, and thus
         reduce nutrient uptake (Ulrich, 1983; Ritter, 1990).

    *     Eutrophication.  Whether ammonium will accumulate in soil or
         not is strongly dependent upon the nitrification rate and the
         deposition levels (Boxman et al., 1988).  In addition to an
         initial growth stimulation and changes in root/shoot ratio,
         ammonium accumulation will lead to an imbalance of the
         nutritional state of the soil and concomitantly of the trees
         (Roelofs et al., 1985; Van Dijk & Roelofs, 1988; Schulze et al.,
         1989; Boxman et al., 1991).  Accumulation of nitrates in the

         ecosystem may also lead to eutrophication.  As a consequence of
         all these processes, the health of the trees declines and their
         sensitivity to drought, frost, insect pests and to pathogens can
         increase markedly (Wellburn, 1988).  These phenomena may also
         play a secondary, but certainly not unimportant, role in the
         dieback of forest trees and have also been reviewed.

         Although many tree species occur in natural forest ecosystems,
    almost all studies on air pollution have concentrated on a few
    forestry tree species from acidic, nutrient-poor soils.  Most of these
    species are conifers ( Picea, Pinus and  Pseudotsuga spp.) and the
    following section concentrates on the long-term soil-mediated effects
    on these trees.  Available data on broad-leaved species ( Fagus,
     Quercus) are also considered.  Long-term effects of nitrogen
    eutrophication on the composition of the tree layer in natural forests
    may be expected but have not yet been quantified.  Soil acidification
     per se has only been briefly reviewed, because the critical load for
    acidity and tree growth is well established (Nilsson & Grennfelt,
    1988; Downing et al., 1993).

    a)  Soil-mediated changes in nutritional status of forest tree species

         It has been shown that in areas with high ammonia/ammonium
    deposition, ammonium accumulates in acid forest soils with little or
    no nitrification.  Van Dijk & Roelofs (1988) found ammonium ion
    accumulation in damaged  Pinus and  Pseudotsuga stands receiving
    60-100 kg nitrogen per ha per year, although the pH of the soil was
    the same as that in healthy stands.  This build-up of ammonium ion
    leads to increased ratios of ammonium to base cations (Roelofs et al.,
    1985; Boxman et al., 1988), a reduction of base cation uptake and,
    eventually, nutritional problems.  Using soil columns with different
    ammonium sulfate spraying treatments, critical ratios of excess
    ammonium to base cations have been determined (Boxman et al., 1988). 
    The nutritional problems of the coniferous species studied have been
    found above values of 5, 10 and 1, respectively, for the NH4/K,
    NH4/Mg and Al/Ca ratios in soil solution.  In soil with zero or a low
    nitrification rate, 10-15 kg nitrogen per ha per year is a reliable
    critical load to prevent critical ammonium to cation ratios, whereas
    in base-cation-rich soil with moderate to high nitrification rates the
    critical loads obtained are higher (20-30 kg nitrogen per ha per
    year).

         The nutritional status of the coniferous trees studied, after
    enhanced nitrogen inputs, is affected by both ammonium accumulation
    and soil acidification.  Base cation concentrations in the soil are
    reduced by leaching, whereas base cation uptake by plants is reduced
    by excess of ammonium and of aluminium.  Furthermore, root growth is
    decreased (see later).  Laboratory, greenhouse and field measurements
    in the Netherlands, Germany and southern Sweden (Van Dijk & Roelofs,
    1988; Van Dijk et al., 1989, 1990, 1992a; Hofmann et al., 1990;

    Schulze & Freer-Smith, 1991; Boxman et al., 1991, 1994; Ericsson et
    al., 1993) have shown that the complex of factors just noted produce
    severe deficiencies of magnesium and potassium in coniferous trees. 
    Most of these studies were in areas, or involved experiments, with
    large inputs (> 40-100 kg nitrogen per ha per year).

         The magnesium and phosphorus concentrations in leaves of oak
    trees  (Fagus sylvatica), a common deciduous tree in Europe,
    decreased significantly from 1984 to 1992 in permanent plots in NW
    Switzerland.  Furthermore, the magnesium concentrations in the leaves
    of young  Fagus sylvatica decreased significantly within a 4-year
    period of fertilizer application at > 25 kg nitrogen per ha per
    year (Flckiger & Braun, 1994).  In Sweden, suboptimal concentrations
    of magnesium and potassium in  Fagus leaves were found in areas with
    the highest nitrogen deposition (Balsberg-Phlsson, 1989) and addition
    of nitrogen enhanced nutritional imbalance in a 120-year-old  Fagus
    stand (Balsberg-Phlsson, 1992).  It is thus clear that this deciduous
    tree species is also sensitive to nutritional imbalance induced by
    enhanced nitrogen supply.

         Base cations are also lost from the canopy by increased leaching,
    linked to high amounts of atmospheric deposition (Wood & Bormann,
    1975; Roelofs et al., 1985; Bobbink et al., 1992b).  As a result of
    high nitrogen inputs, the organic nitrogen concentration in the
    needles of conifers has increased significantly to supra-optimal
    levels (Van Dijk & Roelofs, 1988; De Kam et al., 1991).  Concentrations
    of nitrogen-rich free amino acids, especially arginine, have
    significantly increased in the needles with high nitrogen concentration
    (> 1.5% nitrogen in  Picea abies) (Hllgren & Nsholm, 1988;
    Pietila et al., 1991; Van Dijk et al., 1992) and in  Fagus leaves
    (Balsberg-Phlsson, 1992).

         Although there is clear evidence that high NH3/NH4 loads
    produce adverse changes in the nutritional status and the growth of
    the investigated coniferous and broad-leaved trees, it is difficult to
    obtain a critical load for nitrogen from these studies, because of the
    complexity of the ecosystem.  A quite reliable critical load for
    nitrogen deposition on beech tree health is around 15-20 kg nitrogen
    per ha per year, as demonstrated in the Swiss studies (Flckiger &
    Braun, 1994).

         The results of the EC nitrogen saturation study (NITREX), which
    incorporates long-term experiments in both clean and nitrogen-polluted
    areas and whole ecosystem manipulation of nitrogen inputs, are
    providing important evidence on the effects of nitrogen deposition
    on tree health and ecosystem health.  Atmospheric deposition of
    nitrogen was reduced from 40 to 2 kg nitrogen per ha per year in a
    nitrogen-saturated  Pinus sylvestris stand in the Netherlands (Boxman
    et al., 1994, 1995).  Throughfall water was intercepted with a roof
    and replaced by clean throughfall water from 1989 onwards.  In the

    clean plot a quick response of the soil solution chemistry was
    observed.  The nitrogen concentrations in the upper soil and the
    fluxes of this element through the soil profile decreased.  As a
    result, base cation leaching and the ratios of ammonium to various
    cations also decreased; potassium and magnesium concentrations in the
    needles increased significantly.  The needle nitrogen concentrations
    were only slightly reduced in the "clean" situation, but they were
    significantly lower than in the needles of the control plots.  The
    concentration of arginine decreased significantly in the needles of
    the trees from the clean throughfall plot. Furthermore, tree growth
    became higher after 4 years of clean throughfall than in control plots
    with high nitrogen deposition. No changes in the mycorrhizal status or
    in the undergrowth have so far been observed (Boxman et al., 1994,
    1995).  This study clearly demonstrates the detrimental effects of
    enhanced atmospheric nitrogen deposition on the nutritional balance of
    coniferous trees.

    b)  Nitrogen deposition and tree susceptibility to frost, drought and
        pathogens

         It has been suggested by several authors that sensitivity of
    trees to secondary stress factors is increased by high nitrogen
    loading (Wellburn, 1988; Pitelka & Raynal, 1989).  In field fertilizer
    applications it is often observed that tree growth starts earlier in
    the season, which may increase damage by late frost. Furthermore, it
    has been shown, after nutrient applications, that frost damage to
     Pinus sylvestris increases considerably at needle nitrogen
    concentrations above 1.8% (Aronsson, 1980), although other fertilizer
    studies have demonstrated reverse effects, i.e. improved nitrogen
    status of the plants diminishes frost damage (De Hayes et al., 1989;
    Klein et al., 1989; Cape et al., 1991).

         Only few data are available with respect to frost damage in
    direct relation to airborne nitrogen deposition.  After exposure to
    NH3 and SO2,  Pinus sylvestris saplings became more frost sensitive
    (< -10C) than control plants (Dueck et al., 1990).  Dueck et al.
    (1990) also determined the frost sensitivity of  Pinus sylvestris
    growing in areas with low ammonia/ammonium pollution (approximately
    4 g NH3/m3) and in highly polluted areas (40 g NH3/m3). 
    Surprisingly, the frost sensitivity was not higher in the polluted
    area than in the other investigated sites, and was sometimes even
    lower.  After experimental treatment with ammonia (53 g NH3/m3) the
    growth of the trees had increased, indicating that the observed change
    in frost sensitivity might have occurred as a result of changes in
    physiology and nutrient imbalance.

         The effects of simulated acid mist containing sulfate, ammonium,
    nitrate and H+ on the frost sensitivity of  Picea rubens has been
    studied (Sheppard et al., 1993; Sheppard, 1994).  There was a strong

    correlation between the application of sulfate-containing mist and an
    increase in frost sensitivity, but no such correlation was seen after
    treatment with ammonium or nitrate ions.  Sulfur compounds clearly
    affect the frost sensitivity of coniferous trees, but this effect may
    be a consequence of the nutritional status (nitrogen, base cations) of
    the trees (Sheppard, 1994).  It is concluded that the effects of
    increased nitrogen inputs on frost sensitivity remain uncertain. 
    Insufficient research has been carried out to use the results for
    assessment of a critical load.

         The water uptake of coniferous trees species may be affected by
    increasing nitrogen deposition, owing to an increase in shoot-to-root
    ratio and a reduction in fine-root length.  Indeed, the health of many
    tree species in the regions of the Netherlands with high nitrogen
    deposition was particularly poor in the dry years in the mid-1980s,
    but improved again during the subsequent normal years (Heij et al.,
    1991).  Many authors have mentioned a negative impact of high nitrogen
    supply on the development of fine roots and mycorrhiza, although
    positive effects have also been described (Persson & Ahlstrom, 1991).

         Van Dijk et al. (1990) applied 0, 48, 480 kg nitrogen (as
    ammonium sulfate) per ha per year to young  Pinus sylvestris, Pinus
     nigra and  Pseudotsuga menziesii in a pot experiment.  After seven
    months the coarse root biomass had not changed, but the fine root
    biomass decreased by 36% at the highest nitrogen application.  In
    parallel, a 63% decrease in mycorrhizal infection at the highest
    nitrogen application was found.  In the Dutch EC nitrogen saturation
    study, the fine root biomass and the number of root tips of  Pinus
     sylvestris increased after reduction of the current nitrogen
    deposition to pre-industrial levels, indicating restricted root growth
    and nutrient uptake capacity at the ambient nitrogen load of about
    40 kg nitrogen per ha per year (Boxman et al., 1994, 1995).

         In a hydroculture experiment with  Pinus nigra at pH=4.0, Boxman
    et al. (1991) found an increase in coarse/fine root ratio after
    increasing the ammonium concentration to 5000 M.  Furthermore, a
    clear relation was found between the nitrogen content of the fine
    roots and mycorrhizal infection (as measured as the number of
    dichotomously branched roots). In a hydroculture experiment Jentschke
    et al. (1991) found, however, that 2700 M nitrate had hardly any
    effect on the mycorrhizal development of  Picea abies seedlings
    inoculated with  Lactarius rufus.  Ammonium at 2700 M only had a
    slight negative effect on mycorrhizal development, whereas a reduction
    in root growth was recorded.  In a pot experiment with  Picea abies,
    Meyer (1988) found optimal mycorrhizal development when the mineral
    nitrogen content of the soil was 40 mg nitrogen/kg dry soil, while at
    350 mg nitrogen/kg dry soil a 95% reduction in mycorrhizal development
    was found.  In this study no correlation was found with the soil pH.
    Alexander & Fairly (1983) found, after fertilizer application to a
    35-year-old  Picea sitchensis stand with 300 kg nitrogen (as ammonium

    sulfate) per ha, a 15% reduction in mycorrhizal development in the
    second year after application.  Termorshuizen (1990) applied 0 to
    400 kg nitrogen ha per year either as ammonium or nitrate to young
     Pinus sylvestris inoculated with  Paxillus involutus in a pot
    experiment.  Above application rates of 10 kg nitrogen per ha per year
    there was a decrease in the amount of mycorrhizal root tips and the
    number of sclerotia.

         In addition to the above-mentioned data for coniferous trees, it
    had been shown that the shoot-to-root ratios of young  Fagus
     sylvatica trees, grown in containers with acid forest soil,
    increased significantly from about 1 to between 2 and 3 after a 4-year
    experimental application of nitrogen (25 kg nitrogen per ha per year
    or more) (Flckiger & Braun, 1994).

         It is thus likely that enhanced nitrogen inputs affect drought
    sensitivity through changes in shoot to root ratios, number of fine
    roots and the ectomycorrhizal infection of the roots.  However, the
    data are too few to use for the assessment of a critical load of
    nitrogen, based upon this aspect of reduced tree health.

         There may also be significant effects of fungal pathogens or
    insect pests associated with increasing nitrogen deposition.  The
    foliar concentrations of nitrogen increased markedly in tree needles
    or leaves in experiments with nitrogen additions, and also in forest
    sites with high atmospheric nitrogen loading (Roelofs et al., 1985;
    Van Dijk & Roelofs, 1988; Balsberg-Phlsson, 1992).  Animal grazing
    generally increases with increasing palatability of the leaves or
    shoots.  Nitrogen is of major importance for the palatability of plant
    material, and this certainly holds for insect grazing (Crawley, 1983). 
    Secondary plant chemicals, e.g., phenolics, are important for
    increased resistance of plants. The total amount of phenolics in
     Fagus leaves in a 120-year stand decreased by more than 30% after
    fertilizer application of about 45 kg nitrogen per ha per year,
    compared with the control treatment (Balsberg-Phlsson, 1992).  An
    ecologically important relation between nitrogen enrichment and insect
    pests has been quantified for lowland heathland (Brunsting & Heil,
    1985; Berdowski, 1993, see section 4.1) but not, so far, for forest
    ecosystems.

         From 1982 to 1985 an epidemic outbreak of the pathogenic fungus
     Sphaeropsis sapinea was observed in coniferous forest (mainly
     Pinus nigra) in the Netherlands.  This greatly affected whole
    stands, and was especially severe in the south-east part of the
    Netherlands, where there was high airborne nitrogen deposition
    (Roelofs et al., 1985).  Van Dijk et al. (1992) showed that there was
    a significantly higher foliar nitrogen concentration in the infected
    stands, together with higher soil ammonium levels, than in the
    uninfected stands.  Most of the additional nitrogen in the needles of
    the affected stands was stored as nitrogen-rich free amino acids,

    especially arginine.  Proline concentrations were also higher in the
    infected trees, indicting a relation with water stress (Van Dijk
    et al., 1992).

         The effects of  Sphaeropsis have also been studied by De Kam et
    al. (1991).  Two-year-old plants of  Pinus nigra were grown for
    3 years in pots and given five treatments of ammonium sulfate (very
    low to about 300 kg nitrogen per ha per year), in combination with two
    levels of potassium sulfate.  The 5-year-old plants were then
    inoculated with  Sphaeropsis.  The bark necroses were much more
    frequent in the plants treated with ammonium sulfate than in the
    controls.  Effects of ammonium sulfate upon fungal damage were even
    observed at an addition of 75 kg nitrogen per ha per year, but were
    very significant in the plants treated with 150 kg nitrogen per ha per
    year.  After potassium addition the number of necroses caused by the
    fungus was greatly reduced (De Kam et al., 1991).

         In beech forests in NW Switzerland, a significant positive
    correlation has been found between the nitrogen/potassium ratios in
    the leaves and necroses caused by the beech cancer  Nectria ditissima
    (Flckiger & Braun, 1994).  These authors also experimentally
    inoculated  Fagus sylvatica trees at different applications of
    nitrogen with this beech cancer and observed increased dieback of new
    leaves and shoots.  Furthermore, the infestation of  Fagus sylvatica
    with beech aphids  (Phyllaphis fagi) was also affected by the
    nitrogen availabilities.  The degree of infestation with the aphid
    increased significantly with enhanced leaf nitrogen/potassium ratios
    (Flckiger & Braun, 1994).  Although evidence for nitrogen-mediated
    changes in susceptibility to fungal pests and insect attacks has until
    now been based upon observations of only few species, it is obvious
    that trees became more susceptible to these attacks with increasing
    nitrogen enrichment and this may play a crucial role in the dieback of
    some forest stands.

         A critical load for nitrogen had been established at 10-15 kg
    nitrogen (at no or low nitrification) to 20-30 kg nitrogen per ha per
    year in highly nitrifying soils, based upon nutritional imbalance of
    coniferous species (Boxman et al., 1988).  Recent evidence of  Fagus
     sylvatica tree health in acidic forests indicated a critical load
    of 15-20 kg nitrogen per ha per year, based upon both field and
    experimental observations.  Elevated nitrogen deposition can
    seriously affect tree healthy via a complex web of interactions (e.g.
    susceptibility to frost and drought).  Pathogens may play an important
    role in tree decline, but at this moment it is not possible to combine
    the observed processes and effects to an overall value for a critical
    load of nitrogen for tree health.

    4.2.5.2  Effects on tree epiphytes, ground vegetation and ground fauna
             of forests

    a)  Effects on ground-living and epiphytic lichens and algae

         The effects of SOy as an acidifier on epiphytic lichens have
    been extensively studied (Insarova et al., 1992; Van Dobben, 1993). 
    SOy was previously the dominant airborne pollutant, and it has been
    shown that most (epiphytic) lichens are more negatively affected by
    acidity than by nitrogen compounds (except NOy). Most lichens have
    green algae as photobionts and are affected by acidity but not by
    nitrogen.  Some of them even react positively to nitrogen (Insarova et
    al., 1992).  However, 10% of all lichen species in the world have
    cyanobacteria (blue-green algae) as the photobiont.  These
    cyanobacterial lichens are negatively affected by acidity, and also by
    nitrogen.  Most of the NW European lichens with cyanobacteria live on
    the soil surface or are tree epiphytes.  The most pollution-sensitive
    lichens are among them and they are threatened by extinction in NW
    Europe. This is probably the result of increased nitrogen deposition,
    which inhibits the functioning of the cyanobacteria.  In the
    Netherlands, for example, all cyanobacterial lichens that were present
    at the end of the 19th century are now absent. In Denmark, 96% of the
    lichens with cyanobacteria are extinct or threatened.  Furthermore,
    the cyanobacterial lichens appear frequently on the Red List of the
    European Union countries (Hallingbck, 1991).

         Very few data exist to establish a critical load for nitrogen for
    these lichens with blue-green algae.  Nohrstedt et al. (1988)
    investigated the effects of nitrogen application (as ammonium nitrate
    or calcium nitrate) on ground-living lichens ( Peltigera aphtosa and
     Nephroma arcticum) with blue-green algae as photobionts.  The plots
    were treated once or three or four times with 120, 240 or 360 kg
    nitrogen per ha.  After a short period all  Peltigera and  Nephroma
    lichens were eliminated and even 19 years later no recolonization had
    occurred.  However, it is impossible to transform these very high
    doses to critical loads. The effects of air pollutants on lichens are
    usually related to concentrations in the air or in the precipitation. 
    It is probably more relevant to relate the effects of nitrogen on
    cyanobacterial lichens to deposition than to concentrations.  For tree
    epiphytes stemflow is most relevant, whereas for ground-living lichens
    throughfall will be more important.  Although much research is still
    needed, it has been suggested that a load of 5-15 kg nitrogen per ha
    per year is already critical for the growth of these cyanobacterial
    lichens (Hallingbck, 1991).  These lichens may be the most sensitive
    components of some forest ecosystems and thus determine the critical
    load for these systems.

         Free-living green algae, especially of the genus  Pleurococcus
    ( Protococcus and  Demococcus are synonyms), are strongly stimulated
    by enhanced nitrogen deposition. They cover practically all outdoor

    surfaces which are not subject to frequent desiccation in regions with
    high nitrogen deposition, such as in the Netherlands and in Denmark. 
    The thickness and the colonization rate of spruce needles by green
    algae has been investigated in the Swedish Environmental Monitoring
    Programme (Brakenhielm, 1991).  The Swedish data show that these algae
    do not colonize spruce needles in regions with a total deposition
    (throughfall) lower than about 5 kg nitrogen per ha per year.  In
    areas with deposition above 20 kg nitrogen per ha per year, the green
    algal cover of the needles is so thick and the algae colonize so early
    that they may impede the photosynthesis of the spruce trees.

    b)  Effects on forest ground vegetation

         In the Netherlands the forest vegetation of a site in the central
    part of the country was investigated in 1958 (with about 20 kg
    nitrogen per ha per year) and in 1981 (with about 40 kg nitrogen per
    ha per year).  All lichens had disappeared during this period and a
    considerable increase in  Deschampsia flexuosa and  Corydalis
     claviculata was found.  A large representative sample test (n=2000),
    covering about 90% of the Dutch forests, revealed in the mid-1980s
    that among the 40 most common forest plants were:  Galeopsis
     tetrahit, Rubus species,  Deschampsia flexuosa, Dryoptesis
     cathusiana, Molinia caerulea, Poa trivialis, and  Urtica dioica
    (Dirkse & Van Dobben, 1989; Dirkse, 1993).  In Sweden,  Quercus robur
    stands in two geographical areas with different nitrogen deposition
    were compared with special emphasis on nitrogen indicator species
    (Tyler, 1987).  The stands were quite comparable except for the
    nitrogen inputs: 6-8 kg nitrogen per ha per year and 12-15 kg nitrogen
    per ha per year, respectively.  In the stand with the highest
    deposition, the soil solution was more acidic, probably due to acidic
    deposition as well ( 10 kg sulfur per ha per year), and it was
    estimated that acidification of the soil has accelerated during the
    last 30 to 50 years.  The following species were more common in the
    most polluted site:  Urtica dioca, Epilobium augustifolium, Rubus
     idaeus, Stellaria media, Galium aparine, Aegopodium podagraria and
     Sambucus spp. Thus, both in Sweden and the Netherlands, species
    indicative of nitrogen enrichment became common (Ellenberg, 1988b).

         Comparable observations were reported by Falkengren-Grerup (1986)
    and by Falkengren-Grerup & Eriksson (1990), who examined the changes
    in soil and vegetation in  Quercus and  Fagus stands in southern
    Sweden. They concluded that the exchangeable base cations were reduced
    and that aluminium had doubled over the past 35 years.  They also
    found a decrease in soil pH, with a disappearance of several species
    when pH dropped below a threshold.  In spite of soil acidification
    some species had increased in cover, and the most plausible
    explanation seemed to be increased nitrogen deposition, which was
    about 15-20 kg nitrogen per ha per year in southern Sweden and which
    had doubled since 1955.  A marked increase in cover was found for

     Lactuca muralis, Dryopteris filix-max, Epilobium augustifolium,
     Rubus idaeus, Melica uniflora, Aegopodium podagraria, Stellaria
     holostea and  S. nemorum, some of these species being nitrogen
    indicators.  Despite soil acidification, acid-tolerant species
    ( Deschampsia flexuosa, Maianthemum bifolium and  Luzula pilosa) did
    not increase.  A distinct decrease was observed for  Dentaria
     bulbifera, Pulmonaria officinalis and  Polygonatum multiflorum.
    Furthermore, Rosen et al. (1992) found a significant positive
    correlation between the increase of  Deschampsia flexuosa cover in
    the last 20 years in the Swedish forests and the pattern of nitrogen
    deposition.

         In a large semi-natural  Fagus-Quercus forest in NE France,
    about 50 permanent vegetation plots were investigated in 1972 and
    1991.  The changes in species composition on calcareous soils and in
    moderately acidic habitats were followed.  During the study period a
    significant increase in nitrophilous ground flora was observed in the
    high-pH (6.9) stands.  This indicated that at this location (with
    ambient deposition of 15-20 kg nitrogen per ha per year) there was a
    distinct effect of increasing nitrogen availability (Thimonier et al.,
    1994).

         From 1968 to 1985, three sites in a 30-year-old  Pinus
     sylvestris forest in Lisselbo (central Sweden) were annually
    fertilized with 0, 20, 40 and 60 kg nitrogen per ha per year (as
    NH4NO3 plus ambient deposition of 10 kg nitrogen per ha per year). 
    The original ground vegetation consisted of  Calluna vulgaris,
     Vaccinium vitis-idea, V. myrtillus, Cladonia spp.,  Cladina spp.,
    and the mosses  Dicranum spp.,  Pleurozium spp. and  Hylocomium
    spp.  The first changes were observed within 8 to 15 years and after
    about 20 years the experimental plots were compared and statistically
    analysed.  The original species disappeared at nitrogen applications
    above 20 kg (plus ambient deposition) nitrogen per ha per year and
    were replaced by  Epilobium augustifolium, Rubus idaeus, Deschampsia
     flexuosa, Dryopteris carthusiana and the moss  Brachythecium
     oedipodium (Dirkse et al., 1991; Van Dobben, 1993).  In another
    experiment at Lisselbo the combined effects of acidification (addition
    of H2SO4, pH=2.0) and nitrogen addition (0 and 40 kg nitrogen per ha
    per year) were investigated.  The increased nitrogen level seemed to
    be the more important factor. Acidification was the next most
    discriminating factor: all species disappeared, except for the moss
     Pohlia nutans at high additions of acidity (Dirkse & Van Dobben,
    1989; Dirkse et al., 1991).

         In southern Sweden, Tyler et al. (1992) studied the effects of
    the application of ammonium nitrate (60-180 kg nitrogen per ha per
    year) over a 5-year period on stands of  Fagus sylvatica.  They
    observed a large reduction in biomass of the ground vegetation with
    the application of nitrogen, and the frequency of most herb layer
    species declined significantly.  Soil measurements revealed that, in

    addition to eutrophication effects, the acidification of the soil
    solution was also important for the decline of the original ground
    vegetation.  In an experiment on the effects of nitrogen fertilizer
    application on bryophytes, it appeared that  Brachythecium
     oedipodium, B. reflexum and  B. starkei increased significantly at
    levels up to 60 kg nitrogen per ha per year.  At higher doses these
    species tended to decline, however.  Hylocomium splendens and
     Pleurozium schreberi declined considerably at doses of 30 to 60 kg
    nitrogen per ha per year (Dirkse & Martaki, 1992).

    c)  Effects on macrofungi and mycorrhizas

         During the last two decades many reports have described a
    decrease in species diversity and abundance of macrofungi.  These
    changes can probably be attributed to indirect effects of air
    pollution, in particular to increases in the amount of available
    nitrogen (possibly in combination with acidification), and/or to
    decreased health of trees with concomitant reduction of transport to
    the roots (Arnolds, 1991).

         When comparing sites over time, the number of fruiting bodies of
    macrofungi showed marked differences.  Most studies in western Europe,
    however, have revealed that the number of ectomycorrhizal fungi
    species has declined (Arnolds, 1991).  In the Netherlands the average
    number of ectomycorrhizal species per foray declined significantly
    from 71 in 1912-1954 to 38 in 1973-1982.  Similar changes have been
    observed in Germany: 94 ectomycorrhizal species found in 1950-1979 in
    the Vlklinger area (Saarland) have not been recorded recently.  From
    the 236 species found in 1918-1942 in the Darmstadt area (Germany),
    only 137 were recorded in the early 1970s, a loss of 99 species,
    including many mycorrhizal fungi (Arnolds, 1991).  In contrast to the
    decline in mycorrhizal fungi, the number of saprotrophic species
    remained practically unchanged, while the number of lignocolous
    species increased.  This may be related to soil acidification with a
    increase in aluminium, since the proportion of forest areas in western
    Europe with a soil pH below 4.2 increased from less than 1% in 1960 to
    15% in 1988 (Schneider & Bresser, 1988).

         Arnolds (1988, 1991) concluded that acidification has very little
    effect on the diversity of ectomycorrhizal fungi, but rather triggers
    changes in species composition.  He regarded the increased nitrogen
    flux to the forest floor as the most important factor in the decline
    of mycorrhizal fungi.  Termorshuizen & Schaffers (1987) found a
    negative correlation between the total nitrogen input in mature
     Pinus sylvestris stands and the abundance of fruit bodies of
    ectomycorrhizal fungi.  Similar results were obtained by Schlechte
    (1986) who compared two sites with  Picea abies in the Gttingen area
    of Germany.  An obvious negative relation was found between nitrogen
    input (23 versus 42 kg nitrogen per ha per year) and ectomycorrhizal
    species: 85 basidiomycetes including 21 ectomycorrhizas (25%) at the

    less polluted site compared with 55 basidiomycetes including
    3 ectomycorrhizas (5%) at the most polluted site.  Environmental
    factors other than nitrogen did not differ significantly.  The
    negative impact of nitrogen seems only to hold true for mature forests
    (Termorshuizen & Schaffers, 1987).  Jansen & de Vries (1988) found a
    maximum in fruit-body production in > 20-year-old  Pseudotsuga
     menziesii stands at about 25 kg nitrogen per ha per year.  Meyer
    (1988) found a similar optimum when  Picea abies was planted in soil
    mixed with different amounts of sawdust having a high carbon/nitrogen
    ratio.

         Experiments with nitrogen fertilizer have produced similar
    results.  In a fertilizer trial with simulated nitrogen deposition in
    a  Fagus forest in southern Sweden (ambient deposition 15-20 kg
    nitrogen per ha per year), Ruhling & Tyler (1991) found, after
    applying NH4NO3 (60 and 180 kg nitrogen per ha per year), that
    within 3 to 4 years almost all mycorrhizal species ceased fruit-body
    production. In contrast, several decomposer species increased
    fruit-body production.  Wood decomposers showed no obvious reaction to
    the treatment.  No fruit-bodies were recovered when 300 kg nitrogen
    per ha was applied to  Pinus sylvestris stands as liquid manure
    (Ritter & Tlle, 1978).  The mycorrhizal frequency of the roots,
    however, was still 55% as compared to 87% in the controls. 
    Application of 112 kg nitrogen (as NH4NO3) per ha to 11-year-old
     Pinus taeda stands revealed an 88% reduction in the number of
    fruit-bodies and a 14% decrease in the number of mycorrhizas per unit
    of soil volume (Menge & Grand, 1978).  In the Lisselbo study the
    number of fruit-bodies decreased considerably at each nitrogen
    fertilizer dose (Wasterlund, 1982).  Termorshuizen (1990) applied
    0, 30 and 60 kg nitrogen (as ammonium sulfate or nitrate) per ha per
    year to young  Pinus sylvestris stands.  In general fruit-body
    production was more negatively influenced by the higher ammonium
    levels than nitrate levels.  The mycorrhizal frequency and the number
    of mycorrhizas per unit of soil volume were not influenced.  It was
    concluded by Termorshuizen (1990) that fruit-body production is much
    more sensitive to nitrogen enrichment that mycorrhizal formation. 
    Branderud (1995) found after only 1.5 year a decrease in fruit-body
    production of mycorrhizal species at a nitrogen application of 35 kg
    nitrogen (as NH4NO3) per ha in a  Picea abies stand at the Swedish
    Nitrex stand.

         In contrast, some studies have shown an increase in the number of
    fruit-bodies of insensitive mycorrhizal fungi after nitrogen
    fertilizer application, e.g.,  Paxillus involutes (Hora, 1959),
     Laccaria bicolor (Ohenoja, 1988) and  Lactarius rufus (Hora, 1959).

    d)  Effects on soil fauna of forests

         Almost all studies of changes in faunal species composition due
    to nitrogen enrichment have been conducted in arable fields or

    agricultural grasslands using complete fertilization and thus cannot
    be used to substantiate critical loads for semi-natural forest
    ecosystems (Marshall, 1977).  The relationship between acidity and
    soil fauna has also been studied in northern coniferous forests, but
    only very few studies have incorporated the effects of nitrogenous
    compounds (Grdenfors, 1987).  The abundance of  Nematoda,
     Oligochaeta and microarthropods (especially  Collembola) had
    increased in some studies, but decreased in others, after application
    of high doses of nitrogen fertilizers (> 150 kg nitrogen per ha per
    year) (Abrahamsen & Thompson, 1979; Huhta et al., 1983; Vilkamaa &
    Huhta, 1986).  A reduction in the nitrogen deposition in a  Pinus
     sylvestris stand (Nitrex site Ysselstein) to pre-industrial levels
    increased the species diversity of microarthropods due to a decreased
    dominance of some species (Boxman et al., 1995).  However, it is not
    possible to use these few data to formulate a critical load for
    changes in forest soil fauna due to increased nitrogen deposition.

         On the basis of the results presented in this overview, the
    critical load for changes in the ground vegetation of both coniferous
    and deciduous acidic forest may be 15 to 20 kg nitrogen per ha per
    year.  The critical load for changes in the fruit-body production of
    ectomycorrhizal fungi is probably about 30 kg nitrogen per ha per
    year, while the critical load for changes in mycorrhizal frequency of
    tree roots is hard to estimate, but certainly considerably higher. 
    There is insufficient data on the effects of enhanced nitrogen
    deposition on faunal components of forest ecosystems to allow critical
    loads to be set.  Epiphytic or ground-living lichens with
    cyanobacteria as the photobiont probably form a sensitive part of
    forest ecosystems and have an estimated critical load of 10-15 kg
    nitrogen per ha per year.  A summary of the critical loads for forests
    is given in chapter 8.

    4.2.6  Effects on estuarine and marine ecosystems

         Few topics in aquatic biology have received as much attention
    over the past decade as the debate over whether estuarine and coastal
    ecosystems are limited by nitrogen, phosphorus or some other factor
    (Hecky & Kilham, 1988).  Numerous geochemical and experimental studies
    have suggested that nitrogen limitation is much more common in
    estuarine and coastal waters than in freshwater systems.  Taken as a
    whole, the productivity of estuarine waters in the USA correlates more
    closely with supply rates of nitrogen than with those of other
    nutrients (Nixon & Pilson, 1983).

         Estimation of the contribution of nitrogen deposition to the
    eutrophication of estuarine and coastal waters is made difficult by
    the multiple direct anthropogenic sources (e.g., from agriculture and
    sewage) of nitrogen against which the importance of atmospheric
    sources must be weighed.  Estuaries and coastal areas are common
    locations for cities and ports. The crux of any assessment of the

    importance of nitrogen deposition to estuarine eutrophication lies in
    establishing the relative importance of direct anthropogenic exposure
    (e.g., sewage and agricultural run-off) and indirect effects
    (e.g., atmospheric deposition). 

         The effects of nitrogen deposition in certain estuarine systems
    have been investigated.  Complete nitrogen budgets, as well as
    information on nutrient limitation and seasonal nutrient dynamics,
    have been compiled for two large "estuaries", the Baltic Sea
    (Scandinavia) and the Chesapeake Bay (USA), and for the Mediterranean
    Sea.  In the case of the Mediterranean, Loye-Pilot et al. (1990)
    suggest that 50% of the nitrogen load originates as deposition falling
    directly on the water surface.  In the case of the Baltic and
    Chesapeake, deposition of atmospheric nitrogen has been suggested as a
    major contributor to eutrophication.  Data for other coastal and
    estuarine systems are less complete, but similarities between these
    two systems and other estuarine systems suggest that their results may
    be more widely applicable.  Discussion in this monograph is limited to
    these two case studies, with some speculation about how other
    estuaries may be related.

         The Baltic Sea is perhaps the best-documented case study of the
    effects of nitrogen additions in causing estuarine eutrophication. 
    Like many other coastal waters, the Baltic Sea has experienced a
    rapidly increasing anthropogenic nutrient load. It has been estimated
    that the supply of nitrogen has increased by a factor of 4, and
    phosphorus by a factor of 8, since the beginning of the 20th century
    (Larsson et al., 1985).  The first observable changes attributable to
    eutrophication of the Baltic were declines in the concentration of
    dissolved oxygen in the 1960s (Rosenberg et al., 1990).  Decreased
    dissolved oxygen concentrations result when decomposition in deeper
    waters is enhanced by the increased supply of sedimenting algal cells
    from the surface water layers to the sediment.  In the case of the
    Baltic, the spring algal blooms that now result from nutrient
    enrichment consist of large, rapidly sedimenting algal cells, which
    supply large amounts of organic matter to the sediment for
    decomposition (Enoksson et al., 1990).  Since the 1960s, researchers
    in the Baltic have documented increases in algal productivity,
    increased incidence of nuisance algal blooms, and periodic failures
    and unpredictability in fish and Norway Lobster catches (Fleischer &
    Stibe, 1989; Rosenberg et al., 1990).  It has now been shown by a
    number of methods that algal productivity in nearly all areas of the
    Baltic Sea is limited by nitrogen.  Nitrogen-to-phosphorus ratios
    range from 6:1 to 60:1 (Rosenberg et al., 1990), but the higher ratios
    are only found in the remote and relatively unaffected area of the
    Bothnian Bay (between Sweden and Finland).  Productivity in the spring
    (the season of highest algal biomass) is fuelled by nutrients supplied
    from deeper waters during spring overturn (Graneli et al., 1990); deep
    waters are low in nitrogen and high in phosphorus, resulting in
    nitrogen-to-phosphorus ratios near 5 (Rosenberg et al., 1990),

    suggesting potential nitrogen limitation when deep waters are mixed
    with surface waters.  Low nitrogen-to-phosphorus ratios in deep water
    result from denitrification in the deep sediments (Shaffer & Rnner,
    1984).  Primary productivity measurements in the Kattegat (the portion
    of the Baltic between Denmark and Sweden) correlate closely with
    uptake of NO3-, but not of PO43- (Rydberg et al., 1990).  Level II
    and III nutrient enrichment experiments conducted in coastal areas of
    the Baltic, as well as in the Kattegat, indicate nitrogen limitation
    at most seasons of the year (Graneli et al., 1990).  Growth
    stimulation of algae has also been produced by addition of rain water
    to experimental enclosures, in amounts as small as 10% of the total
    volume (Graneli et al., 1990); rain water in the Baltic is rich in
    nitrogen but poor in phosphorus.  In portions of the Baltic where
    freshwater inputs keep the salinity low, blooms of the nitrogen-fixing
    cyanobacterium  Aphanizomenon flos-aquae are common (Graneli et al.,
    1990); cyanobacterial blooms are common features of nitrogen-limited
    freshwater lakes but are usually absent from marine waters.

         Nitrogen budget estimates indicate that the Baltic Sea as a whole
    receives 7.6  1010 eq of nitrogen per year, of which 2.8  1010 eq
    per year (37%) comes directly from atmospheric deposition (Rosenberg
    et al., 1990).  Fleischer & Stibe (1989) reported that the nitrogen
    flux from agricultural watersheds feeding the Baltic has been
    decreasing since about 1980 but that the nitrogen contribution from
    forested watersheds is increasing. They cite both increases in
    nitrogen deposition and the spread of modern forestry practices as
    causes for the increase.  It should be noted, however, that the Baltic
    also experiences a substantial phosphorus load from agricultural and
    urban lands, and that phosphorus inputs may help to maintain
    nitrogen-limited conditions (Graneli et al., 1990). If the Baltic had
    received consistent nitrogen additions (e.g., from the atmosphere or
    from agricultural run-off) in the absence of phosphorus additions, it
    might well have evolved into a phosphorus-limited system some time
    ago.

         The physical structure of the Baltic Sea, with a shallow sill
    limiting exchange of water with the North Sea contributes to the
    eutrophication of the basin, by trapping nutrients in the basin once
    they reach the deeper waters.  Because the larger algal cells that
    result from nutrient enrichment in the basin provide more nutrients to
    the deep water through sedimentation, and because only shallow waters
    have the ability to exchange with the North Sea, it is estimated that
    less than 10% of nutrients added to the Baltic are exported over the
    sill to the North Sea (Wulff et al., 1990).  Throughout much of the
    year (i.e., especially during the dry months) productivity in the
    Baltic is maintained by nutrients recycled within the water column
    (Enoksson et al., 1990).  The trapping of nutrients within the basin
    and recycling of nutrients from deeper water by circulation patterns

    suggest that eutrophication of the Baltic is a self-accelerating
    process (Enoksson et al., 1990) and has a long time-lag between
    reductions of inputs and improvements in water quality.

         In the USA, a large effort has been made to establish the
    relative importance of sources of nitrogen to Chesapeake Bay (D'Elia
    et al., 1982; Smullen et al., 1982; Fisher et al., 1988; Tyler, 1988). 
    Estimates of the contribution of nitrogen to Chesapeake Bay from each
    individual source are very uncertain; estimating the proportion of
    nitrogen deposition exported from forested watersheds is especially
    problematic but critical to the analysis, because about 80% of the
    Chesapeake Bay basin is forested.  Nonetheless, three attempts at
    determining the proportion of the total nitrate load to the Bay
    attributable to nitrogen deposition all produce estimates in the range
    of 18 to 31%.  Supplies of nitrogen from deposition exceed supplies
    from all other non-point sources to the Bay (e.g., agricultural
    run-off, pastureland run-off, urban run-off), and only point source
    inputs represent a greater input than deposition.

         It is considered that the data from these studies are indicators
    of the impact of anthropogenic nitrogen.  Nevertheless, they are
    insufficient to estimate critical loads for estuarine/marine systems. 
    It may well by that critical loads for these systems differ for
    different climatic regions.

    4.2.7  Appraisal and conclusions

         Atmospheric deposition of nitrogen-containing and acidifying
    compounds have an impact on soil and groundwater quality and on the
    health and species composition of vegetation.  Critical loads for
    these effects are given in Table 26.  Critical loads have been derived
    using empirical data that relate loads directly to effects and
    steady-state soil models that calculate critical loads from critical
    chemical values for ion concentrations or ratios in foliage, soil
    solution and groundwater (De Vries, 1993). Information on the effects
    which occur when critical loads are exceeded is given in Table 27. 
    The values given in Tables 26 and 27 apply to forest vegetation in a
    temperate climate.  Whether they are representative of other climates
    is uncertain.  An overview of the critical loads for atmospheric
    nitrogen deposition in a range of natural and semi-natural ecosystems
    is given in chapter 8.

         Effects of nitrogen and acidifying deposition on soil and
    groundwater chemistry are most evident.  Field studies showed that
    deposited nitrogen is partly retained in the forest soil.  Even at
    high nitrogen deposition rates, as in the Netherlands, soil
    acidification (which is mainly manifested by leaching of aluminium and
    nitrate) is mainly caused by sulfur deposition.  A relatively small
    contribution of nitrogen to acidification does not imply that sulfur
    has a larger impact on the health of forests, since the relationship

    between soil acidification and forest health is not very clear.  The
    eutrophying impact of nitrogen is probably more important than the
    acidifying impact at present.

         There is substantial evidence from field surveys in several
    countries of Europe that exceeding critical loads does not imply
    dieback of the forest trees in the short term (one or two decades). 
    However, it does increase the risk of damage due to secondary stress
    factors and it affects the long-term sustainability of forests.  These
    risks increase with the extent to which present loads exceed critical
    loads and with the duration.

        Table 26.  Critical loads for acidity and nitrogen for forest ecosystems in temperate climates
               (From: De Vries, 1993)
                                                                                                              

                      Effects                       Criteriaa                           Critical loads
                                                                                    (kg per ha per year)
                                                                                        (H for acidity;
                                                                                    N for eutrophication)
                                                                                                              
                                                                                    Coniferous   Deciduous
                                                                                    forests      forests
                                                                                                              

    Acidity           root damage;                  Al < 0.2 mol/m3                 1.1b         1.4b
                      inhibition of uptake;         Al/Ca < 1.0 mol/mol             1.4b         1.1b
                      Al depletion;                 delta Al(OH)3 = 0 mmol/m3       1.2b         1.3b
                      Al pollution                  Al < 0.02 mol/m3                0.5b         0.3b

    Eutrophication    inhibition of uptake of K;    NH4/K < 5 mol/mol               17-70c
                      increased susceptibility;     N < 1.8%                        21-42d
                      vegetation changes;           NO3 < 0.1 mol/m3                7-20e        11-20e
                      nitrate pollution             NO3 < 0.4-0.8 mol/m3            13-21f       24-41f
                                                                                                              

    a    Background information on the various criteria is given in De Vries (1993).  Critical Al and NO3-
         concentrations and critical Al/Ca and NH4/K ratios related to root damage, inhibition of nutrient
         uptake and vegetation changes  refer to the soil solution. Critical Al and NO3- concentrations
         related to pollution refer to phreatic groundwater. Critical nitrogen contents related to an
         increased risk for frost damage and diseases refer to the foliage.
    b    Derived by a steady-state model. Al pollution refers to phreatic groundwater.  For groundwater
         used for the preparation of drinking-water, a critical acid load of 1600 mol/ha per year was derived
         (De Vries, 1993).
    c    Derived by a steady-state model assuming 50% nitrification in the mineral topsoil (second value).
    d    Empirical data on the relation between nitrogen deposition and foliar nitrogen contents.

    Table 26  (Con't)

    e    The first value is derived by a steady-state model (worst case) and the second value is based
         on empirical data.
    f    Derived by a steady-state model using critical NO3- concentrations of 0.4 and 0.8 mol/m3,
         respectively. NO3- pollution refers to phreatric groundwater.  For deep groundwater, the
         critical load will be higher because of denitrification.

    Table 27.  Possible and observed effects when critical loads are exceeded
                                                                                                              

    Possible effects         Average critical load         Observed effects in the field
                             (kg per ha per year)a
                                                                                                              

    Root damage              1.1-1.4 H                     critical Al concentrations
                                                           exceeded greatly

    Inhibition of            1.1-1.4 H                     critical Al/Ca ratios
    uptake                                                 exceeded greatly

                             17-70 N                       critical NH4/K ratios
                                                           exceeded slightly

    Aluminium depletion      1.2-1.3 H                     depletion of secondary Al compounds

    Groundwater              0.3-0.5 H                     critical Al concentrations
    pollution                                              exceeded greatly

                             13-21 N                       critical NO3 concentrations
                                                           exceeded substantially

    Increased                21-42 N                       critical N contents exceeded
    susceptibility                                         substantially; nutrient imbalances;
                                                           increased shoot/root ratios

    Vegetation changes       7-20 N                        strong increase in nitrophilous species
                                                                                                              

    a  H = acidity; N = total nitrogen
    
    5.  STUDIES OF THE EFFECTS OF NITROGEN OXIDES ON EXPERIMENTAL ANIMALS

    5.1  Introduction

         Most of the data reviewed in this chapter concerns the effects of
    NO2, since the bulk of the NOx literature is on NO2.  The results
    of the few comparative NOx studies suggest that NO2 is the most
    toxic species studied so far.  Most of the reports describe the
    effects of NO2 on the respiratory tract, but extrapulmonary effects
    are also briefly discussed.  A broad range of NO2 concentrations has
    been evaluated, but emphasis has been placed primarily on those
    studies with exposure concentrations of 9400 g/m3 (5.0 ppm) or less,
    with the exception of studies on dosimetry and emphysema.  Discussions
    of available literature on the effects of other nitrogen compounds,
    e.g., NO, HNO3, and mixtures containing NO2, also are included.  WHO
    (1987), Berglund et al. (1993) and US EPA (1993) comprise other
    reviews of the animal toxicological literature concerning NOx
    effects.

    5.2  Nitrogen dioxide

    5.2.1  Dosimetry

         It is generally agreed that effects of NO2 observed in several
    laboratory animal species can be qualitatively extrapolated to humans. 
    However, to extrapolate animal data quantitatively to humans,
    knowledge of both dosimetry and species sensitivity must be
    considered.  Dosimetry refers to estimating the quantity of NO2
    absorbed by target sites within the respiratory tract.  Even when two
    species receive an identical local tissue/cellular dose, cellular
    sensitivity to that dose is likely to show interspecies variability
    due to differences in defence and repair mechanisms and other
    physiological/metabolic parameters.  Current knowledge of dosimetry is
    more advanced than that of species sensitivity, impeding quantitative
    animal-to-human extrapolation of effective NO2 concentrations. 
    Nevertheless, information on dosimetry alone can be crucial to
    interpretation of the data base.  Both theoretical (modelling) and
    experimental dosimetry studies are discussed below.

    5.2.1.1  Respiratory tract dosimetry

         The uptake of NO2 in the upper respiratory tract (above the
    larynx) has been experimentally studied in dogs, rats and rabbits. 
    The upper airways of dogs and rabbits exposed to 7520 to 77 080 g/m3
    (4.0 to 41.0 ppm) NO2 removed 42.1% of the NO2 drawn through
    the nose (Yokoyama, 1968).  The uptake of NO2 by isolated
    upper respiratory tracts of naive and previously exposed rats
    (76 000 g/m3, 40.4 ppm NO2) was 28% and 25%, respectively (Cavanagh
    & Morris, 1987).  Kleinman & Mautz (1987) exposed dogs to 1880 or

    9400 g/m3 (1.0 or 5.0 ppm) NO2 and found that more NO2 was
    absorbed in the upper respiratory tract with nasal breathing than with
    oral breathing.  In addition, the percentage uptake of NO2 by the
    upper respiratory tract decreased with increasing ventilation rates. 
    As ventilation increased up to four times resting values, NO2 uptake
    during nasal breathing decreased from approximately 85% to less than
    80% and during oral breathing decreased from about 60% to approximately
    45%.  At rest, about 85% of the inhaled NO2 entering the lungs was
    absorbed by the lower respiratory tract; this increased to 100% with
    high ventilation rates.

         Miller et al. (1982) and Overton (1984) modelled NO2 uptake in
    the lower respiratory tract using the same dosimetry model described
    by Miller et al. (1978) for ozone (O3), but with the diffusion
    coefficient and Henry's law constant appropriate to NO2; however,
    values of the latter constant and reaction chemistry were considered
    uncertain.  For all species modelled (i.e., rat, guinea-pig, rabbit
    and humans), the results indicate that NO2 is absorbed throughout the
    lower respiratory tract, but the major dose to tissue is delivered in
    the centriacinar region (i.e., junction between the conducting and
    respiratory airways), findings consistent with the site of
    morphological effects (see section 5.2.2.4).

         Total respiratory tract uptake has been measured in healthy and
    diseased humans.  In healthy humans exposed to an NO/NO2 mixture
    containing 545 to 13 500 g/m3 (0.29 to 7.2 ppm) NO2 for brief (but
    unspecified) periods, 81 to 90% of the NO2 was absorbed during normal
    respiration; this increased to 91 to 92% with maximal ventilation
    (Wagner, 1970).  Bauer et al. (1986) exposed adult asthmatics to
    564 g/m3 (0.3 ppm) NO2 via a mouthpiece for 30 min, including
    10 min of exercise (30 litres/min) and measured inspired and expired
    NO2 concentrations.  At rest, the average uptake was 72%; during
    exercise, the average uptake was 87%, a statistically significant
    increase.  Because of the large increase in minute ventilation, the
    deposition was 3.1 g/min at rest and 14.8 g/min during exercise.

         As discussed above, increased ventilation increases the quantity
    of NO2 delivered to the respiratory tract and shifts the site of
    deposition.  Typically, the percentage uptake of NO2 in the upper
    respiratory tract decreases, with a consequent increase in uptake by
    the lower respiratory tract owing to the deeper penetration of the
    inspired gas with increased tidal volume. These experimental results
    are qualitatively similar to conclusions for the modelled effects of
    ventilation on O3 dosimetry (Miller et al., 1985; Overton et al.,
    1987a,b).

    5.2.1.2  Systemic dosimetry

         Once deposited, NO2 dissolves in lung fluids and various
    chemical reactions occur, giving rise to products that are found in
    the blood and other body fluids.  Labelled 13NO2 (564 to 1710 g/m3
    (0.3 to 0.91 ppm)) inhaled for 7 to 9 min by rhesus monkeys was
    distributed throughout the lungs (Goldstein et al., 1977b).  These
    investigators also concluded that NO2 probably reacts with water in
    the fluids of the respiratory tract to form nitrous and nitric acids. 
    Saul & Archer (1983) provided support for this pathway using rats
    inhaling NO2.  This study subsequently led to the discovery of
    endogenous NO (Moncada et al., 1988, 1991).

         The current database indicates that once NO2 is absorbed in lung
    fluids, the subsequent reaction products are rapidly taken up and then
    translocated via the bloodstream.  For example, Oda et al. (1981)
    reported a concentration-dependent increase in both NO2- and NO3-
    levels in the blood of mice during 1-h exposures to 9400 to
    75 200 g/m3 (5.0 to 40.0 ppm) NO2.  The blood levels of NO2- and
    NO3- declined rapidly after exposures ended, with decay half-times
    of a few minutes for NO2- and about 1 h for NO3-.

    5.2.2  Respiratory tract effects

    5.2.2.1  Host defence mechanisms

         Respiratory tract defences encompass many interrelated responses;
    however, for simplicity, they can be divided into physical and
    cellular defence mechanisms.  Physical defence mechanisms include the
    mucociliary system of the conducting airways.  Ciliary action moves
    particles and dissolved gases within the mucous layer towards the
    pharynx, where the mucus is swallowed or expectorated.  Both nasal
    and tracheobronchial regions are immunologically active (e.g.,
    nasal-associated lymphoid tissue and bronchial-associated lymphoid
    tissue), but this function has not been studied following NO2
    exposure.  Cellular defence mechanisms (phagocytic and immunological
    reactions) operate in the pulmonary region of the lung.  Alveolar
    macrophages (AMs) are the first line of cellular defence.  The AMs
    perform such activities as detoxifying and/or removing inhaled
    particles, maintaining sterility against inhaled microorganisms,
    interacting with lymphoid cells in a variety of immunological
    reactions, and removing damaged or dying cells from the alveoli
    through phagocytosis.  Polymorphonuclear leukocytes (PMNs), another
    group of phagocytic cells, are present in relatively small numbers
    (i.e., a small percentage of cells obtained from bronchoalveolar
    lavage (BAL) fluid) from normal lungs, but in response to a variety of
    insults, there can be an influx of PMNs from blood into the lung
    tissues and onto the surface of the airways.  Once recruited to the
    lung, PMNs then ingest and kill opsonized microbes and other foreign
    substances by mechanisms similar to those for AMs.

         The responses of PMNs and AMs are frequently studied using BAL,
    the washing of the airways and alveolar spaces with saline.  Cells and
    fluid obtained from this procedure can be used in a variety of ways to
    assess immune responses.

         Humoral and cell-mediated immunity are also active in the
    respiratory tract.  The humoral part of this system primarily involves
    the B cells that function in the synthesis and secretion of antibodies
    into the blood and body fluids.  The cell-mediated component primarily
    involves T lymphocytes, which are involved in delayed hypersensitivity
    and defences against viral, fungal, bacterial and neoplastic disease.

    a)  Mucociliary clearance

         Exposure to NO2 can cause loss of cilia and ciliated epithelial
    cells, as discussed in section 5.2.2.4 on morphological changes.  Such
    changes are reflected in the functional impairment of mucociliary
    clearance at high levels of NO2 (> 9400 g/m3, 5.0 ppm) (Giordano
    & Morrow, 1972; Kita & Omichi, 1974).  At lower exposures (2 h/day for
    2, 7 and 14 days to 564 and 1880 g/m3, 0.3 and 1.0 ppm NO2), the
    mucociliary clearance of inhaled tracer particles deposited in the
    tracheobronchial tree of rabbits was not altered (Schlesinger et al.,
    1987).

    b)  Alveolar macrophages

         Structural, biochemical, and functional changes in AMs observed
    in experimental animal studies to be caused by NO2 exposure are
    summarized in Table 28.  The adversity of these effects is not clearly
    understood at present, but they are taken as hallmarks of adverse
    reactions.  Studies of AMs in humans are discussed in chapter 6.

         Alveolar macrophages isolated from mice continuously exposed to
    3760 g/m3 (2.0 ppm) NO2 or to 940 g/m3 (0.5 ppm) NO2
    continuously with a 1-h peak to 3760 g/m3 (2.0 ppm) for 5 days/week
    showed distinctive morphological changes after 21 weeks of exposure,
    compared to controls (Aranyi et al., 1976).  Structural changes
    included the loss of surface processes, appearance of fenestrae, bleb
    formation and denuded surface areas.  Continuous exposure to a lower
    NO2 level did not result in any significant morphological changes. 
    Numerous morphological studies have shown that NO2 exposure increases
    the number of AMs (see section 5.2.2.4).

         BAL methods have also been used to study AMs.  Mochitate et al.
    (1986) reported a significant increase in the total number of AMs
    isolated from rats during 10 days of exposure to 7520 g/m3 (4.0 ppm)
    NO2, but the number of PMNs did not increase.  The AMs from exposed
    animals also exhibited increased metabolic activity, as measured by
    the activities of glucose-6-phosphate dehydrogenase, glutathione
    peroxidase and pyruvate kinase.  The AMs also showed an increase in

        Table 28.  Effects of nitrogen dioxide (NO2) on alveolar macrophagesa
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    564          0.3          2 h/day,              Rabbits     Increase in alveolar clearance.                             Schlesinger &
    1880         1.0          14 days                                                                                       Gearhart (1987)

    564          0.3          2 h/day, 13 days      Rabbit      Decreased AM phagocytic capacity at 564 g/m3; increase     Schlesinger
    1880         1.0                                            at 1880 g/m3 after 2 days of exposure.  No effect on cell  (1987a,b)
                                                                number or viability; random mobility reduced at 564 g/m3
                                                                only. No effects from 6 days of exposure on.

    564          0.3          2 h/day, 1 or         Rabbit      Acceleration in alveolar clearance at < 1880 g/m3.         Vollmuth et
    1880         1.0          14 days                                                                                       al. (1986)
    5640         3.0

    940 or       0.5 or       Continuous base       Mouse       No observable effects on AM morphology.                     Aranyi et al.
    188 base;    0.1 base;    with 2-h/day peak                                                                             (1976)
    1880 peak    1.0 peak     (5 days/week),
                              24 weeks

    3760 or      2.0 or 0.5   Continuous base       Mouse       Morphological changes, such as loss of surface processes,   Aranyi et al.
    940 base;    base;        with 7 h/day peak                 appearance of fenestrae, bleb formation, and denuded        (1976)
    3760 peak    2.0 peak     (5 days/week),                    surface areas.
                              21 weeks

    1880         1.0          17 h                  Mouse       Bactericidal activity significantly decreased by 6 and      Goldstein et al.
    4320         2.3                                            35% at 4320 and 12 400 g/m3, respectively; no effect at    (1974)
    12 400       6.6                                            1880 g/m3.
                                                                                                                                              

    Table 28.  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    1880 base;   1.0 base;    7 h/day, 5 days       Rat         Accumulation of AMs.  Superimposed spikes produced          Gregory et al.
    9400 peak    5.0 peak     per week base with                changes that may persist with continued exposures.          (1983)
                              one 1.5-h peak/day,
                              15 weeks

    2444-31 960  1.3-17.0          -                Rat         Decreased production of superoxide anion radical.           Amoruso et al.
                                                                                                                            (1981)

    3760         2.0          8 h/day,              Baboon      Impaired AM responsiveness to migration inhibitory          Greene &
                              5 days/week,                      factor.                                                     Schneider (1978)
                              6 months

    5640         3.0          3 h                   Rabbit      Increased swelling of AMs.                                  Dowell et al.
                                                                                                                            (1971)

    6768         3.6          2 h                   Rat         Enhanced AM agglutination with concanavalin A.              Goldstein et al.
                                                                                                                            (1977a)

    7520         4.0          6 h/day, 7, 14,       Rat         Changes in AM morphology; no change in numbers of AMs       Hooftman et al.
                              or 21 days                        or phagocytic capacity.                                     (1988)

    7520         4.0          10 days               Rat         Increase in number of AMs; no increase in PMNs; increased   Mochitate et al.
                                                                metabolic activity, protein and DNA synthesis; all          (1986)
                                                                responses peaked on day 4 and returned to normal on
                                                                day 10.
                                                                                                                                              

    Table 28.  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    7520         4.0          Up to 10 days         Rat         Increase in number of AMs, reaching a peak on days 3 and    Suzuki et al.
                                                                5; no increase in number of PMNs; decrease in AM viability  (1986)
                                                                throughout exposure period. Suppression of phagocytic
                                                                activity on day 7 that returned to normal value at
                                                                day 10.  Decrease in superoxide radical production on
                                                                days 3, 5 and 10.

    9400         5.0          7 days                Mouse       No effect on phagocytic activity.                           Lefkowitz et al.
                                                                                                                            (1986)

    9400         5.0          3 h                   Rabbit      No change in AM resistance to pox virus.                    Acton & Myrvik
                                                                                                                            (1972)
                                                                                                                                              

    a  Modified from US EPA (1993)
    b  AM = alveolar macrophage; PMN = polymorphonuclear leukocyte
        the rate of synthesis of protein and DNA.  All responses peaked on day
    4 and returned to control levels by the tenth day.  Suzuki et al.
    (1986) made similar observations and, in addition, found that the
    viability of AMs was decreased on day 1 and remained depressed for the
    remainder of the exposure period.  Increased numbers and metabolic
    activity of AMs would be expected to have a positive influence on host
    defences.  However, AMs are rich in proteolytic enzymes, and increased
    numbers could result in some tissue destruction when the enzymes are
    released.  Furthermore, as discussed below, although more AMs may be
    present, they often have a decreased phagocytic ability.

         Schlesinger (1987a,b) found no significant changes in the number
    or the viability of AMs in BAL from rabbits exposed to 564 or
    1880 g/m3 (0.3 or 1.0 ppm) NO2, 2 h/day, for 13 days.  Although
    there were no effects on the numbers of AMs that phagocytosed latex
    spheres, 2 days of exposure to 564 g/m3 (0.3 ppm) decreased the
    phagocytic capacity (i.e., number of spheres per cell); the higher
    level of NO2 increased phagocytosis.  Longer exposures had no effect. 
    The phagocytic activity of rat AMs was significantly depressed after
    7 days of exposure to 7520 g/m3 (4.0 ppm) but returned to the
    control value at 10 days of exposure (Suzuki et al., 1986).  There may
    be a species difference in responsiveness because Lefkowitz et al.
    (1986) did not observe a depression in phagocytosis in mice exposed
    for 7 days to 9400 g/m3 (5.0 ppm) NO2.  Suzuki et al. (1986)
    proposed that the inhibition of phagocytosis might be due to NO2
    effects on membrane lipid peroxidation.  Studies by Dowell et al.
    (1971) and Goldstein et al. (1977a) add support to this hypothesis. 
    Acute exposure to 5640-7520 g/m3 (3.0-4.0 ppm) caused swelling of
    AMs (Dowell et al., 1971) and increased AM agglutination with
    concanavalin A (Goldstein et al., 1977a), suggesting damage to the
    membrane function.

         Two independent studies have shown that NO2 exposure decreases
    the ability of rat AMs to produce superoxide anion involved in
    antibacterial activity.  Amoruso et al. (1981) presented evidence
    of such an effect at NO2 concentrations ranging from 2440 to
    32 000 g/m3 (1.3 to 17.0 ppm).  The duration of the NO2 exposure
    was not given; all exposures were expressed in terms of parts per
    million  hours.  A 50% decrease of superoxide anion production began
    after exposure to 54 700 g/m3  h (29.1 ppm  h) NO2.  Suzuki et
    al. (1986) reported a marked decrease in the ability of rat AMs to
    produce superoxide anion following a 10-day exposure to either 7520 or
    15 000 g/m3 (4.0 or 8.0 ppm) NO2.  At the highest concentration,
    the effect was significant each day, but at the lower concentration,
    the depression was significant only on exposure days 3, 5 and 10.

         Alveolar macrophages obtained by BAL from baboons exposed to
    3760 g/m3 (2.0 ppm) NO2 for 8 h/day, 5 days/week, for 6 months had
    impaired responsiveness to migration inhibitory factor produced by
    sensitized lymphocytes (Greene & Schneider, 1978).  This substance

    affects the behaviour of AMs by inhibiting free migration, which, in
    turn, interferes with the functional capacity of these defence cells. 
    In addition, the random mobility of AMs was significantly depressed in
    rabbits following a 2 h/day exposure for 13 days to 564 g/m3
    (0.3 ppm), but not to 1880 g/m3 (1.0 ppm) (Schlesinger, 1987b).

         Vollmuth et al. (1986) studied the clearance of strontium-
    85-tagged polystyrene latex spheres from the lungs of rabbits
    following a single 2-h exposure to 564, 1880, 5640 or 18 800 g/m3
    (0.3, 1.0, 3.0 or 10.0 ppm) NO2.  An acceleration in clearance
    occurred immediately after exposure to the two lowest NO2
    concentrations; a similar effect was found by Schlesinger & Gearhart
    (1987).  At the higher levels of NO2, an acceleration in clearance
    was not evident until midway through the 14-day post-exposure period. 
    Repeated exposure for 14 days (2 h/day) to 1880 or 18 800 g/m3
    (1.0 or 10.0 ppm) NO2 produced a response similar to a single
    exposure at the same concentration.

    c)  Humoral and cell-mediated immunity

         Various humoral and cell-mediated effects are summarized in Table
    29.

         Exposing sheep to 9400 g/m3 (5.0 ppm) NO2, 1.5 h/day for 10 to
    11 days showed that intermittent short-term exposure may temporarily
    alter the pulmonary immune responsiveness (Joel et al., 1982).  One
    technique commonly used in determining the production of specific
    antibody-forming cells is to measure the number of plaque-forming
    cells (PFCs) in the blood or tissues of immunized animals.  In this
    study, the authors assessed immunological response by monitoring the
    daily output of PFCs in the efferent lymph of caudal mediastinal lymph
    nodes of sheep immunized with horse erythrocytes (a T-cell dependent
    antigen).  Although the number of animals used was small and the data
    were not analysed statistically, it would appear that, in the animals
    that were immunized 2 days (but not 4 days) after NO2 exposure
    started, the output of PFC was below control values.  Blastogenic
    responses of T cells from the efferent pulmonary lymph and venous
    blood also appeared to be decreased.

         Hillam et al. (1983) examined the effects of a 24-h exposure to
    9400, 18 800 and 48 900 g/m3 (5.0, 10.0 and 26.0 ppm) NO2 on
    cellular immunity in rats after intratracheal immunization with sheep
    erythrocytes (SRBCs).  Cellular immunity was evaluated by antigen-
    specific lymphocyte stimulation assays of pooled lymphoid cell
    suspensions from either the thoracic lymph nodes or the spleen. 
    Concentration-related elevation of cellular immunity in thoracic lymph
    nodes and spleen were reported after immunizing the lung with SRBCs.


        Table 29.  Effects of nitrogen dioxide (NO2) on the immune systema
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    188 base;    0.1 base;    Continuous base       Mouse       Suppression of splenic T and B cell responsiveness to       Maigetter et al.
    470, 940,    0.25, 0.5,   with 3-h/day peak                 mitogens variable and not related to concentration or       (1978)
    or 1880      or 1.0 peak  (5 days/week), 1, 3,              duration, except for the 940 g/m3 continuous group,
    peak                      6, 9, 12 months                   which had a linear decrease in PHA-induced mitogenesis
                                                                with NO2 duration.
    940          0.5          Continuous

    470          0.25         7 h/day,              Mouse       Reduced percentage of total T-cell population and trend     Richters & Damji
                              5 days/week,          (AKR/cum)   towards reduced percentage of certain T-cell                (1988)
                              7 weeks                           subpopulations; no reduction of mature T cells or natural
                                                                killer cells.

    470          0.25         7 h/day,              Mouse       Reduced percentage of total T-cell population and           Richters & Damji
                              5 days/week,          (AKR/cum)   percentages of T helper/inducer cells on days 37 and 181.   (1990)
                              36 weeks

    658          0.35         7 h/day,              Mouse       Trend towards suppression in total percentage of T-cells.   Richters & Damji
                              5 days/week,          (C57BL/6J)  No effects on percentages of other T-cell subpopulations.   (1988)
                              12 weeks

    752          0.4          24 h/day              Mouse       Decrease in primary PFC response at >752 g/m3.             Fujimaki et al.
    3010         1.6          4 weeks                           Increase in secondary PFC response at 3010 g/m3.           (1982)
                                                                                                                                              

    Table 29.  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    940 base;    0.5 base;    22-h/day base         Rat         No effect on splenic or circulatory B or T cell response    Selgrade et al.
    2820 peak    1.5 peak     (7 days/week);                    to mitogens. After 3 weeks of exposure only, decrease in    (1991)
                              6-h ramped peak                   splenic natural killer cell activity. No histological
                              (5 days/week)                     changes in lymphoid tissues
                              1, 3, 13, 52,
                              78 weeks

    940 base,    0.5 base,    Continuous base       Mouse       Vaccination with influenza A2/Taiwan virus after exposure.  Ehrlich et al.
    3760 peak    2.0 peak     with 1 h/day                      Decrease in serum neutralizing antibody; haemagglutination  (1975)
                              (5 days/week)                     inhibition titres unchanged. Before virus challenge, NO2
    3760         2.0          peak, 3 months                    exposure decreased serum IgA and increased IgG1, IgM, and
                                                                IgG2; after virus, serum IgA unchanged and IgM increased.

    1880         1.0          493 days              Monkey      Monkeys challenged five times with monkey-adapted           Fenters et al.
                                                                influenza virus during NO2 exposure. Haemagglutination      (1973)
                                                                inhibition antibody titres not altered. Compared to
                                                                controls, NO2 caused an earlier and greater increase in
                                                                serum neutralization antibody titres to the virus.

    1880         1.0          6 months              Guinea-pig  Intranasal challenge with K. pneumoniae after exposure.     Kosmider et al.
                                                                Decreased haemolytic activity of complement; decrease in    (1973)
                                                                all immunoelectrophoretic fractions.

    2820         1.5          24 h/day, 6,          Mouse       Reduction in number of splenic PFCs; lowering               Lefkowitz et al.
    9400         5.0          14, or 21 days                    concentration to 2820 g/m3 and extending the length to     (1986)
                                                                14 or 21 days decreased PFCs by 33 and 50%, respectively;
                                                                no effect on cell-mediated immune system or
                                                                haemagglutination titres.
                                                                                                                                              

    Table 29.  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    9400         5.0          1.5 h/day,            Sheep       Reduction in PFCs from pulmonary lymph and in mitogenesis   Joel et al.
                              10-11 days                        of T cells from pulmonary lymph and blood.                  (1982)

    9400         5.0          4 h/day,              Guinea-pig  Serum antibodies against lung tissue increased with         Balchum et al.
    28 200       15.0         5 days/week,                      concentration and duration of exposure.                     (1965)
                              5.52 months

    9400         5.0          Continuous, 169       Monkey      Initial depression in serum neutralization titres with      Fenters et al.
                              days, challenged                  return to normal by day 133; no effect on secondary         (1971)
                              4 x with mouse-                   response on haemmagglutin inhibition titre.
                              adapted influenza
                              virus

    9400         5.0          3-7 days              Mouse       No effect on serum interferon levels.                       Lefkowitz et al.
    47 000       25.0                                                                                                       (1983, 1984)

    9400         5.0          24 h                  Rat         Concentration-related elevation of cellular immunity in     Hillam et al.
    18 800       10.0                                           thoracic lymph nodes and spleen after immunizing the lung   (1983)
    48 900       26.0                                           with sheep RBCs.

                                                                                                                                              

    Table 29.  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    9400         5.0          Continuous,           Monkey      Depressed postvaccination serum neutralizing antibody       Ehrlich &
                              6 months                          formation.                                                  Fenters (1973)

    9400         5.0          12 h                  Mouse       No effect on primary and secondary splenic PFC response.    Fujimaki &
                                                                                                                            Shimizu (1981);
                                                                                                                            Fujimaki et al.
                                                                                                                            (1981)
                                                                                                                                              

    a  Source: Modified from US EPA (1993)
    b  PFC = plaque-forming cell; PHA = phytohaemagglutinin; Ig = immunoglobulin; RBCs = red blood cells
             Fujimaki et al. (1982) investigated the effect of a 4-week
    exposure to 752 and 3000 g/m3 (0.4 and 1.6 ppm) NO2 in mice (i.e.,
    primary and secondary antibody response to SRBCs, using the splenic
    PFC response as the end-point).  The primary PFC response was
    decreased by both NO2 concentrations.  Secondary antibody response
    was not affected at 752 g/m3 (0.4 ppm), but was slightly enhanced at
    3000 g/m3 NO2.  Acute exposure (12 h) of mice to 9400 g/m3
    (5.0 ppm) NO2 caused no such effects (Fujimaki & Shimizu, 1981;
    Fujimaki et al., 1981).

         The effect of exposure pattern was examined by Maigetter et al.
    (1978) by exposing mice for up to 1 year to 940 g NO2/m3 (0.5 ppm)
    continuously or to three regimens having a continuous baseline of
    188 g/m3 (0.1 ppm) with 3-h peaks (5 days/week) of either 470, 940
    or 1880 g/m3 (0.25, 0.5 or 1.0 ppm).  General mitogenic responses of
    splenic lymphocytes to phytohaemagglutinin (PHA) (a T cell dependent
    mitogen) and lipopolysaccharide (a B-cell dependent mitogen)
    decreased, but this was not related to the concentration or duration
    of exposure, with a single exception. The decrease in PHA-induced
    mitogenesis was linearly related to the increased duration of NO2
    exposure to 940 g/m3 (0.5 ppm).

         Shorter exposure (6 days) to 9400 g/m3 (5.0 ppm) NO2 did not
    affect mitogenesis of T cells (Lefkowitz et al., 1986).  Although NO2
    did not affect haemagglutination antibody titres, it did reduce the
    number of splenic PFCs to SRBCs.  The authors stated (data were not
    shown) that mice exposed to 2820 g/m3 (1.5 ppm) NO2 for 14 or
    21 days showed a 33 and 50% decrease, respectively, in the number of
    PFCs.

         Kosmider et al. (1973) exposed guinea-pigs to 1880 g/m3
    (1.0 ppm) NO2 for 6 months and reported a significant reduction in
    all serum immunoglobulin (Ig) fractions and complement.  Decreased
    levels of these substances may lead to an increase in the frequency,
    duration and severity of an infectious disease.  Mice exposed to NO2
    had decreased serum levels of IgA and exhibited nonspecific increases
    in serum IgM, IgG and IgG2 (Ehrlich et al., 1975).

         These effects on lymphocyte function may reflect changes in
    lymphocyte populations.  Richters & Damji (1988) found that the
    percentage of the total T lymphocyte population was reduced in the
    spleens of AKR/cum mice exposed for 7 weeks (7 h/day, 5 days/week) to
    470 g/m3 (0.25 ppm) NO2.  The percentages of mature helper/inducer
    T and T cytotoxic/suppressor lymphocytes were also lower in the
    spleens of exposed animals.  There were no changes in the percentages
    of natural killer cells or mature T cells.  Upon a longer (36-week)
    exposure, Richters & Damji (1990) found that the numbers of T-helper/
    inducer (CD4+) lymphocytes (spleen) were reduced, but no effects
    were observed on T cytotoxic/suppressor cells.  Spontaneously
    developing lymphomas in NO2-exposed animals progressed more

    slowly than those in control animals.  This was attributed to
    the NO2-induced reduction in the T-helper/inducer lymphocytes. 
    C57BL/6J mice exposed to 658 g/m3 (0.35 ppm) for 7 h/day,
    5 days/week for 12 weeks, also showed a suppression in the percentage
    of total matured T lymphocytes, but no effect on any specific
    subpopulation upon longer exposure (36 weeks) to 470 g/m3 (0.25 ppm)
    (Richters & Damji, 1988).  Selgrade et al. (1991) found that chronic
    exposure (up to 78 weeks) to an urban pattern of NO2 (baseline of
    940 g/m3 (0.5 ppm) with a ramped 6-h peak to 2820 g/m3 (1.5 ppm))
    had no effect on splenic or circulating B or T cell mitogenic
    response.  However, there was a transient decrease in splenic natural
    killer cell activity (at 3 weeks only).

         Few studies have been undertaken to assess the effects of NO2 on
    interferon production.  Mice exposed to either 9400 or 47 000 g/m3
    (5.0 or 25.0 ppm) NO2 for 3 to 7 days had serum levels of interferon
    similar to those of controls (Lefkowitz et al., 1983, 1984).

         Induction of autoimmunity was suggested by the work of Balchum
    et al. (1965). Guinea-pigs exposed to 9400 g/m3 (5.0 ppm) and
    28 200 g/m3 (15.0 ppm) NO2 had an increase in the titre of serum
    antibodies against lung tissue, starting after 160 h of NO2 exposure. 
    These antibody titres continued to increase with NO2 concentration
    and duration of exposure.

         The impact of NO2 on the humoral immune response of squirrel
    monkeys to intratracheally delivered influenza vaccine was studied by
    Fenters et al. (1971, 1973) and Ehrlich & Fenters (1973).  In monkeys
    exposed for 493 days to 1880 g/m3 (1.0 ppm) NO2 and immunized with
    monkey-adapted virus (A/PR/8/34), the serum neutralizing antibody
    titres were significantly increased earlier and to a greater degree
    than those of controls (Fenters et al., 1973; Ehrlich & Fenters,
    1973).  In monkeys exposed to 9400 g/m3 (5.0 ppm) NO2 for a total
    of 169 days and immunized with mouse-adapted influenza virus (A/PR/8),
    serum neutralization titres were lower than controls initially; no
    significant difference was observed by 133 days of exposure (Fenters
    et al., 1971; Ehrlich & Fenters, 1973).  In all of these studies, the
    haemagglutination inhibition antibody titres were not affected. 
    Differences between studies might be due to the difference in the
    virus used for immunization, along with exposure differences. Also,
    exposure to 1880 g/m3 (1.0 ppm) NO2 may have increased the
    establishment of infection and the survival of the monkey-adapted
    virus within the respiratory tract, resulting in an increase in
    antibody production.

         Mice that were vaccinated with influenza virus (A-2/Taiwan/ 1/64)
    after 3 months of continuous exposure to 3760 g/m3 (2.0 ppm) or to
    940 g/m3 (0.5 ppm) NO2 with a 1-h daily (5 days/week) spike
    exposure to 3760 g/m3 (2.0 ppm) had mean serum neutralizing antibody
    titres that were four-fold lower than those of clean air controls
    (Ehrlich et al., 1975).  The haemagglutination inhibition antibody
    titres in these animals were unchanged.  This agrees with the Fenters
    et al. (1973) findings in monkeys exposed to 1880 g/m3 (1.0 ppm) for
    over 1 year.

    d)  Interaction with infectious agents

         Various experimental approaches have been employed using animals
    in an effort to determine the overall functional efficiency of the
    host's pulmonary defences following NO2 exposure.  In the most
    commonly used infectivity model, animals are exposed to either NO2
    or filtered air.  After NO2 exposure, the treatment groups are
    combined and exposed briefly to an aerosol of a viable agent, such as
     Streptococcus sp.,  Klebsiella pneumoniae, Diplococcus pneumoniae
    or influenza virus.  The animals are then returned to clean air
    for a holding period (usually 15 days), and the mortality in the
    NO2-exposed and the control groups are compared. If host defences are
    compromised by the NO2 exposure, mortality rates will be higher
    (Ehrlich, 1966; Henry et al., 1970; Coffin & Gardner, 1972; Ehrlich et
    al., 1979; Gardner, 1982).  Although the end-point is mortality, it is
    a sensitive indicator of the depression of the defence mechanisms used
    to control infection.  Because these specific defence mechanisms are
    common to laboratory animals and humans, the increased susceptibility
    to infection can be qualitatively extrapolated to humans, even though
    mortality would not be an expected outcome in humans receiving
    appropriate medical treatment.  However, different exposure levels of
    NO2 and infectious agents may be required to produce changes in human
    host defences.  Effects of NO2 on pulmonary infectious disease in
    humans are discussed in chapters 6 and 7.  Table 30 summarizes effects
    of exposure to NO2 and infectious agents observed in animals.

         An enhancement in mortality following exposure to NO2 in
    combination with a pathogenic microorganism could be due to several
    factors. Goldstein et al. (1973) showed decreases in pulmonary
    bactericidal activity following NO2 exposure.  In their first
    experiments, mice breathed aerosols of  Staphylococcus aureus
     (S. aureus) labelled with radioactive phosphorus and were then
    exposed to NO2 for 4 h. Physical removal of the bacteria was not
    affected by any of the NO2 concentrations used up to 27 800 g/m3
    (14.8 ppm).  Concentrations > 13 200 g/m3 (7.0 ppm) NO2 lowered
    the bactericidal activity by > 7%.  Lower concentrations (3570 and
    7140 g/m3 (1.9 and 3.8 ppm)) had no significant effect.  In another
    experiment (Goldstein et al., 1974), mice breathed 1800, 4320 and
    12 400 (1.0, 2.3 and 6.6 ppm) NO2 for 17 h and then were exposed
    to an aerosol of  S. aureus.  Four hours later, the animals were
    examined for the number of organisms present in the lungs.  No
    difference in the number of bacteria inhaled was found in the
    NO2-exposed animals. Concentrations of 4320 and 12 400 g/m3
    (2.3 and 6.6 ppm) NO2 decreased pulmonary bactericidal activity by
    6 and 35%, respectively, compared to controls. Exposure to 1880 g/m3
    (1.0 ppm) NO2 had no significant effect. Goldstein et al. (1974)
    hypothesized that the decreased bactericidal activity was due to
    defects in AM function.  Jakab (1987) confirmed these findings and
    found that the concentration of NO2 required to suppress pulmonary
    bactericidal activity in mice depended on the specific organism.  For
    example, exposure to > 7520 g/m3 (> 4.0 ppm) NO2 for 4 h
    after bacterial challenge depressed  bactericidal activity
    against  S. aureus, but it required a concentration of 18 800 to
    37 600 g/m3 (10.0 to 20.0 ppm) before the lung's ability to kill
    deposited  Pasteurella and  Proteus was impaired. Parker et al.
    (1989) made similar observations in mice exposed for 4 h to 9400 or
    18 800 g/m3 (5.0 or 10.0 ppm) NO2 and infected with  Mycoplasma
     pulmonis.  The higher concentration of NO2 increased mortality.
    Both concentrations: (1) reduced lung bactericidal activity and
    increased bacterial growth, without affecting deposition or physical
    clearance; and (2) increased the incidence of lung lesions as well as
    their severity. Davis et al. (1991) found no effects of lower NO2
    concentrations on bactericidal activity using the same model system.

        Table 30.  Interaction of nitrogen dioxide (NO2) with infectious agentsa
                                                                                                                                              

    NO2 concentration
                            

    g/m3          ppm            Exposure                  Species    Infective agent   Effects                              Reference
                                                                                                                                              

    100 base,      0.05 base,     Continuous, with          Mouse      Streptococcus     No effect                            Gardner (1980);
    188 peak       0.1 peak       1 h peak, twice/day                  sp.                                                    Gardner et al.
                                  (5 days/week),                                                                              (1982); Graham
                                  15 days                                                                                     et al. (1987)

    940 +          0.5 +                                                                 Increased mortality
     1880 peak     1.0 peak

    2260 +         1.2 +                                                                 Increased mortality
     4700 peak     2.5 peak

    376 base,      0.2 base,      Continuous base           Mouse      Streptococcus     Spike plus baseline caused           Miller et al.
    1500 peak      0.8 peak       with 1-h peak                        sp.               significantly greater mortality      (1987)
                                  twice/day                                              than baseline.
                                  (5 days/week),
                                  1 year

    564-940        0.3-0.5        Continuous,               Mouse      A/PR/8            High incidence of adenomatous        Motomiya et al.
                                  3 months                             virus             proliferation of peripheral and      (1973)
                                                                                         bronchial epithelial cells; NO2
                                                                                         alone and virus alone caused less
                                                                                         severe alterations.
                                                                                                                                              

    Table 30  (Con't)
                                                                                                                                              

    NO2 concentration
                            

    g/m3          ppm            Exposure                  Species    Infective agent   Effects                              Reference
                                                                                                                                              

                                  Continuous,                                            No enhancement of effect of NO2
                                  6 months                                               and virus.

    940            0.5            3 h/day,                  Mouse      Streptococcus     Increase in mortality with           Ehrlich et al.
                                  3 months                             sp.               reduction in mean survival time.     (1979)

    940            0.5            Intermittent,             Mouse      Klebsiella        Increased mortality after 6 months   Ehrlich &
                                  6 or 18 h/day,                       pneumoniae        intermittent exposure or after       Henry (1968)
                                  up to 12 months                                        3, 6, 9 or 12 months continuous
                                                                                         exposure; following 12 months
                                                                                         exposure, increased mortality was
                                  Continuous,                                            significant only in continuously
                                  24 h/day up to                                         exposed mice.
                                  12 months

    940-1880       0.5-1.0        Continuous,               Mouse      A/PR/8            Increased susceptibility to          Ito (1971)
                                  39 days                   (female)   virus             infection
    18 800         10.0           2 h/day, 1, 3,
                                  and 5 days

    940-52 600     0.5-28.0       Varied                    Mouse      Streptococcus     Increased mortality with increased   Gardner et al.
                                                                       sp.               time and concentration;              (1977a,b); Coffin
                                                                                         concentration is more important      et al. (1977)
                                                                                         than time.

    940            0.5            24 h/day,                 Mouse      K. pneumoniae     Significant increase in mortality    McGrath &
    1880           1.0            7 days/week,                                           after 3-day exposure to 9400 g/m3;  Oyervides (1985)
    2820           1.5            3 months                                               no effect at other concentrations,
                                                                                                                                              

    Table 30  (Con't)
                                                                                                                                              

    NO2 concentration
                            

    g/m3          ppm            Exposure                  Species    Infective agent   Effects                              Reference
                                                                                                                                               

    9400           5.0            3 days                                                 but control mortality was very
                                                                                         high.

    1880           1.0            17 h                      Mouse      Staphylococcus    No difference in number of bacteria  Goldstein et al.
    4320           2.3                                                 aureus after      deposited, but at 4320 and           (1974)
    12 400         6.6                                                 NO2 exposure      12 400 g/m3, there was a decrease
                                                                                         in pulmonary bactericidal activity
                                                                                         of 6 and 35%, respectively;
                                                                                         no effect at 1880 g/m3.

    1880-4700      1.0-2.5        4 h                       Mouse      S. aureus         Impaired bactericidal activity       Jakab (1988)
                                                                                         between 1800 and 4700 g/m3 in
                                                                                         animals injected with
                                                                                         corticosteroids

    4320           2.3                                                                   6% decrease in bactericidal
                                                                                         activity

    12 400         6.6                                                                   35% decrease in bactericidal
                                                                                         activity

    1880           1.0            48 h                      Mouse      Streptococcus     Increase proliferation of            Sherwood et al.
                                                                       sp.; S. aureus    Streptococcus sp., but not           (1981)
                                                                                         S. aureus, in lung

    1880           1.0            3 h                       Mouse      Streptococcus     Exercise on continuously moving      Illing et al.
    5640           3.0                                                 sp.               wheels during exposure; increased    (1980)
                                                                                         mortality at 5640 g/m3
                                                                                                                                              

    Table 30  (Con't)
                                                                                                                                              

    NO2 concentration
                            

    g/m3          ppm            Exposure                  Species    Infective agent   Effects                              Reference
                                                                                                                                              

    2820           1.5            Continuous or             Mouse      Streptococcus     After 1 week, mortality with         Gardner et al.
                                  intermittent                         sp.               continuous exposure was greater      (1979)
                                  (7 h/day), 7 days                                      than that for intermittent; after
                                  per week, 2 weeks                                      2 weeks, no significant difference
                                                                                         between continuous and intermittent
                                                                                         exposure.

    6580           3.5                                                                   Increased mortality with increased
                                                                                         duration of exposure; no
                                                                                         significant difference between
                                                                                         continuous and intermittent
                                                                                         exposure; with data adjusted for
                                                                                         total difference in the production
                                                                                         of concentration and time,
                                                                                         mortality essentially the same.

    2820 base,     1.5 base,      Continuous 60 h           Mouse      Streptococcus     Mortality increased with 3.5- and    Gardner (1980);
    8460 peak      4.5 peak       then peak for 1,                     sp.               7-h single spike when bacterial      Garnder et al.
                                  3.5 or 7 h, then                                       challenge was immediate, and         (1982); Graham
                                  continuous 18 h                                        18 h after the spike                 et al. (1987)

    8460           4.5            1, 3.5, or 7 h                                         Mortality proportional to duration
                                                                                         when bacterial challenge was
                                                                                         immediate, but not 18 h
                                                                                         post-exposure.

    2820           1.5            7 h/day, 4, 5,            Mouse      Streptococcus     Elevated temperature (32C)          Gardner et al.
                                  and 7 days                           sp.               increased mortality after 7 days.    (1982)
                                                                                                                                              

    Table 30  (Con't)
                                                                                                                                              

    NO2 concentration
                            

    g/m3          ppm            Exposure                  Species    Infective agent   Effects                              Reference
                                                                                                                                              

    2820           1.5            2 h                       Mouse      K. pneumoniae     Increased mortality only at          Purvis &
    4700           2.5                                                                   > 6580 g/m3.  Increase in           Ehrlich (1966);
                                                                                         mortality                  
    
    6580           3.5                                                                   K. pneumoniae challenge 1 and 6 h    Ehrlich (1979)
    9400           5.0                                                                   after 9400 or 18 800 g/m3;
    18 800         10.0                                                                  when K. pneumoniae challenge 27 h
    28 200         15.0                                                                  following NO2 exposure, effect
                                                                                         only at 28 200 g/m3.

    3570           1.9            4 h                       Mouse      S. aureus         Physical removal of bacteria         Goldstein et al.
    7140           3.8                                                 prior to NO2      unchanged at 3570 to 27 800 g/m3.   (1973)
                                                                       exposure

    13 160         7.0                                                                   7% lower bactericidal activity
    17 300         9.2                                                                   14% lower bactericidal activity
    27 800         14.8                                                                  50% lower bactericidal activity

    3760           2.0            3 h                       Mouse      Streptococcus     Increased mortality                  Ehrlich et al.
                                                                       sp.                                                    (1977);
                                                                                                                              Ehrlich (1980)

    4700           2.5-30.0       4 h                       Mouse      S. aureus,        Concentration-related decrease       Jakab (1987)
    56 400                                                             Pasteurella and   in bactericidal activity, starting
                                                                       Proteus           at > 7500 g/m3 with S. aureus when
                                                                                         NO2 exposure was after bacterial
                                                                                         challenge; when NO2 was before
                                                                                         bacterial challenge, effect at
                                                                                         18 800 g/m3.  Higher concentration
                                                                                         required to affect other organisms.
                                                                                                                                              

    Table 30  (Con't)
                                                                                                                                              

    NO2 concentration
                            

    g/m3          ppm            Exposure                  Species    Infective agent   Effects                              Reference
                                                                                                                                              

    6580           3.5            2 h                       Mouse      K. pneumoniae     Increased mortality of all species   Ehrlich (1975)

    65 830         35.0           2 h                       Hamster
    94 050         50.0           2 h                       Squirrel
                                                             monkey

    9400           5.0            6 h/day,                  Mouse      Cytomegalovirus   Increase in virus susceptibility     Rose et al.
                                  6 days                                                                                      (1988)

    9400           5.0            Continuous,               Squirrel   K. pneumoniae     Increased viral-induced mortality    Henry et al.
                                  2 months                   monkey    or A/PR/8         (1/3).  Increase in Klebsiella-      (1970)
                                                                       influenza virus   induced mortality (2/7); no
                                                                       deaths.           control

    19 000         10.0           Continuous,                                            Increased virus-induced mortality
                                  1 month                                                (6/6) within 2-3 days after
                                                                                         infection; no control deaths.
                                                                                         Increase in Klebsiella-induced
                                                                                         mortality (1/4); no control deaths.

    9400           5.0            4 h                       Mouse      Mycoplasma        NO2 increased incidence and          Parker et al.
    19 000         10.0                                                pulmonis          severity of pneumonia lesions and    (1989)
                                                                                         decreased the number of organisms
                                                                                         needed to induce pneumonia; no
                                                                                         effect on physical clearance,
                                                                                         decreased mycoplasmal killing
                                                                                         and increased growth; no effect on
                                                                                         specific IgM in serum;
                                                                                                                                              

    Table 30  (Con't)
                                                                                                                                              

    NO2 concentration
                            

    g/m3          ppm            Exposure                  Species    Infective agent   Effects                              Reference
                                                                                                                                              

                                                                                         C57Bl/6N mice generally more
                                                                                         sensitive than C3H/HeN mice.
                                                                                         At 19 000 g/m3, one strain
                                                                                         (C57BL/6N) of mice had increased
                                                                                         mortality.

    9400           5.0            2 months                  Squirrel   K. pneumoniae     Mortality 2/7; bacteria present      Henry et al.
                                                             monkey                      in lung of survivors at autopsy.     (1969)

    65 800         35.0           1 month                                                Mortality 1/4; bacteria present
                                                                                         in lungs of survivors at autopsy.

    94 000         50.0           2 h                                                    Mortality 3/3
                                                                                                                                              

    a  Modified from US EPA (1993)
             Differences in species susceptibility to NO2 or to a pathogen
    may play a role in the enhancement of mortality seen in experimental
    animals.  An enhancement in mortality was noted in mice, hamsters and
    monkeys acutely exposed to NO2 for 2 h followed by a challenge
    of  K. pneumonia (Ehrlich, 1975).  However, differences in
    susceptibility were noted between the species.  Ehrlich found
    increased mortality occurred in monkeys only at 94 000 g/m3
    (50.0 ppm), whereas, lower NO2 levels increased mortality in mice
    (6580 g/m3, 3.5 ppm) and hamsters (65 800 g/m3, 35.0 ppm). The
    mouse model was the most sensitive to NO2 exposure, as shown by
    enhanced mortality from  K. pneumoniae following exposure to
    6580 g/m3 (3.5 ppm) but not to 2820-4700 g/m3 (1.5-2.5 ppm) NO2
    for 2 h (Purvis & Ehrlich, 1963; Ehrlich, 1975).  With prolonged
    (2 month) exposure, Henry et al. (1969) found that lower levels of
    NO2 (9400 g/m3, 5.0 ppm) increased  susceptibility to bacterial
    infections in monkeys than the 50.0 ppm concentration found to be
    effective by Ehrlich (1975) with acute (2 h) exposure.  The
    sensitivity is also affected by the test organism.  For example, when
     Streptococcus sp. was the infectious agent, a 3-h exposure to
    3760 g/m3 (2.0 ppm) NO2 caused an increased in mortality in mice
    (Ehrlich et al., 1977).  Sherwood et al. (1981) illustrated that
    exposure to 1880 g/m3 (1.0 ppm) NO2 for 48 h increased the
    propensity of virulent group-C streptococci, but not  S. aureus, to
    proliferate within mouse lungs and cause earlier mortality.

         Additional factors can influence the interaction of NO2 and
    infectious agents.  Mice placed on continuously moving exercise wheels
    during exposure to 5640 g/m3 (3.0 ppm) NO2, but not 1880 g/m3
    (1.0 ppm), for 3 h showed enhanced mortality over non-exercised
    NO2-exposed mice using the streptococcal infectivity model (Illing et
    al., 1980).  The presence of other environmental factors, such as O3
    (Ehrlich et al., 1977; Gardner, 1980; Gardner et al., 1982; Graham et
    al., 1987) or elevated temperatures (Gardner et al., 1982), also
    exacerbated the effects of NO2.

         The influence of a wide variety of exposure regimens has been
    evaluated using the infectivity model.  For example, Gardner et al.
    (1977b) examined the effect of varying durations of continuous
    exposure on the mortality of mice exposed to six concentrations of
    NO2 (940 to 52 600 g/m3 (0.5 to 28.0 ppm)) for durations ranging
    from 15 min to 1 year.   Streptococcus sp. was used for all
    concentrations, except 940 g/m3, in which case  K. pneumoniae was
    used.  Mortality increased linearly with increasing duration of
    exposure to a given concentration of NO2.  Mortality also increased
    with increasing concentration of NO2 as indicated by the steeper
    slopes with higher concentrations.  When the product of concentration
    and time (C  T) was held constant, the relationship between
    concentration and time produced significantly different mortality
    responses.  At a constant C  T of approximately 21 ppm-h, a 14-h
    exposure to 2820 g/m3 (1.5 ppm) NO2 increased mortality by 12.5%,
    whereas a 1.5-h exposure to 27 300 g/m3 (14.0 ppm) NO2 enhanced
    mortality by 58.5%.  These findings demonstrate that concentration is
    more important than time in determining the degree of injury induced
    by NO2 in this model, and they were confirmed at additional C  T
    values (Gardner et al., 1977a,b, 1982; Coffin et al., 1977).

         Gardner et al. (1979) also compared the effect of continuous
    versus intermittent exposure to NO2 followed by bacterial challenge
    with  Streptococcus sp.  Mice were exposed either continuously or
    intermittently (7 h/day, 7 days/week) to 2820 or 6580 g/m3 (1.5 or
    3.5 ppm) NO2.  The continuous exposure of mice to 2820 g/m3 NO2
    increased mortality after 24 h of exposure.  During the first week of
    exposure, the mortality was significantly higher in mice exposed
    continuously to NO2 than in those exposed intermittently.  By the
    14th day of exposure, the difference between intermittent and
    continuous exposure became indistinguishable.  At the higher
    concentration, there was essentially no difference between continuous
    and intermittent regimens.  This suggests that fluctuating levels of
    NO2 may ultimately be as toxic as sustained high levels (Gardner et
    al., 1979).

         Mice were exposed continuously or intermittently (6 or 18 h/day)
    to 940 g/m3 (0.5 ppm) NO2 for up to 12 months (Ehrlich & Henry,
    1968).  None of the exposure regimens affected resistance to
     K. pneumoniae infection during the first month.  Those exposed
    continuously exhibited decreased resistance to the infectious agent,
    as demonstrated by a significant enhancement in mortality at 3, 6, 9
    and 12 months.  In another experiment, a significant enhancement did

    not occur at 3 months, but was observed after 6 months of exposure. 
    After 6 months, mice exposed intermittently (6 or 18 h/day) to NO2
    showed significant increases in mortality over controls (18%).  Only
    the continuously exposed animals showed increased mortality (23%) over
    controls following 12 months of exposure.  After 12 months of
    exposure, mice in the three experimental groups showed a reduced
    capacity to clear viable bacteria from their lungs.  This was first
    observed after 6 months in the continuously exposed group and after
    9 months in the intermittently exposed groups.  These changes,
    however, were not statistically tested for significance.  Although it
    is not possible to compare directly the results of the studies using
     Streptococcus sp. to those using  K. pneumoniae, the data suggest
    that, as the concentration of NO2 is decreased, a longer exposure
    time is necessary for the intermittent exposure regimen to produce a
    level of effect equivalent to that of a continuous exposure.  McGrath
    & Oyervides (1985) did not confirm these findings in mice exposed to
    940, 1880 and 2820 g/m3 (0.5, 1.0 and 1.5 ppm) NO2 for 3 months. 
    The inconsistency may be attributed to the fact that the McGrath &
    Oyervides (1985) study had 95% mortality in the control groups, making
    it virtually impossible to detect a further enhancement in mortality
    due to NO2.

         Gardner (1980), Gardner et al. (1982) and Graham et al. (1987)
    reported extensive investigations on the response to airborne
    infections in mice breathing NO2 spike exposures superimposed on a
    lower continuous background level of NO2, which simulated the pattern
    (although not the NO2 concentrations) of exposure in the urban
    environment in the USA.  Mice were exposed to spikes of 8460 g/m3
    (4.5 ppm) for 1, 3.5 or 7 h and then were challenged with
     Streptococcus sp. either immediately or 18 h after exposure. 
    Mortality was proportional to the duration of the spike when the mice
    were challenged with bacteria immediately after exposure, but mice had
    recovered from the exposure by 18 h.  Similar findings were reported
    by Purvis & Ehrlich (1963) using  K. pneumoniae.  When a spike of
    8460 g/m3 (4.5 ppm) was superimposed on a continuous background of
    2820 g/m3 (1.5 ppm) for 62 h preceding and 18 h following the spike,
    mortality was significantly enhanced by a spike lasting 3.5 or 7 h
    when the infectious agent was administered 18 h after the spike
    (Gardner, 1980; Gardner et al., 1982; Graham et al., 1987).  Possible
    explanations for these differences due to the presence or absence of a
    background exposure are that mice continuously exposed are not capable
    of recovery or that new AMs or PMNs recruited to the site of infection
    are impaired by the continuous exposure to NO2.  The effect of
    multiple spikes was examined by exposing mice for 2 weeks to two
    daily 1-h spikes (morning and afternoon, 5 days/week) of 8460 g/m3
    (4.5 ppm) superimposed on a continuous background of 2820 g/m3
    (1.5 ppm) NO2.  Mice were challenged with the infectious agent either
    immediately before or after the morning spike.  When the infectious
    agent was given before the morning spike, the increase in mortality

    did not closely approach that of a continuous exposure to 2820 g/m3
    (1.5 ppm) NO2.  However, in mice challenged after the morning spike,
    by 2 weeks of exposure, the increased mortality over controls
    approached that equivalent to continuous exposure to 2820 g/m3
    (1.5 ppm) NO2.  Thus, the magnitude of the effect of the base-plus-
    spike group, which had a higher C  T than the continuous groups, did
    not exceed the effect of the continuous group.  These findings
    demonstrate that the pattern of exposure determines the response and
    that the response is not predictable based on a simple C  T
    relationship.

         Further investigations into the effects of chronic exposure to
    NO2 spikes on murine antibacterial lung defences have been conducted
    using a spike-to-baseline ratio of 4:1, which is not uncommon in the
    urban environment in the USA (Miller et al., 1987).  For 1 year, mice
    were exposed 23 h/day, 7 days/week, to a baseline of 376 g/m3
    (0.2 ppm) or to this baseline level on which was superimposed a 1-h
    spike of 1500 g/m3 (0.8 ppm) NO2, twice a day, 5 days/week.  The
    animals exposed to the baseline level did not exhibit any significant
    effects; however, the streptococcal-induced mortality of the mice
    exposed to the baseline plus spike regimen was significantly greater
    than that of either the NO2-background-exposed mice or the control
    mice.  Human epidemiological studies in chapter 7 indicate increased
    risk of respiratory infection.  Data from experimental animals support
    the epidemiological responses in humans.

         Antiviral defences are also compromised by NO2.  Squirrel
    monkeys exposed to 9400 or 18 800 g/m3 (5.0 or 10.0 ppm) NO2 for
    2 or 1 month, respectively, had an increased susceptibility to a
    laboratory-induced viral influenza infection (Henry et al., 1970). 
    All six animals exposed to the highest concentration died within 2
    to 3 days of infection with the influenza virus; at the lower
    concentration, one out of three monkeys died.

         Mice exposed continuously for 3 months to 564-940 g/m3
    (0.3-0.5 ppm) NO2 followed by a challenge with A/PR/8 influenza virus
    exhibited significant pulmonary pathological responses (Motomiya et
    al., 1973).  A greater incidence of adenomatous proliferation of
    bronchial epithelial cells resulted from the combined exposures of
    virus plus NO2 than with either the viral or NO2 exposures alone. 
    Continuous NO2 exposure for an additional 3 months did not enhance
    the effect of NO2 or the subsequent virus challenge.

         Ito (1971) challenged mice with influenza A/PR/8 virus after
    continuous exposure to 940 to 1880 g/m3 (0.5 to 1.0 ppm) NO2 for
    39 days and to 18 800 g/m3 (10.0 ppm) NO2, 2 h daily for 1, 3 and
    5 days.  Acute and intermittent exposure to 18 800 g/m3 (10.0 ppm)
    NO2 as well as continuous exposure to 940 to 1880 g/m3 (0.5 to
    1.0 ppm) NO2 increased the susceptibility of mice to influenza virus
    as demonstrated by increased mortality.

         The lower respiratory tract of mice became significantly more
    susceptible to murine cytomegalovirus infection after 6-h exposures
    for 6 days to 9400 g/m3 (5.0 ppm) NO2 (Rose et al., 1988).  No
    effects occurred at levels < 4700 g/m3 (2.5 ppm).  Exposure to
    9400 g/m3 (5.0 ppm) NO2 did not significantly alter the course of
    a parainfluenza (murine sendai virus) infection in mice as measured by
    the infectious pulmonary virus titres in the lungs.  However, this
    concentration of NO2, when combined with the virus exposure, did
    increase the severity of the pulmonary disease process (viral
    pneumonitis) (Jakab, 1988).

    5.2.2.2  Lung biochemistry

         Studies of lung biochemistry in animals exposed to NO2 have
    focused on either the putative mechanisms of toxic action of NO2 or
    on detection of indicators of tissue and cell damage.  One theory of
    the mechanism underlying NO2 toxicity is that NO2 initiates lipid
    peroxidation in unsaturated fatty acids in membranes of target cells,
    thereby causing cell injury or death (Menzel, 1976).  Another theory
    is that NO2 oxidizes water-soluble, low molecular weight reducing
    substances and proteins, resulting in a metabolic dysfunction that
    manifests itself in toxicity (Freeman & Mudd, 1981).  It is likely
    that NO2 acts by both means. Several potential biochemical mechanisms
    related to detoxification of NO2 or to responses to NO2 intoxication
    have been proposed and summarized below according to impacts on
    lipids, proteins, and antioxidant metabolism and antioxidants.  The
    following discussion focuses on inhalation studies because they are
    more interpretable for risk assessment purposes;  in vitro exposure
    studies have been reviewed elsewhere (US EPA, 1993).

    a)  Lipid peroxidation

         Animal toxicology studies evaluating effects of NO2 on lipid
    peroxidation are summarized in Table 31.

         Lipid peroxidation induced by NO2 exposure has been detected at
    exposure levels as low as 75 g/m3 (0.04 ppm).  Lipid peroxidation,
    measured as ethane exhalation, was detected after 9 months of exposure
    of rats to 75-750 g/m3 (0.04-0.4 ppm) (Sagai et al., 1984).  Lipid
    peroxidation has also been evaluated by measuring the content of lipid
    peroxides or substances reactive to thiobarbituric acid in alveolar
    lavage fluid and lung tissue after exposure to similar NO2
    concentrations (Ichinose & Sagai, 1982; Ichinose et al., 1983).  Acute
    or subacute exposure to higher concentrations of NO2 has also been
    shown to cause a rapid increase in lung peroxide levels in several
    species.

        Table 31.  Effects of nitrogen dioxide (NO2) on lung lipid metabolisma
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    75           0.04         Continuous, 9,        Rat         Increased TBA products at 7520 g/m3 after 9 months and     Sagai et al.
    752          0.4          18 or 27 months                   at > 752 g/m3 after 18 months; increased ethane            (1984)
    7520         4.0                                            exhalation at all levels. No changes in total lipid,
                                                                phospholipid, total cholesterol or triglyceride contents.

    75           0.04         Continuous, 6,                    Increased ethane exhalation after 9 and 18 months.
    225          0.12         9 and 18 months
    752          0.4

    752          0.4          2 weeks               Rat         Changes in TBA-reactive substances, exhaled ethane and      Ichinose et
    2260         1.2          1-16 weeks                        enzyme activities in lung homogenates, dependent on         al. (1983)
    7520         4.0                                            concentration and duration of exposure.
    18 800       10.0

    75           0.04         9, 18,
    752          0.4          27 months
    7520         4.0

    752          0.4          4 months              Rat         Duration-dependent increase in ethane exhalation and        Ichinose &
    2260         1.2                                            TBA-reactive substances; peak increase in early weeks of    Sagai (l982)
    7520         4.0                                            exposure, return towards control in mid-exposure, and
                                                                increase late in exposure.

    752          0.4          72 h                  Guinea-pig  No effect at 752 g/m3; increase in lung lipid content in   Selgrade et
    1880         1.0                                            BAL of vitamin C-depleted, but not normal, animals at       al. (1981)
    5640         3.0                                            1880 g/m3 or more.
    9400         5.0
                                                                                                                                              

    Table 31  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    9400         5.0          3 h                               Increased lung lipid content in vitamin C-depleted
                                                                guinea-pigs 18-h after exposure.

    752          0.4          1 week                            No effects in normal or vitamin C-depleted animals.

    1880         1.0          Continuous,           Rabbit      Decrease in lecithin synthesis after 1 week; less marked    Seto et al.
                              2 weeks                           depression after 2 weeks.                                   (1975)

    1880         1.0          4 h/day,              Rat         Vitamin E supplement reduced the lipid peroxidation.        Thomas et al.
                              6 days                                                                                        (1967)

    5450         2.9          Continuous,           Rat         Increase in lung wet weight (l2.7%) and decrease in total   Arner &
                              5 days/week                       lipid (8.7%); decrease in saturated fatty acid content of   Rhoades (1973)
                              9 months                          lung lavage fluid and tissue; increase in surface tension
                                                                of lung lavage fluid; and decrease in lung compliance.

    1880         1.0          2 h                   Rabbit      1800 g/m3: elevated thromboxane B2. 5640 g/m3:            Schlesinger
    5640         3.0                                            depressed thromboxane B2. 18 800 g/m3: depressed           et al. (1990)
    18 800       10.0                                           6-keto-prostaglandin F1alpha and thromboxane B2.

    5640         3.0          Continuous,           Rat         Decrease in linoleic and linolenic acid content of BAL.     Menzel et al.
                              17 days                                                                                       (1972)

    5640         3.0          7 days                Rat         Increased TBA reactants with vitamin E deficiency.          Sevanian et al.
                                                                                                                            (1982)
                                                                                                                                              

    a  Modified from US EPA (1993)
    b  TBA = Thiobarbituric acid; BAL = Bronchoalveolar lavage
             Lipid peroxidation results in an alteration in phospholipid
    composition.  Exposure of either mice or guinea-pigs to an NO2
    level of 750 g/m3 (0.4 ppm) for a week resulted in a decreased
    concentration of phosphatidyl ethanolamine and a relative increase in
    the phosphatidyl choline concentration (Sagai et al., 1987).

         Several investigators have also demonstrated NO2-induced lipid
    peroxidation in  in vitro systems.  The cell type most commonly used
    is the endothelial cell from either pig arteries or aorta.  Studies
    using these cell types have recently attempted to relate the effect on
    lipid metabolism to functional parameters such as membrane fluidity
    and enzyme activation or inactivation.

         Membrane fluidity changes are related to lipid peroxidation. 
    NO2-induced changes in membrane fluidity have been demonstrated in
    alveolar macrophages and endothelial cells in culture.  Endothelial
    cells exposed to a NO2 level of 9400 g/m3 (5 ppm), for instance,
    exhibit decreased membrane fluidity after 3 h.  Thus, NO2 changes the
    physical state of the membrane lipids, perhaps through initiating
    lipid peroxidation, and hence impairs membrane functions (Patel et
    al., 1988).

         Lipid peroxidation can also activate phospholipase activities. 
    Activation of phospholipase A1 in cultured endothelial cells by NO2
    has been demonstrated.  This activation, which is specific for
    phospholipase A1 occurs at an NO2 concentration of 9400 g/m3
    (5 ppm) after 40 h of exposure and is speculated to depend on a
    specific NO2-induced increase in phosphatidyl serine in the plasma
    membranes (Sekharam et al., 1991).

         One function of phospholipases is the release of arachidonic
    acid.  The effect of NO2 on the release and metabolism of arachidonic
    acid has been studied both  in vivo and  in vitro.  Both an increase
    and a decrease in the metabolism of arachidonic acid has been observed
    in several species.   In vivo exposure of rats to 18 800 g/m3
    (10 ppm) for 2 h resulted in decreased levels of prostaglandins E2
    and F2alpha, as well as thromboxane B2, in lavage fluid.  On the
    other hand, at an exposure level of 1880 g/m3 (1 ppm), the
    concentrations of thromboxane B2 were increased (Schlesinger et al.,
    1990).

    b)  Effects on lung proteins and enzymes

         Nitrogen dioxide can cause lung inflammation (associated with
    concomitant infiltration of serum protein, enzymes and inflammatory
    cells) and hyperplasia of Type 2 cells. Thus, some changes in lung
    enzyme activity and protein content may reflect inflammation and/or
    changes in cell types, rather than direct effects of NO2 on lung cell
    enzymes.  Some direct effects of NO2 on enzymes are possible because

    NO2 can oxidize various reducible amino acids or side chains of
    proteins in aqueous solution (Freeman & Mudd, 1981).  These effects
    are summarized in Table 32.

         Nitrogen dioxide can increase the protein content of BAL in
    vitamin-C-deficient guinea-pigs (Sherwin & Carlson, 1973; Selgrade et
    al., 1981; Hatch et al., 1986; Slade et al., 1989).  Selgrade et al.
    (1981) found effects at NO2 levels as low as 1880 g/m3 (1.0 ppm)
    after a 72-h exposure, but a 1-week exposure to 752 g/m3 (0.4 ppm)
    did not increase protein levels.  The results of the 1-week exposure
    apparently conflict with those of Sherwin & Carlson (1973), who found
    increased protein content of BAL from vitamin-C-deficient guinea-pigs
    exposed to 752 g/m3 (0.4 ppm) NO2 for 1 week.  Differences in
    exposure techniques,  protein measurement methods, and/or degree of
    vitamin C deficiencies may explain the difference between the two
    studies.  Hatch et al. (1986) found that the NO2-induced increase in
    BAL protein in vitamin-C-deficient guinea-pigs was accompanied by an
    increase in lung content of non-protein sulfhydryls and ascorbic acid
    and a decrease in vitamin E content.  The increased susceptibility to
    NO2 was observed when lung vitamin C was reduced (by diet) to levels
    below 50% of normal.  A depletion of lung non-protein sulfhydryls also
    enhances susceptibility to a high level (18 800 g/m3, 10.0 ppm) of
    NO2 (Slade et al., 1989).

         The effects of NO2 on structural proteins of the lungs has been
    of major interest because elastic recoil is lost after exposure
    (section 5.2.2.3).  Last et al. (1983) examined collagen synthesis
    rates by lung minces from animals exposed to NO2.  In rats
    continuously exposed to 9400 to 47 000 g/m3 (5.0 to 25.0 ppm) NO2
    for 7 days, there was a linear concentration-related increase in
    collagen synthesis rate.  In a subsequent paper, Last & Warren (1987)
    confirmed that 9400 g/m3 (5.0 ppm) increased collagen synthesis. 
    Such biochemical changes are typically interpreted as reflecting
    increases in total lung collagen, which, if sufficient, could result
    in pulmonary fibrosis after longer periods of exposure.  However, such
    correlations have not been made directly after NO2 exposure.

        Table 32.  Effects of nitrogen dioxide (NO2) on lung proteins and enzymesa
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    75           0.04         Continuous,           Rat         NPSHs increased at the 2 higher NO2 levels after 9 or 18    Sagai et al.
    752          0.4          9 and 18 months                   months; GSH peroxidase activity decreased at 752 g/m3      (1984)
    7520         4.0                                            after 18 months and at 7520 g/m3 after 9 or 18 months;
                                                                GSH reductase activity increased after a 9-month exposure
                                                                to 7520 g/m3; G-6-PD was increased after a 9- or 18-month
                                                                exposure to 7520 g/m3; no effects on 6-phosphogluconate
                                                                dehydrogenase, superoxide dismutase, or disulfide
                                                                reductase; some GSH S-transferases had decreased
                                                                activities after an 18-month exposure to 752 or
                                                                7520 g/m3.

    752          0.4          72 h                  Guinea-pig  No effect at 752 g/m3; increase in BAL protein in          Selgrade et
    1880         1.0                                            vitamin-C-depleted but not normal animals at > 1880 g/m3.  al. (1981)
    5640         3.0
    9400         5.0

    9400         5.0          3 h                               Increased BAL protein in vitamin-C-depleted guinea-pigs
                                                                15-h post-exposure.

    752          0.4          Continuous,                       No effect on BAL protein in vitamin-C-depleted guinea-pigs.
                              1 week

    752          0.4          Continuous,           Guinea-pig  Increase in BAL protein content of guinea-pigs with an      Sherwin &
                              1 week                            unquantified vitamin C deficiency.                          Carlson (1973)
                                                                                                                                              

    Table 32  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    752          0.4          1 to 14 weeks         Rat         Complex concentration and duration dependence of effects.   Takahashi et
    2260         1.2                                            Example: at 752 g/m3, cytochrome P-450 levels decreased    al. (1986)
    7520         4.0                                            at 2 weeks, returned to control level by 5 weeks. At
                                                                2260 g/m3, cytochrome P-450 levels decreased initially,
                                                                increased at 5 weeks, and decreased at 10 weeks. Effects
                                                                on succinate-cytochrome c reductase also.

    752          0.4          4 months              Rat         Duration-dependent pattern for increase in activities of    Ichinose &
    2260         1.2                                            antioxidant enzymes; increase, peaking at week 4, and       Sagai (1982)
    7520         4.0                                            then decreasing; concentration-dependent effects.

    752          0.4          2 weeks               Rat         No effect on TBA reactants, antioxidants or antioxidant     Ichinose &
                                                    Guinea-pig  enzyme activities.                                          Sagai (1989)

    752          0.4          7 days                Rat         Decrease in cytochrome P-450 level at > 2260 g/m3.         Mochitate et
    2260         1.2                                                                                                        al. (1984)
    7520         4.0

    846          0.45         7 h/day               Mouse       No changes in lung serotonin levels.                        Sherwin et
                              4 weeks                                                                                       al. (1986)

    884          0.47         Continuous, 10,       Mouse       Increased content of serum proteins in homogenized whole    Sherwin &
                              12, 14 days                       lung tissue.                                                Layfield (1974)

    940          0.5          Continuous,           Mouse       Decrease in lung GSH peroxidase activity at 1880 g/m3      Ayaz &
    1880         1.0          17 months                         in vitamin-E-deficient mice. Increased activity in          Csallany (1978)
                                                                vitamin-E-supplemented mice at > 940 g/m3.
                                                                                                                                              

    Table 32  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    1880         1.0          Continuous,           Rat         Activities of GSH reductase and G-6-PD increased at         Chow et al.
    4320         2.3          4 days                            11 700 g/m3 proportional to duration of exposure; no       (1974)
    11 700       6.2                                            effect on GSH peroxidase. No effects at < 4320 g/m3.

    1880         1.0          15 weeks              Rat         Changes in BAL fluid and lung tissue levels of enzymes      Gregory et al.
    9400         5.0                                            early in exposure; resolved by 15 weeks.                    (1983)

    3760         2.0          3 days                Rat         Decreased superoxide dismutase activity.                    Azoulay-Dupuis
    18 800       10.0                               Guinea-pig                                                              et al. (1983)

    3760         2.0          Continuous,           Rat         Increased activities of several glycolytic enzymes.         Mochitate et
    7520         4.0          7, 10, 14 days                    At < 7520 g/m3, pyruvate kinase increased on days          al. (1985)
                                                                4 and 7; recovery occurred by day 14. G-6-PD increased
                                                                at all levels; at 3760 g/m3, 14 days of exposure needed.

    3760         2.0          1-7 days              Rat         Increased lung protein content; increase in microsomal      Mochitate et
    7520         4.0                                            succinate cytochrome c reductase activity.                  al. (1984)
    18 800       10.0

    5640         3.0          7 days                Rat         Various changes in lung homogenate protein and DNA          Elsayed &
                                                                content and enzyme activities; changes more severe in       Mustafa (1982)
                                                                vitamin-E-deficient rats.

    5640         3.0          7 days                Rat         No effects on antioxidant metabolism or oxygen              Mustafa et al.
    9400         5.0          4 days                            consumption enzymes at < 9400 g/m3.                        (1979)

    7520         4.0          7, 14 and             Rat         Increased gamma-glutamyl transferase on days 14 and 21;     Hooftman et al.
    18 800       10.0         21 days                           no consistent effect on alkaline phosphatase, lactate       (1988)
    47 000       25.0                                           dehydrogenase or total protein.
                                                                                                                                              

    Table 32  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    9020         4.8          3 h                   Guinea-pig  Increased BAL protein content in vitamin-C-deficient        Hatch et al.
                                                                guinea-pigs.                                                (1986)

    8460         4.5          16 h                              Increased lung wet weight, alterations in lung
                                                                antioxidant levels in vitamin-C-deficient guinea-pigs.

    9020         4.8          7 days                Mouse       No significant changes in lung homogenate parameters.       Mustafa et al.
                                                                                                                            (1984)

    9400         5.0          14-72 h               Mouse       Increase in lung protein (14 to 58 h) by radioactive        Csallany (1975)
                                                                label incorporation.

    9400-47 000  5.0-25.0     Continuous,           Rat         Concentration-related increase in rate of collagen          Last et al.
                              7 days                            synthesis; 125% increase at 9400 g/m3.                     (1983)

    9400         5.0          3 h                   Rabbit      Benzo[a]pyrene hydroxylase activity of tracheal mucosa      Palmer et al.
    37 600       20.0                                           not affected.                                               (1972)
    94 000       50.0
                                                                                                                                              

    a  Modified from US EPA (1993)
    b  NPSHs = Non-protein sulfhydryls; GSH = Glutathione; G-6-PD = Glucose-6-phosphate dehydrogenase; BAL = Bronchoalveolar lavage
             Alterations in lung xenobiotic metabolism follow a complex
    duration of exposure pattern in rats exposed to 752, 2260 and
    7520 g/m3 (0.4, 1.2 and 4.0 ppm) NO2 (Takahashi et al., 1986).  At
    the lowest NO2 concentration tested, cytochrome P-450 levels
    decreased initially (at 2 weeks) and then returned to control levels
    by 5 weeks, where they remained throughout exposure.  At 2260 g/m3
    (1.2 ppm), cytochrome P-450 levels decreased initially, then increased
    after 5 weeks of exposure and decreased again by 10 weeks.  A similar
    pattern of response occurred at the highest concentration.  Only
    7520 g/m3 (4.0 ppm) NO2 affected other microsomal electron-
    transport systems.  The activity of succinate-cytochrome c reductase
    was decreased by 14 weeks of exposure to 752 g/m3 (0.4 ppm), but at
    the higher NO2 levels, the activity was decreased sooner.  In
    contrast, Mochitate et al. (1984) also found a decrease in levels of
    cytochrome P-450 at > 2260 g/m3 (1.2 ppm) in rats exposed for
    7 days.

         Glycolytic pathways are also increased by NO2 exposure,
    apparently due to a concurrent increase in Type 2 cells (Mochitate et
    al., 1985).  The most sensitive enzyme was pyruvate kinase, exhibiting
    an increased activity after a 14-day exposure to 3760 g/m3 (2.0 ppm)
    NO2.  At higher NO2 concentrations (e.g., 7520 g/m3, 4.0 ppm),
    pyruvate kinase activity increased sooner (4 and 7 days) and then
    decreased to control levels by 14 days.

    c)  Antioxidant defence systems

         Since NO2 is an oxidant and lipid peroxidation is believed to be
    a major molecular event responsible for the toxic effects of NO2,
    much attention has been focused on the effect of the  antioxidant
    defence system in the epithelial lining fluid and in pulmonary cells. 
    Investigations with subacute and chronic NO2 exposure levels of 75
    to 62 040 g/m3 (0.04-33 ppm) have been performed both  in vivo
    and  in vitro and focussed on effects on low molecular weight
    antioxidants such as glutathione, vitamin E and vitamin C, as well as
    on some enzymes involved in the synthesis and catabolism of
    glutathione.  Experiments made  in vitro using human plasma have
    shown a rapid depletion of vitamin C and glutathione and a loss of
    vitamin E.  This result was achieved with a concentration of
    26 320 g/m3 (14 ppm) (Halliwel et al., 1992).

         Menzel (1970) proposed that antioxidants might protect the lung
    from NO2 damage by inhibiting lipid peroxidation.  Data related to
    this hypothesis have been reported (Thomas et al., 1968; Menzel et
    al., 1972; Fletcher & Tappel, 1973; Csallany, 1975; Ayaz & Csallany,
    1978; Slade et al., 1989).  Several laboratories have observed changes
    in the activity of enzymes in the lungs of NO2-exposed animals that
    regulate levels of glutathione (GSH), the major water-soluble
    reductant in the lung.  Chow et al. (1974) exposed rats to 1880, 4320
    or 11 700 g/m3 (1.0, 2.3 or 6.2 ppm) NO2 continuously for 4 days to
    examine the effect on activities of GSH reductase, glucose-6-phosphate
    dehydrogenase and GSH peroxidase in the soluble fraction of exposed
    rat lungs.  Linear regression analysis of the correlation between the
    NO2 concentration and enzymatic activity showed a significant
    positive correlation coefficient of 0.63 for GSH reductase and of 0.84
    for glucose-6-phosphate dehydrogenase.  No correlation was found
    between the GSH peroxidase activity and the NO2 concentration.  The
    activities of GSH reductase and glucose-6-phosphate dehydrogenase were
    significantly increased during exposure to 11 700 g/m3 (6.2 ppm)
    NO2; GSH peroxidase activity was not affected.  The possible role of
    oedema and cellular inflammation in these findings was not examined. 
    These researchers concluded that after a slightly longer exposure
    (14 days), 3760 g/m3 (2.0 ppm) NO2 increased the activity of
    glucose-6-phosphate dehydrogenase in rats (Mochitate et al., 1985). 
    There is evidence from recent studies that glutathione and vitamins C
    and E are all involved in normal protection of the lung from NO2
    (Rietjens et al., 1986; Hatch et al., 1986; Slade et al., 1989).

         Sagai et al. (1984) studied the effects of prolonged (9 and 18
    months) exposure to 75, 752 and 7520 g/m3 (0.04, 0.4 and 4.0 ppm)
    NO2 on rats.  After both exposure durations, non-protein sulfhydryl
    levels were increased at > 752 g/m3; exposure to 7520 g/m3
    (4.0 ppm) decreased the activity of GSH peroxidase and increased
    glucose-6-phosphate dehydrogenase activity.  Glutathione peroxidase
    activity was also decreased in rats exposed to 752 g/m3 NO2 for
    18 months.  Three GSH  S-transferases were also studied, two of which
    (aryl  S-transferase and aralkyl  S-transferase) exhibited decreased
    activities after 18 months of exposure to > 752 g/m3 NO2.  No
    effects were observed on the activities of 6-phosphogluconate
    dehydrogenase, superoxide dismutase or disulfide reductase.  When
    effects were observed, they followed a concentration and exposure-
    duration response function.  The decreases in antioxidant metabolism

    were inversely related to the apparent formation of lipid peroxides
    (see lipid peroxidation subsection).  Shorter exposures (4 months) to
    NO2 between 752 and 7520 g/m3 (0.4 and 4.0 ppm) also caused
    concentration- and duration-dependent effects on antioxidant enzyme
    activities (Ichinose & Sagai, 1982).  For example, glucose-6-phosphate
    dehydrogenase increased, reaching a peak at 1 month, and then
    decreased towards the control value.  Briefer (2-week) exposures to
    752 g/m3 (0.4 ppm) NO2 caused no such effects in rats or
    guinea-pigs (Ichinose & Sagai, 1989).

         Ayaz & Csallany (1978) exposed vitamin-E-deficient and vitamin-E-
    supplemented mice continuously for 17 months to 940 or 1880 g/m3
    (0.5 or 1.0 ppm) NO2 and assayed them for GSH peroxidase activity. 
    Exposure to 1880 g/m3 (1.0 ppm) NO2 decreased enzyme activity in
    the vitamin-E-deficient mice.  However, in vitamin-E-supplemented
    mice, GSH peroxidase activity increased at 940 g/m3 (0.5 ppm) NO2.

    5.2.2.3  Pulmonary function

         Animal studies of NO2 effects on pulmonary function are
    summarized in Table 33.  NO2 concentrations in many urban areas of
    the USA and elsewhere consist of spikes superimposed on a relatively
    constant background level.  Miller et al. (1987) evaluated this urban
    pattern of NO2 exposure in mice using continuous 7 days/week,
    23 h/day exposures to 376 g/m3 (0.2 ppm) NO2 with twice daily
    (5 days/week) 1-h spike exposures to 1500 g/m3 (0.8 ppm) NO2 for
    32 and 52 weeks.  Mice exposed to clean air and to the constant
    background concentration of 376 g/m3 (0.2 ppm) served as controls.
    Vital capacity tended to be lower (p = 0.054) in mice exposed to NO2
    with diurnal spikes than in mice exposed to air.  Lung distensibility,
    measured as respiratory system compliance, also tended to be lower in
    mice exposed to diurnal spikes of NO2 compared with constant NO2
    exposure or air exposure.  These changes suggest that up to 52 weeks
    of low-level NO2 exposure with diurnal spikes may produce a subtle
    decrease in lung distensibility, although part of this loss in
    compliance may be a reflection of the reduced vital capacity.  Vital
    capacity appeared to remain suppressed for at least 30 days after
    exposure.  Lung morphology in these mice was evaluated only by light
    microscopy (a relatively insensitive method) and showed no exposure-
    related lesions.  The decrease in lung distensibility suggested by
    this study is consistent with the thickening of collagen fibrils in
    monkeys (Bils, 1976) and the increase in lung collagen synthesis rates
    of rats (Last et al., 1983) after exposure to higher levels of NO2.

         Tepper et al. (1993) exposed 60-day-old rats to 940 g/m3
    0.5 ppm) NO2, 22 h/day, 7 days/week, with a 2-h spike of 2820 g/m3
    (1.5 ppm) NO2, 5 days/week for up to 78 weeks. There were no effects
    on pulmonary function between 1 and 52 weeks of exposure.  Following
    78 weeks of exposure, flow at 25% forced vital capacity was decreased,
    perhaps indicating airway obstruction.  A significant decrease in the
    frequency of breathing was also observed at 78 weeks that was
    paralleled by a trend toward increased expiratory resistance and
    expiratory time.  Taken together, these results suggest that few, if
    any, significant effects were seen that suggest incipient lung
    degeneration.

         The age sensitivity to exposure to diurnal spikes of NO2 was
    studied by Stevens et al. (1988), who exposed 1-day- and 7-week-old
    rats to continuous baselines of 940, 1880 and 3760 g/m3 (0.5, 1.0
    and 2.0 ppm) NO2 with twice daily 1-h spikes at 3 times these
    baseline concentrations for 1, 3 and 7 weeks.  In neonatal rats, vital
    capacity and respiratory system compliance increased following 3
    weeks, but not 6 weeks, of exposure to the 1880 and 3760 g/m3 NO2
    baselines with spikes.  In young adult rats, respiratory system
    compliance decreased following 6 weeks of exposure, and body weight
    decreased following 3 and 6 weeks of exposure to the 3760 g/m3
    baseline with spike.  In the young adult rats, pulmonary function
    changes returned to normal values 3 weeks after exposure ceased.  A
    correlated morphometric study (Chang et al., 1986) is summarized in
    section 5.2.2.4.

         Lafuma et al. (1987) exposed 12-week-old hamsters with and
    without laboratory-induced (elastase) emphysema to 3760 g/m3
    (2.0 ppm) NO2, 8 h/day, 5 days/week for 8 weeks.  Vital capacity
    and pulmonary compliance were not affected by NO2 exposure.

    5.2.2.4  Morphological studies

         Inhalation of NO2 produces morphological alterations in the
    respiratory tract, as summarized in Tables 34 and 35.  This
    discussion is generally limited to those studies using NO2 levels
    < 9400 g/m3 (5.0 ppm), but results of studies of emphysema
    conducted at higher concentrations are also discussed. Examination of
    the tables shows variability in responses at similar exposure levels
    in different studies.  This may be due to differences in animal
    species or strain, age, diet, microbiological status of the animals,

    or aspects of experimental protocol.  The latter includes the
    methodology used to evaluate the morphological response.  For example,
    simple light microscopic examination may reveal no effect, whereas
    other techniques, such as quantitative morphological (morphometric)
    procedures with electron microscopy, can detect more subtle structural
    changes.

         There is a large degree of interspecies variability in
    responsiveness to NO2; this is clearly evident from those few studies
    where different species were exposed under identical conditions
    (Wagner et al., 1965; Furiosi et al., 1973; Azoulay-Dupuis et al.,
    1983).  Variability in response may be due to differences in effective
    dose of NO2 reaching target sites, but other species differences are
    likely to play a role.  Guinea-pigs, hamsters and monkeys all appear
    to be more severely affected morphologically by equivalent exposure to
    NO2 than are rats, the most commonly used experimental animal.
    However, in most cases, similar types of histological lesions are
    produced when similar effective concentrations are used.

    a)  Sites affected and time course of effects

         The anatomic region most sensitive to NO2 and within which
    injury is first noted is the centriacinar region.  This region
    includes the terminal conducting airways (terminal bronchioles),
    respiratory bronchioles, and adjacent alveolar ducts and alveoli. 
    Within this region, those cells that are most sensitive to
    NO2-induced injury are the ciliated cells of the bronchiolar
    epithelium and the Type 1 cells of the alveolar epithelium, which are
    then replaced with non-ciliated bronchiolar (Clara) cells and Type
    2 cells, respectively.  In addition to these dynamic changes,
    permanent alterations resembling emphysema-like disease may result
    from chronic exposure.

         The temporal progression of early events due to NO2 exposure has
    been described best in the rat (e.g., Freeman et al., 1966, 1968c,
    1972; Stephens et al., 1971a, 1972; Evans et al., 1972, 1973a,b, 1974,
    1975, 1976, 1977; Cabral-Anderson et al., 1977; Rombout et al., 1986)
    and guinea-pig (Sherwin et al., 1973).  The earliest alterations
    resulting from exposure to concentrations of > 3760 g/m3
    (2.0 ppm) are seen within 24 to 72 h of exposure and include increased
    AM aggregation, desquamation of Type 1 cells and ciliated bronchiolar
    cells, and accumulation of fibrin in small airways.  However, repair
    of injured tissue and replacement of destroyed cells can begin within
    24 to 48 h of continuous exposure.  Hyperplasia of nonciliated
    bronchiolar (Clara) cells occurs in the bronchioli, whereas in the
    alveoli, the damaged Type 1 cells are replaced with Type 2 cells. 
    These new cells are relatively resistant to effects of continued NO2
    exposure.

        Table 33.  Effects of nitrogen dioxide (NO2) on pulmonary functiona
                                                                                                                                              
    NO2 Concentration
                      
    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    376          0.2          23 h/day base         Mouse       Decreased vital capacity following base + spike             Miller et
                              (7 days/week), 1-h                NO2 exposures compared with control and base NO2            al. (1987)
    376 base,    0.2 base,    peaks twice/day,                  exposures. Tendency toward decreased respiratory
    1500 peak    0.8 peak     32 and 52 weeks                   system compliance following spike NO2 exposures
                                                                compared and control and base NO2 exposures.

    940 base,    0.5 base,    23 h/day              Rat (1-day  Increased lung volume and compliance in neonates            Stevens et
    2820 peak    1.5 peak     (7 days/week) base,   and         following 3-week, but not 6-week, exposure to the           al. (1988)
                              1-h peaks twice/day   7-weeks     two higher exposure levels. Decreased body weight
    1880 base,   1.0 base,    (5 days/week);        old)        and lung compliance in adult rats following 6-week
    5640 peak    3.0 peak     1, 3 and 6 weeks                  exposure to 3760 g/m3 + spike. Adults recovered
                                                                3 weeks after exposure.
    3760 base,   2.0 base,
    11 300 peak  6.0 peak

    940 base,    0.5 base,    22 h/day (7 days per  Rat         Decreased delta FEF25 and frequency of breathing            Tepper et al.
    2820 peak    1.5 peak     week), 2-h peak                   following 78-week NO2 exposure.                             (1993)
                              (5 days/week); 1,
                              3, 12, 52 and
                              78 weeks

    3760         2.0          8 h/day,              Hamster     No change in vital capacity or lung compliance              Lafuma et al.
                              5 days/week,                      following NO2 exposures in both normal and                  (1987)
                              8 weeks                           elastase-treated animals.

    10 200       5.4          3 h/day for 7, 14     Rat         Tendency toward increased lung volume at low                Yokoyama et
                              or 30 days                        inflation pressures.                                        al. (1980)
                                                                                                                                              
    a  Modified from: US EPA (1993)
    b  PaO2 = Arterial oxygen tension; delta FEF25 = Change in forced expiratory flow at 25% of forced vital capacity;
       PaCO2 = Arterial carbon dioxide tension

    Table 34.  Effects of acute and subchronic exposure to nitrogen dioxide (NO2) on lung morphologya
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    207          0.11         Continuous,           Rat (1,     Various morphometric changes, depending on age              Kyono & Kawai
    865          0.46         1 month               3, 12,      and exposure level. Multiphasic pattern (e.g.,              (1982)
    5260         2.8                                21 months   decrease in air-blood barrier thickness from 1 to
    16 500       8.8                                old)        12 months of age, and increase in 21-month-old
                                                                rats).

    639          0.34         6 h/day, 5 days       Mouse       Type 2 cell hypertrophy and hyperplasia; increase           Sherwin &
                              per week, 6 weeks                 in mean linear intercept and amount of alveolar             Richters (1982)
                                                                wall area.

    940          0.5          4 h                   Rat         Loss of cytoplasic granules in and rupture of               Thomas et al.
                                                                mast cells.                                                 (1967)

    940          0.5          Continuous, up        Rat         Increased number of mast cells in trachea as exposure       Hayashi et al.
                              to 6 days                         duration increased.                                         (1987)
                                                                                                                                              

    Table 34  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    940 base,    0.5 base,    23 h/day (7 days      Rat (1 day  In proximal alveolar region: base (940 g/m3) + peak        Crapo et al.
    2820 peak    1.5 peak     per week) base, 1-h   and         caused Type 2 cells to become spread over more              (1984); Chang
                              peaks twice/day       6 weeks     surface area in neonates and adults; Type 2 cell            et al. (1986,
    3760 base,   2.0 base,    (5 days/week);        old)        hypertrophy and increase in number of AMs in adults;        1988)
    11 280 peak  6.0 peak     6 weeks                           Type 2 cells thinner in neonates. Base (3760 g/m3)
                                                                + peak (only adults studied) caused similar changes
                                                                plus an increase in numbers of Type 1 cells, which
                                                                were smaller than normal Type 1 cells.
                                                                In terminal bronchiolar region: base (940 g/m3) +
                                                                peak caused no effects on percentage distribution of
                                                                ciliated cells and Clara cells in neonates or adults,
                                                                but neonates (only) had a increase in ciliated cell
                                                                surface area and mean luminal surface area of Clara
                                                                cells. Base (3760 g/m3) + peak (only adults studied)
                                                                had fewer ciliated cells with a reduced surface area
                                                                and alterations in the shape of Clara cells.

    1000         0.53         Continuous            Rat         At < 2500 g/m3: no pathology. At 5000 g/m3: focal         Rombout et al.
    2500         1.33         (24 h/day)                        thickening of centriacinar septa by 2 days; progressive     (1986)
    5000         2.66         28 days                           loss of cilia and abnormal cilia in trachea and main
                                                                bronchi at > 4 days; hypertrophy of bronchiolar
                                                                epithelium at > 8 days.
                                                                At days 16 and 28, all epithelial cells hypertrophied.
                                                                                                                                              

    Table 34  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    1000         0.53         24 h/day,             Guinea-     No pathology                                                Steadman et al.
                              90 days               pig,                                                                    (1966)
                                                    rabbit,
                                                    dog,
                                                    monkey,
                                                    rat

    1320-1500    0.7-0.8      Continuous,           Mouse       Mucous hypersecretion; focal degeneration and               Nakajima et al.
                              1 month                           desquamation of mucous membrane; terminal                   (1980)
                                                                bronchiolar epithelial hyperplasia; some alveolar
                                                                enlargement; shortening of cilia.

    1880         1-1.5        Continuous,           Mouse       Terminal bronchiolar epithelial hyperplasia; some           Nakajima et al.
    2820                      1 month                           alveolar enlargement.                                       (1980)

    1880         1.0          1 h                   Rat         Degranulation and decreased number of mast cells.           Thomas et al.
                                                                                                                            (1967)

    3760         2.0          3 days                Rat         No historical changes                                       Azoulay-Dupuis
                                                                                                                            et al. (1983)

    3760         2.0          3 days                Guinea-pig  Thickening of alveolar walls; oedema; increase in           Azoulay-Dupuis
                                                                AM numbers; loss of bronchiolar cilia; inflammation.        et al. (1983)
                                                                                                                                              

    Table 34  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    3760         2.0          8 h/day,              Hamster     Moderate alveolar enlargement, primarily at                 Lafuma et al.
                              5 days/week,                      bronchiolar-alveolar duct junction; increase in mean        (1987)
                              8 weeks                           linear intercept; decrease internal surface area of
                                                                lung; no lesions in bronchial, bronchiolar, alveolar
                                                                duct, or alveolar epithelium; no change in
                                                                macrophage number.

    3760         2.0          Continuous,           Guinea-pig  Type 2 cell hypertrophy at 7 or 21 days.                    Sherwin et al.
                              7-21 days                                                                                     (1973)

    3760         2.0          Continuous,           Guinea-pig  Increase in number of LDH-positive cells with time          Sherwin et al.
                              1-3 weeks                         of exposure. Correlated to increase in Type 2 cells         (1973)
                                                                (LDH positive).

    3760         2.0          Continuous,           Rat         Minimal effect: some cilia loss in terminal bronchioles;    Azoulay et al.
                              6 weeks                           some distended or disrupted alveolar walls.                 (1978)

    9400         5.0          Continuous,           Cynomolgus  Bronchiolar epithelia hyperplasia; some focal               Busey et al.
    18 800       10.0         90 days               monkey      pulmonary odema.                                            (1974)
                                                                                                                                              

    a  Modified from US EPA (1993)
    b  AMs = Alveolar macrophages; LDH = Lactate dehydrogenase

    Table 35.  Effects of chronic exposure to nitrogen dioxide (NO2) on lung morphologya
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    75           0.04         Continuous,           Rat         At 75 g/m3: no significant change, but some tendency       Kubota et al.
    752          0.4          9-27 months                       towards increase in arithmetic mean thickness of air-blood  (1987)
    7520         4.0                                            barrier.  At 752 g/m3: slight increase in arithmetic
                                                                mean thickness of air-blood barrier by 18 months, becoming
                                                                significant by 27 months; some interstitial oedema and
                                                                slight change in bronchiolar and alveolar epithelium by
                                                                27 months.  At 7520 g/m3: hypertrophy and hyperplasia of
                                                                bronchiolar epithelium and increase in arithmetic mean
                                                                thickness of air-blood barrier by 9 months, which became
                                                                significant at 18 months and decreased slightly by
                                                                27 months; Clara cell hyperplasia. By 27 months:
                                                                interstitial fibrosis and hypertrophy of Type 1 and
                                                                Type 2 cells.

    188 base;    0.1 base;    Continuous            Mouse       Dilated airspaces and aveolar wall destruction (small       Port et al.
    1880 peak    1.0 peak     baseline; 2-h                     sample size).                                               (1977)
                              daily peak;
                              6 months
                                                                                                                                              

    Table 35  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    940          0.5          Continuous,           Rat         At 940 g/m3: swelling of terminal bronchiolar cilia and    Yamamoto &
    1880         1.0          7 months                          hyperplasia of Type 2 cells.                                Takahashi
    7520         4.0                                            At 1880 g/m3: cilia loss in terminal bronchioles;          (1984)
                                                                hyperplasia of Type 2 cells; and interstitial oedema.
                                                                At 7520 g/m3: cilia loss in terminal bronchioles;
                                                                hyperplasia of Type 2 cells, interstitial oedema; decrease
                                                                in number of lamellar bodies in Type 2 cells; lysosomes
                                                                with osmiophilic lamellar structure in ciliated cells of
                                                                terminal bronchioles.

    940          0.5          Continuous, up        Rat         Type 2 cell hypertrophy and interstitial oedema by          Hayashi et al.
                              to 19 months                      4 months; increased thickness of alveolar septa by          (1987)
                                                                6 months; fibrous pleural thickening by 19 months.

    940          0.5          6-24 h/day,           Mouse       3 months: pneumonitis and alveolar size increase; loss of   Blair et al.
                              3-12 months                       cilia in respiratory bronchioles and bronchiolar            (1969)
                                                                inflammation with 24 h/day.
                                                                6-12 months: pneumonitis; cilia loss; bronchial and
                                                                bronchiolar inflammation; alveolar size increase.

    1500         0.8          Continuous,           Rat         Minimal changes: slight enlargement of alveoli and          Freeman et
                              lifetime (up                      alveolar ducts; some rounding of bronchial and bronchiolar  al. (1966)
                              to 33 months)                     epithelial cells; increase in elastic fibers around
                                                                alveolar ducts.

    1880         1.0          Continuous,           Squirrel    No pathology                                                Fenters et
                              16 months             monkey                                                                  al. (1973)
                                                                                                                                              

    Table 35  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    1880         1.0          6 h/day,              Dog         At 1880 g/m3 - 6 months: no pathology; 12 months:          Wagner et
                              5 days/week, up                   dilated alveoli and alveolar ducts; 18 months:              al. (1965)
                              to 18 months                      dilated alveoli, oedema, thickening alveolar septa
                                                                due to inflammation.
    9400         5.0                                            At 9400 g/m3 - 6 months: no pathology; 12 months:
                                                                dilated alveolar ducts; 18 months: oedema, congestion,
                                                                and thickened alveolar septa due to inflammatory cells.

    1880         1.0          6 h/day               Guinea-pig  Mild thickening of alveolar septa due to inflammation;      Wagner et
                              5 days/week,                      some alveolar dilatation.                                   al. (1965)
                              18 months

    1880         1.0          7 h/day,              Rat         No pathology                                                Gregory et
                              5 days/week,                                                                                  al. (1983)
                              15 weeks

    3760         2.0          Continuous,           Rat         Loss of cilia in terminal bronchioles; abnormal             Stephens et
                              2 years                           ciliogenesis; crystalloid inclusions in bronchiolar         al. (1971a,b)
                                                                epithelial cells; increased thickness of collagen fibrils
                                                                and basement membrane in terminal bronchioles.

    3760         2.0          Continuous, up        Rat         Hypertrophy of ciliated cells and cilia loss by 72 h;       Stephens et
                              to 12 months                      decreased number of ciliated cells by 7 days; normal        al. (1972)
                                                                ciliated cells from 21 days-12 months.

    3760         2.0          Continuous, up        Rat         No change in turnover of terminal bronchiolar epithelial    Evans et al.
                              to 360 days                       cells; increase in turnover of Type 2 cells in peripheral   (1972)
                                                                alveoli by 1 day, but normal by 7 days.
                                                                                                                                              

    Table 35  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    3760         2.0          Continuous,           Monkey      Bronchiolar epithelial hypertrophy, especially adjacent     Furiosi et al.
                              14 months             (Macaca     to alveolar ducts; change to cuboidal cells in proximal     (1973)
                                                    peciosa)    bronchiolar epithelium.

    3760         2.0          Continuous,           Rat         Minimal effect: some terminal bronchiolar epithelial        Furiosi et al.
                              14 months                         hypertrophy.                                                (1973)

    3760         2.0          Continuous,           Rat         Alveolar distension, especially near alveolar duct level;   Freeman et
                              lifetime (up to                   increased variability in alveolar size; loss of cilia and   al. (1968b)
                              763 days); 1500                   hypertrophy in terminal bronchiolar cells; no
                              g/m3 for 1st                     inflammation.
                              69 days, then
                              3760 g/m3

    7520         4.0          Continuous,           Rat         Bronchial epithelial hyperplasia                            Haydon et al.
                              16 months                                                                                     (1965)

    9400         5.0          6 h/day,              Mouse       No pathology                                                Wagner et al.
                              5 days/week,                                                                                  (1965)
                              14 months

    9400         5.0          4-7.5 h/day,          Guinea-pig  Some dilatation of terminal bronchioles; tracheal           Balchum et al.
                              5 days/week,                      inflammation; pneumonitis.                                  (1965)
                              5.5 months
                                                                                                                                              

    Table 35  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    9400         5.0          7 h/day,              Rat         Focal hyperinflation and areas of subpleural accumulation   Gregory et al.
                              5 days/week,                      of macrophages.                                             (1983)
                              15 weeks
                                                                                                                                              

    a  Modified from US EPA (1993)
             The time course of alveolar lesions over a chronic exposure was
    examined by Kubota et al. (1987) in small groups of rats exposed to
    7520 g/m3 (4.0 ppm) NO2, 24 h/day for up to 27 months.  One phase,
    which lasted for 9 to 18 months of exposure, consisted of a decrease
    in number and an increase in cell volume of Type 1 epithelium, an
    increase in the relative ratio of Type 2 to Type 1 cells, and an
    increase in the number and volume of Type 2 cells.  A second phase,
    between 18 to 27 months of exposure, showed some recovery of the
    alveolar epithelium, but the total volume of interstitial tissue
    decreased and collagen fibres in the interstitium increased.  Thus,
    some lesions resolved with continued exposure, whereas others
    progressed.  At 752 g/m3 (0.4 ppm), Kubota et al. (1987) found that
    the lesion typically was milder and its initiation delayed, compared
    to the higher concentration.  In general, most NO2-induced lesions
    were resolved following a recovery period.  This period may be as
    short as 30 days for exposures at < 9400 g/m3 (5.0 ppm).  With
    continuous exposure, early morphological damage may also be resolved. 
    For example, in rats exposed continuously for 7 months to 940 g/m3
    (0.5 ppm) NO2, resolution of epithelial lesions occurred by 4 to
    6 months of exposure (Yamamoto & Takahashi, 1984).

    b)  Effects of nitrogen dioxide as a function of exposure pattern

         Several morphological studies were designed to evaluate ambient
    NO2 patterns consisting of a low baseline level with transient spikes
    of NO2.  However, in some cases, there was no group at the baseline
    exposure only, preventing evaluation of the contribution of peaks to
    the responses.  Gregory et al. (1983) exposed rats (14 to 16 weeks
    old) for 7 h/day, 5 days/week for up to 15 weeks to atmospheres
    consisting of the following concentrations of NO2: (1) 1880 g/m3
    (1.0 ppm), (2) 9400 g/m3 (5.0 ppm), or (3) 1880 g/m3 (1.0 ppm)
    with two 1.5-h spikes of 9400 g/m3 (5.0 ppm) per day.  After
    15 weeks of exposure, histopathology was minimal, with focal
    hyperinflation and areas of subpleural accumulation of macrophages
    found in some of the animals exposed either to the baseline of
    9400 g/m3 (5.0 ppm) or to 1880 g/m3 (1.0 ppm) with the 9400 g/m3
    (5.0 ppm) spikes.

         Port et al. (1977) observed dilated respiratory bronchioles and
    alveolar ducts in mice exposed to 188 g/m3 (0.1 ppm) NO2 with daily
    2-h peaks to 1880 g/m3 (1.0 ppm), for 6 months.  Miller et al.
    (1987) found no morphological effects in mice exposed for 1 year,
    although host defence and functional changes were noted (see sections
    5.2.2.1 and 5.2.2.3).

         Crapo et al. (1984) and Chang et al. (1986) used quantitative
    morphometric analyses to examine the proximal alveolar and terminal
    bronchiolar regions of rats exposed for 6 weeks to a baseline
    concentration of 940 or 3760 g/m3 (0.5 or 2.0 ppm) NO2, 23 h/day
    for 7 days/week, onto which were superimposed two daily 30 min spikes
    of 3 times the baseline concentration for 5 days/week.  At the lower
    exposure level, the volumes of the Type 2 epithelium, interstitial
    matrix, and AMs increased, whereas the volume of the fibroblasts
    decreased.  The surface area of Type 2 cells increased.  Most of these
    changes also occurred at the higher exposure level, and in some cases
    the change was greater than that at the lower level (i.e., increase in
    Type 1 and Type 2 epithelial volume).  At both levels of exposure, the
    volume of Type 2 cells and interstitial fibroblasts increased, with no
    significant changes in their numbers, and the number of AMs decreased. 
    The number of Type 1 cells decreased and their average surface area
    increased in the highest exposure group.  Generally, there was a
    spreading and hypertrophy of Type 2 cells.  A correlation between
    decreased compliance (Stevens et al., 1988) and thickening of the
    alveolar interstitium was found (see section 5.2.2.3 for details of
    the pulmonary function portion of the study).  Examination of the
    terminal bronchiolar region revealed no effects at the lower exposure
    level.  At the higher level, there was a 19% decrease in ciliated
    cells per unit area of the epithelial basement membrane and a
    reduction in the mean ciliated surface area.  The size of the dome
    protrusions of non-ciliated bronchiolar (Clara) cells was decreased,
    giving the bronchial epithelium a flattened appearance, but there was
    no change in the number of cells.

    c)  Factors affecting susceptibility to morphological changes

         Age-related responsiveness to an urban pattern of NO2 was
    evaluated by Chang et al. (1986, 1988) using 1-day- or 6-week-old rats
    exposed for 6 weeks to a baseline of 940 g/m3 (0.5 ppm) NO2 for
    23 h/day, 7 days/week, with two 1-h spikes (given in the morning and
    afternoon) of 2820 g/m3 (1.5 ppm) 5 days/week.  Electron microscopic
    morphometric procedures were used.  In the proximal alveolar region,
    only the older animals showed an increase in the surface density of
    the alveolar basement membrane.  The increase in the mean cellular
    volume of Type 2 cells was greater in the young adult animals,
    although the neonates also exhibited this effect.  Although there was
    no qualitative evidence of morphological injury in the terminal
    bronchioles of the neonatal rats, there was a 19% increase in the
    average ciliated cell surface and a 13% increase of the mean luminal

    surface area of non-ciliated bronchiolar (Clara) cells that was not
    evident in the young adult rats.  Generally, the neonatal rats were as
    sensitive or more susceptible than young adults, depending upon the
    end-point.  However, the terminal bronchioles of the neonatal rats
    were more susceptible than those of young adults (Chang et al.,
    1988).  For example, the lower exposure altered ciliated cells and
    non-ciliated bronchiolar (Clara) cells in the neonates but not the
    young adults.  Other indices were unaffected.  Pulmonary function was
    also altered in similarly exposed rats (Stevens et al., 1988) (see
    section 5.2.2.3).  Interpretation of the neonatal effects is
    difficult.  Assuming that rats prior to weaning are more resistant to
    NO2 (Stephens et al., 1978) (see below), effects observed after a
    6-week exposure from birth may have resulted from the last 3 weeks of
    exposure, as the first 3 weeks may constitute a more resistant period. 
    In contrast, effects observed in young adults probably reflect the
    impact of the entire 6-week exposure.

         In one of the more extensive studies, Kyono & Kawai (1982)
    exposed rats at 1, 3, 12, and 21 months of age continuously for
    1 month to 207 g/m3, 865 g/m3, 5260 g/m3 or 16 500 g/m3
    (0.11, 0.46, 2.8 or 8.8 ppm) NO2.  Various morphometric parameters
    were assessed, including arithmetic mean thickness of the air-blood
    barrier and the volume density of various alveolar wall components. 
    Quantitative estimations deliberately excluded the site of main damage
    (i.e., the peripheral alveolar wall was examined).  Analysis of
    individual results was complex, but depending upon the animal's age
    and the specified end-point, exposure levels as low as 207 g/m3
    (0.11 ppm) changed specific morphometric parameters.  There was a
    trend towards a concentration-dependent increase in air-blood barrier
    thickness in all age groups, with evidence of age-related differences
    in response.  At any concentration, the response of this end-point
    decreased in rats from 1 to 12 months old, but increased again in
    21-month-old animals.  Type 1 and 2 cells showed various degrees of
    response, depending on both age at onset of exposure and exposure
    concentration.  The response of each lung component did not always
    show a simple concentration-dependent increase or decrease, but
    suggested a multiphasic reaction pattern.

         The above studies with rats may not have used the most
    susceptible animal model, as demonstrated by Azoulay-Dupuis et al.
    (1983), who exposed both rats and guinea-pigs aged 5 to > 60 days
    old to 3760 (2.0 ppm) for 3 days.  Rats at all ages and guinea-pigs
    < 45 days old were not affected.  The 45-day-old guinea-pigs showed
    thickening of alveolar walls, alveolar oedema, and inflammation,
    whereas animals older than 45 days showed similar, but more frequent,
    alterations that seemed to increase with age.  Adults also had focal
    loss of cilia in bronchioli.

         In general, it appears that neonates, prior to weaning, are
    relatively resistant to NO2, and that responsiveness then increases
    (Stephens et al., 1978).  Furthermore, the responsiveness of mature
    animals appears to decline somewhat with age, until an increase in
    responsiveness occurs at some point in senescence.  However, the
    morphological response to NO2 in animals of different ages involves
    similarities in the cell types affected and in the nature of the
    damage incurred.  Age-related differences occur in the extent of
    damage and in the time required for repair, the latter taking longer
    in older animals.  The reasons for age differences in susceptibility
    are not known, but may involve differences in doses to the target
    cells and variable sensitivity of target cells during different growth
    phases.

         The database regarding the effects of levels of NO2
    < 9400 g/m3 (5.0 ppm) on animals with pre-existing respiratory
    disease is very limited and only includes animals with laboratory-
    induced emphysema or infections.  Lafuma et al. (1987) exposed both
    normal and elastase-induced emphysematous hamsters (2 months old) to
    3760 g/m3 (2.0 ppm) NO2 for 8 h/day, 5 days/week, for 8 weeks. 
    Morphometric analyses indicated that emphysematous lesions were
    exacerbated by NO2 (i.e., NO2 increased pulmonary volume and
    decreased internal alveolar surface area).  The investigators
    suggested that these results may imply a role for NO2 in enhancing
    pre-existing emphysema.  Acute infectious (influenza) lung disease
    enhanced the morphological effects of NO2 in squirrel monkeys
    exposed continuously to 1880 g/m3 (1.0 ppm) NO2 for 16 months
    (Fenters et al., 1973).

    d)  Emphysema following nitrogen dioxide exposure

         Numerous investigators have observed morphological lesions that
    led them to the diagnosis of NO2-induced emphysema.  However, to
    evaluate these reports independently, it is necessary to apply the
    current definition of emphysema, especially because the definition
    changed after several of the reports were published.  Such an
    evaluation is described in detail by the US EPA (1993), based upon the
    most recent definition of emphysema from the report of the US National
    Heart, Lung and Blood Institute (NHLBI), Division of Lung Diseases
    Workshop (National Institutes of Health, 1985).  According to this
    document, in human lungs: "Emphysema is defined as a condition of the
    lung characterized by abnormal, permanent enlargement of airspaces
    distal to the terminal bronchiole, accompanied by destruction of their
    walls, and without obvious fibrosis".  Destruction in emphysema is
    further defined as "non-uniformity in the pattern of respiratory
    airspace enlargement so that the orderly appearance of the acinus and
    its components is disturbed and may be lost".  The report further
    indicates: "Destruction...may be recognized by subgross examination of
    an inflation-fixed lung slice...".  However, emphysema in animal

    models was defined differently.  An animal model of emphysema is
    defined as "an abnormal state of the lungs in which there is
    enlargement of the airspaces distal to the terminal bronchiole. 
    Airspace enlargement should be determined qualitatively in appropriate
    specimens and quantitatively by stereologic methods".  Thus, in animal
    models of emphysema, airspace wall destruction need not be present. 
    "Appropriate specimens" presumably refers to lungs fixed in the
    inflated state.   When reports of emphysema following NO2 exposures
    of animals are to be extrapolated to potential hazards for humans, the
    definition of human emphysema, rather than that for emphysema in
    experimental animals, should be used.  The presence of airspace wall
    destruction, critical to the definition of human emphysema, can only
    be determined independently in published reports by careful review of
    the authors' description of the lesions or by examining the
    micrographs that the author selected for publication.  Because
    descriptions in some reports are inadequate for independent
    evaluation,  more evidence may exist for emphysema than is summarized
    here.  All reports reviewed are summarized in Table 36, but only those
    showing emphysema of the type seen in human lungs are discussed in the
    text that follows.

         Haydon et al. (1967) reported emphysema in rabbits exposed
    continuously (presumably 24 h/day) for 3 to 4 months to 15 000 or
    22 600 g/m3 (8.0 or 12.0 ppm) NO2.  They reported enlarged lungs
    that failed to collapse when the thorax was opened.  The lungs were
    fixed in an expanded state via the trachea.  In 100-m thick sections
    from formaldehyde-fixed dried lungs they reported "dilated" airspaces
    with "distorted architecture."  In those and other tissue
    preparations, they reported that the airspaces appeared "grossly
    enlarged and irregular, which appears to be due to disrupted alveoli
    ... and the absence of adjacent alveolar collapse."  Thus, in
    appropriately fixed lungs, they reported evidence of enlarged
    airspaces with destructive changes in alveolar walls.  Although no
    stereology was performed, this appears to be emphysema of the type
    seen in human lungs.

         Freeman et al. (1972) exposed rats to 37 600 g/m3 (20.0 ppm)
    NO2, which was reduced during the exposure to 28 200 g/m3
    (15.0 ppm) or to 18 800 g/m3 (10.0 ppm), for varying periods up to
    33 months. Following removal at necropsy, the lungs were fixed via the
    trachea at 25 cm of fixative pressure. Morphometry of lung and
    alveolar size was performed in a suitable, although unconventional,
    manner. The morphometry indicated enlargement of alveoli and reduction
    in alveolar surface area.  The authors also both reported alveolar
    destruction and illustrated alveolar destruction in their figures. 
    They correctly concluded that they had demonstrated emphysema in their
    NO2-exposed rats.  However, it is not entirely clear whether both
    experimental groups or only the group exposed to 28 200 g/m3
    (15.0 ppm) had emphysema.

        Table 36.  Effects of nitrogen dioxide (NO2) on the development of emphysemaa
                                                                                                                                              

    NO2 concentration
                                              

    g/m3                         ppm                  Exposure                           Species         Emphysemab    Reference
                                                                                                                                              

    188 with 2-h peaks to 1880    0.1 with             Daily, 6 months                    Mouse                        Port et al. (1977)
                                  peaks to 1.0

    263 plus 2050 g/m3 NO        0.14                 16 h/day, 68 months                Beagle dog      -             Hyde et al. (1978)
    1200 plus 310 g/m3 NO        0.64                                                                    +

    940                           0.5                  6, 18 or 24 h/day, 1-12 months     Mouse           -             Blair et al. (1969)

    1500                          0.8                  51-813 days                        Rat             -             Haydon et al. (1965)
    7520                          4.0

    1880 (with and without viral  1.0                  16 months                          Squirrel                     Ehrlich & Fenters (1973)
    challenge)                                                                            monkey

    3760                          2.0                  Continuous, 112-763 days           Rat             -             Freeman et al. (1968c)

    3760                          2.0                  8 h/day, 5 days/week               Hamster         -             Lafuma et al. (1987)
                                                       for 8 weeks

    9400                          5.0                  3 months                           Squirrel                     Ehrlich & Fenters (1973)
    18 800                        10.0                                                    monkey
                                                                                                                                              

    Table 36  (Con't)
                                                                                                                                              

    NO2 concentration
                                              

    g/m3                         ppm                  Exposure                           Species         Emphysemab    Reference
                                                                                                                                              

    9400                          5.0                  Up to 18 months                    Dog,            -             Wagner et al. (1965)
                                                                                          rabbit,
                                                                                          guinea-pig,
                                                                                          rat, hamster,
                                                                                          mouse

    15 000                        8.0                  3-4 months (presumably             Rabbit          +             Haydon et al. (1967)
    22 560                        12.0                 24 h/day)

    28 200                        15.0                 3-5 months                         Rat             -             Stephens et al. (1976)

    28 200                        15.0                 Continuously for 35 days then      Rat                          Port et al. (1977)
                                                       intermittently for at least
                                                       2 years

    33 800                        18.0                 24 h/day for 1-6 days or           Rat                          Freeman et al. (1968a)
                                                       4 weeks

    37 600 reduced to either      20.0 reduced to      Up to 33 months                    Rat             +             Freeman et al. (1972)
    28 200 or 18 800              15.0 or 10.0

    47 000                        25.0                 32-65 days                         Rat             -             Freeman & Haydon (1964)

    56 400                        30.0                 22 h/day, 12 months                Hamster         -             Kleinerman et al. (1985)

    56 400                        30.0                 Continuous, up to 140 days         Rat                          Glasgow et al. (1987)

    56 400                        30.0                 Continuous, up to 8 weeks          Rat             -             Blank et al. (1978)
                                                                                                                                              

    Table 36  (Con't)
                                                                                                                                              

    NO2 concentration
                                              

    g/m3                         ppm                  Exposure                           Species         Emphysemab    Reference
                                                                                                                                              

    56 400 to 65 800              30.0-35.0            23 h/day for 7 days                Hamster         -             Lam et al. (1983)

    65 800                        35.0                 6 h/day for 25 days                Rat             -             Stavert et al. (1986)

    75 200                        40.0                 6 or 8 weeks                       Mouse           -             Buckley & Loosli (1969)

    94 000 to 169 200 for         50-90 reduced        2 h/day, 5 days/week,              Hamster,                     Gross et al. (1968)
    4 weeks, reduced to 56 400    to 30-50             12 months                          guinea-pig
    to 94 000

    84 600 to 103 400             45-55                22-23 h/day, 10 weeks              Hamster         -             Kleinerman & Cowdrey
                                                                                                                        (1968)
                                                                                                                                              

    a  Modified from US EPA (1993)
    b  + = emphysema; - = no emphysema;  = equivocal
       Emphysema is defined according to the 1985 US National Heart, Lung, and Blood Institute Workshop criteria for human emphysema.
       Although many of the papers reviewed (US EPA, 1993) reported finding emphysema, some of these studies were reported according
       to previous, different criteria; some reports did not fully describe the methods used; and/or the results obtained were not in
       sufficient detail to allow independent confirmation of the presence of emphysema.  Thus, a "-" (i.e. no emphysema) should only be
       interpreted as lack of proof of emphysema, because it is conceivable that if the study were repeated with current methods and
       the current criteria applied, it might be judged to be positive.
             Hyde et al. (1978) studied beagle dogs that had been exposed 16 h
    daily for 68 months to either filtered air or to 1200 g/m3
    (0.64 ppm) NO2 with 310 g/m3 (0.25 ppm) NO or to 263 g/m3
    (0.14 ppm) NO2 with 2050 g/m3 (1.67 ppm) NO.  The dogs then
    breathed clean air during a 32- to 36-month post-exposure period.  The
    right lungs were fixed via the trachea at 30-cm fixative pressure
    in a distended state.  Semiautomated image analysis was used for
    morphometry of air spaces.  The dogs exposed to 1200 g/m3 NO2 with
    310 g/m3 NO had significantly larger lungs with enlarged air spaces
    and evidence of destruction of alveolar walls.  These effects were not
    observed in dogs exposed to 270 g/m3 NO2 with 2050 g/m3 NO,
    implying a significant role of the NO2 in the production of the
    lesions.  The lesions in dogs exposed to the higher NO2 concentration
    meet the criteria of the 1985 NHLBI workshop for emphysema of the type
    seen in human lungs.

    5.2.3  Genotoxicity, potential carcinogenic or co-carcinogenic effects

         NO2 forms nitrous and nitric acids in aqueous solutions, which
    are in equilibrium with the nitrite (NO2-) and nitrate (NO3-) ions
    that constitute the main metabolites of NO2.  Nitrous acid/NO2- is
    mutagenic  in vitro, causing deamination of bases in DNA.  The
    formation of  N-nitroso compounds from secondary amines and amides is
    another mechanism for indirect mutagenic activity (Zimmermann, 1977).

          In vitro studies with NO2 have demonstrated mutations in
    bacteria (Salmonella strain TA100) (Isomura et al., 1984; Victorin &
    Stahlberg, 1988) but not in a mammalian cell culture (Isomura et al.,
    1984).  Other experiments using cell cultures were positive concerning
    chromatid-type chromosome abberations, sister chromatid exchanges
    (SCE) and DNA single strand breaks (Tsuda et al., 1981; Shiraishi &
    Bandow, 1985; Gorsdorf et al., 1990).

         NO2 did not induce recessive lethal mutations or somatic
    mutations in Drosophila (Inoue et al., 1981; Victorin et al., 1990)
    and was negative in  in vivo studies with mice concerning chromosome
    abberations in peripheral lymphocytes or spermatocytes (Gooch et al.,
    1977) and micronuclei in bone marrow cells in mice (Victorin et al.,
    1990).

         Two studies have dealt with genotoxic effects in the relevant
    target organ, i.e. the lung, and both were positive at high
    concentrations. In the first one, Isomura et al. (1984) demonstrated
    the induction of mutations and chromosome abberations in lung cells of
    rats exposed to 27 ppm (50 000 g/m3) for 3 h.  In the other (Walles
    et al., 1995), DNA single strand breaks were induced in lung cells of
    mice exposed to 54 000 g/m3 (30 ppm) for 16 h or 94 000 g/m3
    (50 ppm) for 5 h.

         Several studies have evaluated the issue of carcinogenesis and
    co-carcinogenesis, but results are often unclear or conflicting
    (Table 37).  However, there do not appear to be any published reports
    on studies using classical carcinogenesis whole-animal bioassays.  An
    excellent critical review and discussion of some of the important
    theoretical issues in interpreting these types of studies has been
    published (Witschi, 1988).  Although lung epithelial hyperplasia
    (section 5.2.2.4) and enhancement of endogenous retrovirus expression
    (Roy-Burman et al., 1982) have been thought by some to suggest
    increased carcinogenic potential, such findings are not conclusive
    (see US EPA, 1993).

         Wagner et al. (1965) suggested that NO2 may accelerate the
    production of tumours in CAF1/Jax mice (a strain that has
    spontaneously high pulmonary tumour rates) after continuous exposure
    to 9400 g/m3 (5.0 ppm) NO2.  After 12 months of exposure, 7 out of
    10 mice in the exposed group had tumours, compared to 4 of 10 in the
    controls.  No differences in tumour production were observed after 14
    and 16 months of exposure.  A statistical evaluation of the data was
    not presented.  The frequency and incidence of spontaneously
    occurring pulmonary adenomas was increased in strain A/J mice (with
    spontaneously high tumour rates) after exposure to 18 800 g/m3
    (10.0 ppm) NO2 for 6 h/day, 5 days/week, for 6 months (Adkins et al.,
    1986).  These small, but statistically significant, increases were
    only detectable when the control response from nine groups (n = 400)
    were pooled.  Exposure to 1880 and 9400 g/m3 (1.0 and 5.0 ppm) NO2
    had no effect.  In contrast, Richters & Damji (1990) found that an
    intermittent exposure to 470 g/m3 (0.25 ppm) NO2 for up to 26 weeks
    decreased the progression of a spontaneous T cell lymphoma in
    AKR/ cum mice and increased survival rates.  The investigators
    attribute this effect to an NO2-induced decrease in the proliferation
    of T cell subpopulation (especially T-helper/inducer lymphocytes) that
    produce growth factors for the lymphoma.

         Whether NO2 facilitates metastases has been the subject of
    several experiments by Richters & Kuraitis (1981, 1983), Richters &
    Richters (1983) and Richters et al. (1985). Mice were exposed to
    several concentrations and durations of NO2 and were injected
    intravenously with a cultured-derived melanoma cell line (B16) after
    exposure; subsequent tumours in the lung were counted.  Although
    some of the experiments showed an increased number of lung
    tumours, statistical methods were inappropriate.  Furthermore, the 
    experimental technique used in these studies probably did not evaluate
    metastases formation, as the term is generally understood, but more
    correctly, colonization of the lung by tumour cells.

        Table 37.  Effects of nitrogen dioxide (NO2) on carcinogenesis or co-carcinogenesisa
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    188-18 800     0.1-10.0     0.5-4 h               Mouse     Mice exposed to DMA had whole-body concentration-           Iqbal et al.
                                                                related increase in DMN.                                    (1981)

    470            0.25         7 h/day,              Mouse     NO2 slowed progression of spontaneous T cell                Richters & Damji
                                5 days/week,                    lymphomas in AKR/cum mice, increased survival, and          (1990)
                                up to 26 weeks                  decreased number of splenic CD4+ T cells.

    752            0.4          7-8 h/day,            Mouse     Increased lung tumors and mortality in mice injected        Richters &
    940            0.5          5 days/week,                    with melanoma cells after NO2 exposure.                     Kuraitis (1981,
    1500           0.8          12 weeks                                                                                    1983); Richters
                                                                                                                            et al. (1985)

    940-1500       0.5-0.8      Continuous,           Mouse     Hyperplastic foci identical to that observed after          Nakajima et
                                30 days                         exposure to known carcinogens.                              al. (1972)

    1500           0.8          8 h/day,              Mouse     Enhanced retrovirus expression in two strains of            Roy-Burman
                                5 days/week,                    mice.                                                       et al. (1982)
                                18 weeks

    1880           1.0          6 h/day,              Mouse     No effect at 1880 or 9400 g/m3. At 18 800 g/m3,           Adkins et al.
    9400           5.0          5 days/week,                    spontaneous adenomas in strain A/J mice increased           (1986)
    18 800         10.0         6 weeks                         only when compared to pooled control group.

    2000           1.1          Continuous,           Rat       DMA plus NO2 did not produce tumors. Design and             Benemansky
    3010           1.6          lifetime                        statistical analyses not appropriate; exposure              et al. (1981)
                                                                methods not described.
                                                                                                                                              

    Table 37  (Con't)
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    9400-18 800    5.0-10.0     2 h/day,              Mouse     Mice given 4-nitroquinoline-1-oxide during NO2              Ide & Otsu
                                5 days/week,                    exposure; NO2 had no effect on tumor production.            (1973)
                                50 weeks

    18 800         10.0         2 h/day,              Mouse     Mice given 4-nitroquinoline-1-oxide and NO2.                Otsu & Ide
                                5 days/week,                    NO2 decreased incidence of lung tumors.                     (1975)
                                50 weeks

    28 200-94 000  15.0-50.0    1-4 h                 Mouse     Mice gavaged with morpholine had concentration-             Iqbal et al.
                                                                dependent increase in whole-body content of NMOR.           (1980)

    31 020-38 500  16.5-20.5    5-6 h/day,            Mouse     In vivo production of NMOR when 1 g/kg of morpholine        Van Stee et
                                4 days; plus                    was administered each day prior to exposure.                al. (1983)
                                3 h on 5th day

    84 600         45.0         2 h                   Mouse     Mice gavaged with morpholine had an in vivo increase        Norkus et al.
                                                                in NMOR production.                                         (1984)

    199 000        106.0        0.5-4 h               Rat       Rats given morpholine in their diets or by gavage had       Mirvish et al.
                                                      Mouse     no NMOR detected in their bodies.                           (1981)
                                                                In mice morpholine, by gavage, yielded no significant
                                                                in vivo NMOR production.
                                                                                                                                              

    a  Modified from US EPA (1993)
    b  DMA = Dimethylamine; DMN = Dimethylnitrosamine; NMOR = N-nitrosomorpholine
             Ide & Otsu (1973) did not find that chronic exposure to high
    concentrations of NO2 (somewhere between 9400 and 18 800 g/m3,
    5.0 and 10.0 ppm) enhanced tumour production in conventional mice
    receiving five weekly injections of 0.25 mg 4-nitroquinoline-1-oxide
    (a lung-tumour-specific carcinogen).  Benemansky et al. (1981) used a
    known carcinogen, nitrosodimethylamine or its precursor dimethylamine
    (DMA) to test for interactions with a chronic exposure to NO2.
    However, appropriate statistical techniques and control groups were
    not employed and the methods of exposure and monitoring of NO2 were
    not reported, thus precluding accurate evaluation.  In another study,
    rats were injected with  N-bis (2-hydroxy-propyl)nitrosamine (BHPN)
    and continuously exposed to 75, 750 or 7500 g/m3 (0.04, 0.4 or
    4.0 ppm) NO2 for 17 months.  Although the data indicated five times
    as many lung adenomas or adenocarcinomas in the rats injected with
    BHPN and exposed to 7500 g/m3 NO2 (5/40 compared to 1/10), the
    results failed to achieve statistical significance (Ichinose et al.,
    1991).

         Because of evidence that NO2 could produce NO2- and NO3- in
    the blood and the fact that NO2- is known to react with amines to
    produce animal carcinogens (nitrosamines), the possibility that NO2
    could produce cancer via nitrosamine formation has been investigated. 
    Iqbal et al. (1980) was the first to demonstrate a linear time-
    dependent and concentration-dependent relationship between the amount
    of  N-nitrosomorpholine (NMOR) (an animal carcinogen) found in
    whole-mouse homogenates after the mice were gavaged with 2 mg of
    morpholine (an exogenous amine that is rapidly nitrosated) and
    exposure to 28 200 to 94 000 g/m3 (15.0 to 50.0 ppm) NO2 for
    between 1 and 4 h.  In a follow-up study, Iqbal et al. (1981) used
    DMA, an amine that is slowly nitrosated to dimethylnitrosamine (DMN). 
    They reported a concentration-related increase in biosynthesis of DMN
    at NO2 concentrations as low as 188 g/m3 (0.1 ppm); however, the
    rate was significantly greater at concentrations above 18 800 g/m3
    (10.0 ppm) NO2.  Increased length of exposure also increased DMN
    formation between 0.5 and 2 h, but synthesis of DMN was less after 3
    or 4 h of exposure than after 0.5 h.

         Mirvish et al. (1981) conducted analogous research and concluded
    that the results of Iqbal et al. (1980) were technically flawed, but
    found that  in vivo exposure to NO2 could produce a nitrosating
    agent (NSA) that would nitrosate morpholine only when morpholine was
    added  in vitro.  Further experiments showed that the NSA was
    localized in the skin (Mirvish et al., 1983) and that mouse skin
    cholesterol was a likely NSA (Mirvish et al., 1986).  It has also been
    reported that only very lipid-soluble amines, which can penetrate the
    skin, would be available to the NSA.  Compounds such as morpholine,
    which are not lipid-soluble, could only react with NO2 when it was
    painted directly on the skin (Mirvish et al., 1988).  Iqbal (1984),
    responding to the Mirvish et al. (1981) criticisms, verified their
    earlier studies (Iqbal et al., 1980).   In vivo nitrosation was also
    demonstrated by Norkus et al. (1984) after morpholine administration
    and a 2-h exposure to 84 600 g/m3 (45 ppm) NO2.

         Another study (Van Stee et al., 1983) reported that mice gavaged
    with 1 g of morpholine/kg body weight per day and then exposed (5-6 h
    daily for 5 days) to 31 000 to 38 500 g/m3 (16.5 to 20.5 ppm) NO2
    revealed that NMOR could be produced  in vivo.  The single site
    containing the greatest amount of NMOR was the gastrointestinal tract.

         Shoaf et al. (1989) studied the uptake and nitrosation of primary
    amines by NO2 in isolated ventilated rat lungs.  The rate of
    nitrosation was very low because the nitrosation of primary amines is
    a general acid/base catalysed reaction that would be at a minimum at
    pH 7.  The authors could not replicate the previous nitrosation
    studies.  At a maximum, only 0.0001% of an amine would be nitrosated. 
    Such a rate is at or below the detection limit for nitrosamine.  The
    studies reporting nitrosation may be seriously in error.  Nitrosation
    may be a very minimal reaction and of little consequence.

         Victorin (1994) reviewed the genotoxicity of nitrogen oxides and
    concluded that there is no clear evidence of a carcinogenic potential
    of NO2.  Victorin (1994) also directed attention to the possibility
    that NOx compounds in photochemical smog may contribute secondarily
    to formation of other genotoxic compounds.  For example, it was noted
    that strongly mutagenic nitro-PAH compounds are easily formed and
    mutagenic reaction products may be formed from alkenes through
    photochemical reactions.

         Overall, the above critical evaluation indicates that there is no
    evidence establishing that tumours can be directly induced by NO2
    exposure alone.  Also, the available evidence for NO2 promoting or
    enhancing the production or growth of tumours caused by other agents
    is quite limited and conflicting.  It must therefore be concluded that
    the evidence for carcinogenicity of nitrogen oxides is at present
    inadequate, but the issue should be addressed by further research.

    5.2.4  Extrapulmonary effects

         Exposure to NO2 produces a wide array of health effects beyond
    the confines of the lung.  Thus, NO2 and/or some of its reactive
    products penetrate the lung or nasal epithelial and endothelial layers
    to enter the blood and produce alterations in blood and various other
    organs (Shoaf et al., 1989).  Effects on the systemic immune system
    are discussed under section 5.2.2.1.  Information regarding the
    effects of NO2 on animal behaviour and brain enzymes is limited to a
    few studies that cannot be readily interpreted in terms of human risks
    and will not be discussed.  The summary of other systemic effects is
    quite brief because the database suggests that effects on the
    respiratory tract are of more concern.  A more detailed discussion of
    extrapulmonary responses can be found in US EPA (1993).

         Results of research on the number of erythrocytes and leukocytes,
    haemoglobin concentration, and contents of erythrocyte membranes are
    inconsistent.  In the only such study conducted below 9400 g/m3
    (5.0 ppm) NO2, Nakajima & Kusumoto (1968) found that the amount of
    methaemoglobin was not increased when mice were exposed to 1500 g/m3
    (0.8 ppm) NO2 for 5 days.  This topic was of interest because some
    (but not all)  in vitro studies and high concentration  in vivo NO2
    studies found methaemoglobin effects (US EPA, 1993).

         Several studies have examined hepatic function either directly or
    indirectly after NO2 exposure.  Changes in serum chemistry (e.g.,
    plasma cholinesterase, Drozdz et al., 1976; Menzel et al., 1977)
    suggest that NO2 exposure may affect the liver.  Xenobiotic
    metabolism appears to be affected by NO2.  A 3-h exposure to NO2
    concentrations as low as 470 g/m3 (0.25 ppm) increased
    pentobarbital-induced sleeping times in female, but not male, mice
    (Miller et al., 1980; Graham et al., 1982).  Higher exposures
    (9400 g/m3, 5.0 ppm; 3 h) did not affect the level of hepatic
    cytochrome P-450 or the activities of several mixed-function oxidases
    in mice (Graham et al., 1982).  Other authors found mixed effects
    (i.e. increase or decrease depending on exposures) on liver cytochrome
    P-450 levels in rats (Takano & Miyazaki, 1984; Takahashi et al.,
    1986).  Significant decreases in cytochrome P-450 from rat liver
    microsomes were also found after 7 days of exposure to 752 or
    7520 g/m3 (0.4 or 4.0 ppm) NO2, but not after exposure to
    2260 g/m3 (1.2 ppm) NO2 (Mochitate et al., 1984).  NADPH-cytochrome
    C reductase was reduced with 5 days of exposure to 7520 and
    18 800 g/m3 (4.0 and 10.0 ppm) NO2.  Drozdz et al. (1976) found
    decreased total liver protein and sialic acid, but increased protein-
    bound hexoses in guinea-pigs exposed to 2000 g/m3 (1.05 ppm) NO2,
    8 h/day for 180 days.  Liver alanine and aspartate aminotransferase
    activity was increased in the mitochondrial fraction but decreased in
    the cytoplasmic fraction of the liver.  Electron micrographs of the
    liver showed intracellular oedema and inflammatory and parenchymal
    degenerative changes.

         Takahashi et al. (1986) found that continuous exposure to 2260
    and 7520 g/m3 (1.2 and 4.0 ppm) NO2 increased the amount of
    cytochrome P-450 and cytochrome b5 in the kidney after 8 weeks of
    exposure.  Continued exposure for 12 weeks resulted in less
    substantial increases in the amount and activity of the microsomal
    electron-transport enzymes.  This is in contrast to the decreased
    activity in the liver.

         Increases in urinary protein and specific gravity of the urine
    were reported by Sherwin & Layfield (1974) in guinea-pigs exposed
    continuously to 940 g/m3 (0.5 ppm) NO2 for 14 days.  Proteinuria
    was detected in another group of animals when the exposure was reduced
    to 752 g/m3 (0.4 ppm) NO2 for 4 h/day.  Disc electrophoresis of the

    urinary proteins demonstrated the presence of albumin and alpha-,
    beta-, and gamma-globulins.  The presence of high molecular weight
    proteins in urine is characteristic of the nephrotic syndrome. 
    Differences in water consumption or in the histology of the kidney
    were not found.

         Few studies have examined the effects of NO2 on reproduction and
    development or the heritable mutagenic potential of NO2.  Exposure to
    1800 g/m3 (1.0 ppm) NO2 for 7 h/day (5 days/week for 21 days)
    resulted in no alterations in spermatogenesis, germinal cells or
    interstitial cells of the testes of six rats (Kripke & Sherwin, 1984). 
    Similarly, breeding studies by Shalamberidze & Tsereteli (1971) found
    that long-term NO2 exposure had no effect on fertility.  However,
    there was a statistically significant decrease in litter size and
    neonatal weight when male and female rats exposed to 2440 g/m3
    (1.3 ppm) NO2, 12 h/day for 3 months were bred.   In utero death due
    to NO2 exposure resulted in smaller litter sizes, but no direct
    teratogenic effects were observed in the offspring.  In fact, after
    several weeks, NO2-exposed litters approached weights similar to
    those of controls.

         Inhalation exposure of pregnant Wistar rats to NO2
    concentrations of 1000 and 10 000 g/m3 for 6 h/day throughout
    gestation (21 days) was found to have maternal toxic effects and to
    induce developmental disturbances in the progeny (Tabacova et al.,
    1984; Balabaeva & Tabacova, 1985; Tabacova & Balabaeva, 1988). The
    maternal weight gain during gestation was significantly reduced at
    10 000 g/m3 (5.3 ppm).  Pathomorphological changes, manifested at
    the higher exposure level, were found in maternal organs, e.g.,
    desquamative bronchitis and bronchiolitis in the lung, mild
    parenchymal dystrophy and reduction of glycogen in the liver, and
    blood stasis and inflammatory reaction in the placenta.  At gross
    examination, the placentae of the dams exposed to 10 000 g/m3 were
    smaller in size than those of control rats.  A marked increase of
    lipid peroxides was found in maternal lungs and particularly in the
    placenta at both exposure levels by the end of gestation (Balabaeva &
    Tabacova, 1985).  Disturbances in the prenatal development of the
    progeny were registered, such as two- to four-fold increase in late
    post-implantation lethality at 1000 and 10 000 g/m3 (0.5 and
    5.3 ppm), respectively, as well as reduced fetal weight at term and
    stunted growth at 10 000 g/m3 (Tabacova et al., 1984).  These
    effects were significantly related to the content of lipid peroxides
    in the placenta, which was suggestive of a pathogenetic role of
    placental damage (Tabacova & Balabaeva, 1988).  Teratogenic effects
    were not observed, but dose-dependent morphological signs of
    embryotoxicity and retarded intrauterine development, such as
    generalized oedema, subcutaneous haematoma, retarded ossification
    and skeletal aberrations, were found at both exposure levels.

         In the only study that has examined postnatal development, a
    significant delay in eye opening and incisor eruption was observed in
    the progeny of maternally exposed Wistar rats (Tabacova et al., 1985). 
    The dams were exposed to 50, 100, 1000 or 10 000 g/m3 (0.03, 0.05,
    0.53 or 5.3 ppm) NO2 for 6 h/day, 7 days/week throughout gestation,
    and the offspring were studied for 2-month post-exposure.  Significant
    deficits in the onset of normal neuromotor development and reduced
    open field activity were detected in the offspring of dams exposed to
    1000 and 10 000 g/m3 NO2.

    5.3  Effects of mixtures containing nitrogen dioxide

         Humans are exposed to pollutant mixtures in the ambient air, and,
    because pollutant interactions do occur, it is difficult to predict
    the effects of NO2 in a mixture based upon the effects of NO2 alone.
    Epidemiological studies (chapter 7), by their very nature, evaluate
    ambient air mixtures, but the presence of confounding variables makes
    it difficult to demonstrate a cause-effect relationship.  In contrast,
    controlled animal and human clinical studies can often demonstrate the
    cause of a response, but are typically limited to binary or tertiary
    mixtures, which do not truly reflect ambient air exposures.  When
    combinations of air pollutants are studied, there are a number of
    possible outcomes on human or animal responses.  The result of
    exposure to two or more pollutants may be simply the sum of the
    responses to individual pollutants; this is referred to as additive. 
    Another possibility is that the resultant response may be greater than
    the sum of the individual responses, suggesting some type of
    interaction or augmentation of the response; this is referred to as
    synergism.  Finally, responses may be less than additive; this is
    often called antagonism. Generally, such human clinical studies, which
    focused on pulmonary function, have not found that acute exposures to
    NO2 has any impact on the response to other co-occurring pollutants
    (e.g., O3) or that additive effects occur.  Animal toxicological
    studies, with a wider array of designs and end-points, have shown an
    array of interactions, including no interaction, additivity and
    synergism.  Because no clear understanding of NO2 interactions has
    yet emerged from this database, only a brief overview is provided
    here.  A more substantive review can be found in US EPA (1993).  Other
    animal studies sought to understand the effects of ambient air
    mixtures containing NO2 or vehicular combustion exhausts containing
    NOx.  Generally these studies provide useful information on the
    mixtures, but lack NO2-only groups, making it impossible to discern
    the influence of NO2.  Therefore, this class of research is not
    described here, but is reviewed elsewhere (US EPA, 1993).

         The vast majority of interaction studies have involved NO2 and
    O3.  For lung morphology end-points, NO2 had no interaction with O3
    (Freeman et al., 1974) or with sulfur dioxide (SO2) (Azouley et al.,
    1980) after a subchronic exposure. Some biochemical responses to NO2

    plus O3 display no positive interaction or synergism.  For example,
    Mustafa et al. (1984) found synergism for some end-points (e.g.,
    increased activities of O2 consumption and antioxidant enzymes),
    but no interaction for others (e.g., DNA or protein content)
    in rats exposed for 7 days.  Ichinose & Sagai (1989) observed a
    species-dependence in regard to the interaction of O3 (752 g/m3,
    0.4 ppm) and NO2 (752 g/m3, 0.4 ppm) after 2 weeks of exposure. 
    Guinea-pigs, but not rats, had a synergistic increase in lung lipid
    peroxides.  Rats, but not guinea-pigs, had synergistic increases in
    antioxidant factors (e.g., non-protein thiols, vitamin C, glucose-6-
    phosphate dehydrogenase, GSH peroxidase).  Schlesinger et al. (1990)
    observed a synergistic increase in prostaglandin E2 and F2 alpha in the
    lung lavage of acutely exposed rabbits; the response appeared to have
    been driven by O3.  However, with 7 or 14 days of repeated 2-h
    exposures, only prostaglandin E2 was decreased and appeared to have
    been driven by NO2; there was no synergism (Schlesinger et al.,
    1991).

         The infectivity model has been frequently used to study NO2-O3
    mixtures.  In this model, mice are exposed to O3 and NO2 alone or in
    mixtures for various durations.  The mice are then challenged with an
    aerosol of viable bacteria.  An increase in mortality indicates
    detrimental effects on lung host-defence mechanisms.  Ehrlich et al.
    (1977) found additivity after acute exposure to mixtures of NO2 and
    O3.  They reported synergism after subchronic exposures.  Exposure
    scenarios involving NO2 and O3 have also been performed using a
    continuous baseline exposure to one concentration or mixture, with
    superimposed short-term peaks to a higher level.  This body of work
    (Ehrlich et al., 1979; Gardner, 1980; Gardner et al., 1982; Graham et
    al., 1987) shows that differences in the pattern and concentrations of
    the exposure are responsible for the increased susceptibility to
    pulmonary infection, without indicating clearly the mechanism
    controlling the interaction.

         Some aerosols may potentiate response to NO2 by producing local
    changes in the lungs that enhance the toxic action of co-inhaled NO2. 
    The impacts of NO2 and H2SO4 on lung host defences have been
    examined by Schlesinger & Gearhart (1987) and Schlesinger (1987a).  In
    the former study, rabbits were exposed for 2 h/day for 14 days to
    either 564 g/m3 (0.3 ppm) or 1880 g/m3 (1.0 ppm) NO2, or
    500 g/m3 H2SO4 alone, or to mixtures of the low and high NO2
    concentrations with H2SO4.  Exposure to either concentration of NO2
    accelerated alveolar clearance, whereas H2SO4 alone retarded
    clearance.  Exposure to either concentration of NO2 with H2SO4
    resulted in retardation of clearance in a similar manner to that seen
    with H2SO4 alone.

         Schlesinger (1987a) used a similar exposure design, but different
    end-points.  Exposure to 1800 g/m3 (1.0 ppm) NO2 with acid resulted
    in an increase in the numbers of PMNs in lavage fluid at all time
    points (not seen with either pollutant alone), and an increase in
    phagocytic capacity of AMs after two or six exposures.  In contrast,
    exposure to 564 g/m3 (0.3 ppm) NO2 with acid resulted in depressed
    phagocytic capacity and mobility.  The NO2/H2SO4 mixture was
    generally either additive or synergistic, depending on the specific
    cellular end-point being examined.

         Last et al. (1983) and Last & Warren (1987) found that exposure
    to high levels of NO2 (< 9400 g/m3, 5.0 ppm) with very high
    concentrations of H2SO4 (1 mg/m3) caused a synergistic increase in
    collagen synthesis rate and protein content of the lavage fluid of
    rats.

         Dogs were exposed for 68 months (16 h/day) to raw or
    photochemically reactive vehicle exhaust which included mixtures of
    NOx  one with a high NO2 level and a low NO level (1200 g/m3,
    0.64 ppm, NO2; 310 g/m3, 0.25 ppm, NO), and one with a low NO2
    level and a high NO level (270 g/m3, 0.14 ppm, NO2; 2050 g/m3,
    1.67 ppm, NO) (Stara et al., 1980).  Following the end of exposure,
    the animals were maintained for about 3 years in normal indoor air. 
    Numerous pulmonary function, haematological and histological
    end-points were examined at various times during and after exposure. 
    The lack of an NO2-only or NO-only group precludes determination of
    the nature of the interaction.  Even so, the main findings are of
    interest.  Pulmonary function changes appeared to progress after
    exposure ceased.  Dogs in the high NO2 group had morphological
    changes considered to be analogous to human centrilobular emphysema
    (see section 2.2.2.4).  Because these morphological measurements were
    made after a 2.5- to 3-year holding period in clean air, it cannot be
    determined with certainty whether these disease processes abated or
    progressed during this time.  This study suggests progression of
    damage after exposure ends.

    5.4  Effects of other nitrogen oxide compounds

    5.4.1  Nitric oxide

         The toxicological database for NO is small, relative to NO2.  It
    is often difficult to obtain pure NO in air without some contamination
    with NO2.  An excellent review on the effects of NO on animals and
    humans has been prepared by Gustafsson (1993) for the Swedish
    Environmental Protection Agency.  The following sections are based on
    the information in this review.

    5.4.1.1  Endogenous formation of NO

         Endogenous NO synthesis occurs by NO formation from physiological
    substrate (the amino acid L-arginine) in cells of many of the organ
    systems, such as nerve tissue, blood vessels and the immune system. 
    NO has been found to be produced by at least three different
    oxygen-utilizing NO synthases, for purposes such as signalling in the
    nervous system, mediating vasodilation in both systemic and pulmonary
    circulation, and mediating cytotoxicity and host defence reactions in
    the immune system (Garthwaite, 1991; Barinaga, 1991; Moncada et al.,
    1991; McCall & Valance, 1992; Snyder & Bredt, 1992; Moncada, 1992). 
    The impact of these findings for an understanding of the toxicological
    effects of NO is still difficult to assess.

         The actions of endogenous NO can be divided into two main groups. 
    The first group involves low concentrations of NO (nano- to picomolar)
    formed by constitutive enzymes in nerve and endothelial cells.  Nitric
    oxide has local discrete actions exerted via activation of an enzyme,
    guanylate cyclase, in the target cell (Ignarro, 1989).  The second
    group involves high concentrations of NO (micro- to nanomolar) formed
    by enzymes that can increase in amount through the induction of these
    enzymes upon exposure to bacterial toxins or to growth-regulating
    factors (cytokinins).  The inducible NO formation occurs especially in
    macrophages and neutrophil leukocytes and is important for the killing
    of bacteria and parasites, and possibly also for cytostasis in
    antitumour reactions (Hibbs et al., 1988; Ignarro, 1989; Moncada et
    al., 1991; Moncada, 1992).

         For effects of inhaled NO it is important to consider that 
    endogenous NO regulates pulmonary vascular resistance; it is found in
    small amounts in exhaled air and has been suggested to be necessary
    for normal oxygenation of the blood (Persson et al., 1990; Gustafsson
    et al., 1991).

    5.4.1.2  Absorption of NO

         Yoshida et al. (1981) found that < 10% of the NO "inhaled" by
    isolated perfused lungs of rabbits was absorbed.  In normally
    breathing humans, 85 to 92% of NO was absorbed at concentrations
    ranging from 400 to 6100 g/m3 (0.33 to 5.0 ppm) (Wagner, 1970;
    Yoshida & Kasama, 1987); values for NO2 were 81 to 90% (Wagner,
    1970).  Absorption of NO with exercise was 91 to 93% in humans
    (Wagner, 1970).  Yoshida et al. (1980) found the percentage of
    absorption of NO in rats acutely exposed to 169 300 g/m3 (138 ppm),
    331 300 g/m3 (270 ppm) and 1 079 800 g/m3 (880 ppm) to be 90%, 60%
    and 20%, respectively.  The progressive decrease in absorption was
    ascribed to an exposure-induced decrease in ventilation.  In dogs
    exposed to vehicle exhaust mixtures, 73% of the constituent NO was
    removed by the nasopharyngeal region; this compared to 90% removal for

    NO2 (Vaughan et al., 1969).  Thus, respiratory tract absorption of NO
    has some similarities to that for NO2, in spite of solubility
    differences.  The lower solubility of NO may, however, result in
    greater amounts reaching the pulmonary region, where it may then
    diffuse into blood and react with haemoglobin (Yoshida & Kasama,
    1987).   In vivo exposures seem to indicate that NO has a faster rate
    of diffusion through tissue than NO2 (Chiodi & Mohler, 1985).

    5.4.1.3  Effects of NO on pulmonary function, morphology and host lung
             defence function

         No change in respiratory function was found in guinea-pigs
    exposed to NO at 19 600 g/m3 (16 ppm) or 61 300 g/m3 (50 ppm) for
    4 h (Murphy et al., 1964).  Increased airway responsiveness to
    acetylcholine was observed in guinea-pigs exposed to 6130 g/m3
    (5 ppm) NO for 30 min, twice a week for 7 weeks.  In sheep,
    significant reversal of vasoconstriction to an infused thromboxane
    analogue was seen with acute exposure to 6130 g/m3 NO (Fratacci et
    al., 1991).  At the same exposure level, hypoxic vasoconstriction was
    significantly diminished and was nearly abolished at 49 000 g/m3
    (40 ppm) NO in inhaled air (Frostell et al., 1991).

         Reversal of methacholine-induced bronchoconstriction by NO has
    been reported in guinea-pigs at 6130 g/m3 (5 ppm) (Dupuy et
    al., 1992), while in rabbits full reversal of methacholine
    bronchoconstriction was seen at 98 100 g/m3 (80 ppm) (Hgman et
    al., 1993).  Relaxation of bronchial smooth muscle can be exerted
     in vitro by mechanisms dependent on an intact airway epithelium.  An
    endogenous muscle-relaxing factor released by the epithelium has been
    suggested, but it is not clear whether it is endogenous NO (Barnes,
    1993).

         The few studies that have examined histological response to
    non-lethal levels of NO are outlined in Table 38.  With chronic
    exposure, the morphological changes seen are similar to those with
    NO2 (see section 5.2.2.4 on morphological effects of NO2), except
    that NO levels needed to produce them are higher.  Additionally, Hugod
    (1979) noted that the absence of  NO-induced alterations in the
    alveolar epithelium suggested that the observed responses occurred
    after absorption of NO; that is, they were not caused by direct action
    of deposited NO.  Perhaps higher exposure concentrations of NO are
    needed for direct toxic action (e.g., results of Holt et al., 1979). 
    Some of the effects seen by Oda et al. (1976) with 12 270 g/m3
    (10.0 ppm) NO may have been due to the presence of 1880 to 2820 g/m3
    (1.0 to 1.5 ppm) NO2 in the exposure atmosphere.

         It is important to note that in all existing studies of NO
    toxicity in the lungs, histological evaluation of the lungs was
    rudimentary and no quantitative measurements were carried out to test
    for airspace enlargement or destruction.

        Table 38.  Effects of nitric oxide (NO) on respiratory tract morphologya
                                                                                                                                              

    NO2 Concentration
                      

    g/m3        ppm          Exposure              Species     Effectsb                                                    Reference
                                                                                                                                              

    2460           2                        Continuous,         Rat       Slight emphysema-like alterations of alveoli.     Azoulay et
                   (NO2 = 0.08 ppm)b        6 weeks                                                                         al. (1977)

    2950           2.4                      Continuous,         Mouse     No difference from control.                       Oda et al.
                   (NO2 = 0.01-0.04 ppm)b   for lifetime                                                                    (1980b)
                                            (23-29 months)

    6150           5                        Continuous,         Rabbit    Oedema; thickening of alveolo-capillary           Hugod (1979)
                   (NO2 = < 0.1 ppm)b       14 days                       membrane due to fluid in interstitial space;
                                                                          fluid-filled vacuoles seen in arteriolar
                                                                          endothelial cells and at junctions of
                                                                          endothelial cells; no changes in alveolar
                                                                          epithelium; no inflammation.

    12 300         10                       2 h/day, 5 days     Mouse     Enlarged air spaces in lung periphery;            Holt et al.
                                            per week, up to               paraseptal emphysema; some haemorrhage;           (1979)
                                            30 weeks                      some congestion in alveolar septa; increased
                                                                          concentration of goblet cells in bronchi.

    12 300         10                       Continuous,         Mouse     Bronchiolar epithelial hyperplasia; hyperaemia;   Oda et al.
                   (NO2 = 1-1.5 ppm)b       6.5 months                    congestion; enlargement of alveolar septum;       (1976)
                                                                          increase in ratio of lung to body weight.
                                                                                                                                              

    a  Modified from US EPA (1993)
    b  This represents reported nitrogen dioxide (NO2) levels measured during exposure
             A recent study (Mercer et al., 1995) suggests that NO may be more
    potent than NO2 in introducing certain changes in lung morphology. 
    More specifically, male rats were exposed to either NO or NO2 at
    0.5 ppm with twice daily 1-h spikes of 1.5 ppm for 9 weeks.  The
    number of pores of Kohn and detached alveolar septa were evaluated by
    electron microscopy, using stereological procedures for the study of
    lung structure that involved morphometric analyses of electron
    micrographs.  The average number of pores per lung for the NO group
    exceeded by approx. 2.5 times the mean number for the NO2 groups,
    which was more than 10 times that for controls.  Analogously, the
    average number of detached septa per lung was significantly higher for
    the NO group (X = 117) than the NO2 group (X = 20) or the controls
    (X = 4).  There was also a statistically significant 30% reduction in
    interstitial cells in the NO group, but no significant differences in
    the other parenchymal cell types between the controls and the NO- or
    NO2-exposed groups.  Lastly, the thickness of the interstitial space
    was reduced for the NO group (X = 0.24 m versus 0.32 m for controls)
    but not for the NO2 group (X = 0.29 m), and epithelial cell
    thickness did not differ between the groups.

         The effects of NO on host defence function of the lungs has been
    examined in two studies.  Holt et al. (1979) found immunological
    alterations in mice exposed to 12 270 g/m3 (10 ppm) NO for 2 h/day
    (5 days/week for 30 weeks).  However, interpretation is complicated by
    the duration dependence of some of the responses (e.g., an enhancement
    of the humoral immune response to SRBCs was seen at 10 weeks, but this
    was not evident at the end of the exposure series).  The effects of NO
    on bacterial defences were examined by Azoulay et al. (1981).  Male
    and female mice were exposed continuously to 3760 g/m3 (2.0 ppm) NO
    for 6 h to 4 weeks to assess the effect on resistance to infection
    induced by a bacterial aerosol administered after each NO exposure. 
    There were no statistically significant effects for either sex at any
    of the time points studied.

    5.4.1.4  Metabolic effects

         Mice exposed to NO concentrations of 12 300 to 25 800 g/m3
    (10 to 21 ppm) for 3 h daily for 7 days showed no change in the
    levels of reduced glutathione in their lungs (Watanabe et al., 1980). 
     In vitro data indicate that NO stimulates guanylate cyclase and
    therefore leads to smooth muscle relaxation and vasodilation and
    functional effects on the nervous system (Katsuki et al., 1977;
    Ignarro, 1989; Garthwaite, 1991; Moncada et al., 1991).  These effects
    are probably responsible for vasodilation in the pulmonary circulation
    and an acute bronchodilator effect of inhaled NO.  However, it is
    unclear whether other effects might be exerted from ambient NO via
    this pathway.  Due to the rapid inactivation of NO in haemoglobin,
    internal organs other than the lungs are unlikely to be affected
    directly by cyclic GMP-mediated vasodilator influence from ambient
    concentrations of NO.

         Methaemoglobin formation, via the formation of nitrosylhaemoglobin
    (Oda et al., 1975, 1979, 1980a,b; Case et al., 1979; Nakajima et al.,
    1980) and subsequent oxidation with oxygen, is well known (Kon et al.,
    1977; Chiodi & Mohler, 1985).  During NO exposure of mice to 24 500 to
    98 100 g/m3 (20-80 ppm), the levels of methaemoglobin were found
    to increase exponentially with the NO concentration (Oda et al.,
    1980b).  After the cessation of NO exposure, methaemoglobin decreased
    rapidly, with a half-time of only a few minutes.  In humans the ability
    to reduce methaemoglobin varies genetically and is lower in infants. 
    Of the NO reaction products with haemoglobin, methaemoglobin attains
    higher levels than nitrosylhaemoglobin (Maeda et al., 1987).  Exposure
    of mice to 2940 g/m3 (2.4 ppm) NO for 23-29 months resulted in
    nitrosylhaemoglobin levels at 0.01%, while the maximal methaemoglobin
    level was 0.3% (Oda et al., 1980b).  At 12 300 g/m3 for 6.5
    months the nitrosylhaemoglobin level was 0.13% and the level of
    methaemoglobin was 0.2% (Oda et al., 1976).  Rats exposed to
    2450 g/m3 (2 ppm) continuously for six weeks showed no detectable
    methaemoglobin (Azoulay et al., 1977).

    5.4.1.5  Haematological changes

         Mice exposed to NO at 11 070 g/m3 (9 ppm) for 16 h had
    decreased iron transferrin (Case et al., 1979), and when exposed to
    12 300 g/m3 (10 ppm) for 6.5 months had increased leukocyte count
    and proportion of polymorphonuclear cells (Oda et al., 1976).  Red
    blood cell morphology, spleen weight and bilirubin were also affected. 
    A slight increase in haemolysis was seen in mice exposed to
    2940 g/m3 (2.4 ppm) of NO (Oda et al., 1980a).

    5.4.1.6  Biochemical mechanisms for nitric oxide effects: reaction
             with iron and effects on enzymes and nucleic acids

         NO has an affinity for haem-bound iron which is two times higher
    than that of carbon monoxide.  This affinity leads to the formation of
    methaemoglobin and the stimulation of guanylate cyclase.  Furthermore,
    NO reacts with thiol-associated iron in enzymes and eventually
    displaces the iron.  This is a possible mechanism for the cytotoxic
    effects of NO (Hibbs et al., 1988; Weinberg, 1992).   In vitro, the
    NO donor sodium nitroprusside has been shown to mobilize iron from
    ferritin (Reif & Simmons, 1990).  NO might possibly modulate
    arachidonic acid metabolism via interference with iron (Kanner et al.,
    1991a,b).

         NO inhibits aconitase, an enzyme in the Krebs cycle, and also
    complex 1 and 2 of the respiratory chain (Hibbs et al., 1988; Persson
    et al., 1990; Stadler et al., 1991).  Permanent modification of
    haemoglobin has been found; possibly via deamination (Moriguchi
    et al., 1992).  NO can also deaminate DNA, evoke DNA chain breaks,

    and inhibit DNA polymerase and ribonucleotide reductase (Wink et al.,
    1991; Lepoivre et al., 1991; Kwon et al., 1991; Nguya et al., 1992). 
    NO might be antimitogenic and inhibit T cell proliferation in rat
    spleen cells (Fu & Blankenhorn, 1992), and NO donors inhibit DNA
    synthesis, cell proliferation, and mitogenesis in vascular tissue
    (Garg & Hassid, 1989; Nakaki et al., 1990).  ADP (adenosine
    diphosphate) ribosylation is stimulated by NO-generating agents
    (Nakaki et al., 1990).

         Substantial  in vitro evidence has recently been published
    describing other effects of NO in tissues.  These include:  inhibition
    of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) via ADP
    ribosylation (Alheid et al., 1987; Dimmeler et al., 1992); macrophage
    mediated-nitric oxide dependent mechanisms which include inhibition of
    the electron transport chain (Nathan, 1992); inhibition of DNA
    synthesis (Hibbs et al., 1988); inhibition of protein synthesis
    (Curran et al., 1991) and decrease in cytosolic free calcium by a
    cGMP-independent mechanism (Garg & Hassid, 1991).

    5.4.2  Nitric acid

         There have been only a few toxicological studies of HNO3, which
    exists in ambient air generally as a highly water-soluble vapour.  A
    few investigators have examined the histological response to instilled
    HNO3 (usually 1%), a procedure used in developing models of
    bronchiolitis obliterans in various animals, namely dogs, rabbits and
    rats (Totten & Moran, 1961; Greenberg et al., 1971; Gardiner &
    Schanker, 1976; Mink et al., 1984).  However, the relevance of such
    instillation studies is questionable, except to provide information
    for the design of inhalation studies.

         Only two studies have been designed specifically to examine the
    pulmonary response to pure HNO3 vapour.  Abraham et al. (1982)
    exposed both normal sheep and allergic sheep (i.e., having airway
    responses similar to those occurring in humans with allergic airway
    disease) for 4 h to 4120 g/m3 (1.6 ppm) HNO3 vapour.  The exposure,
    using a "head-only" chamber, decreased specific pulmonary flow
    resistance in both groups of sheep; this indicated the absence of any
    bronchoconstriction.  Allergic, but not normal, sheep showed increased
    airway reactivity to carbachol, both immediately and 24 h after HNO3
    exposure.  In another study, rats exposed for 4 h to 1000 g/m3
    (0.38 ppm) HNO3 vapour or for 4 h/day for 4 days to 250 g/m3
    (0.1 ppm) HNO3 showed a decrease in stimulated or unstimulated
    respiratory burst activity of alveolar macrophages (AMs) obtained by
    lavage, as well as an increase in elastase inhibitory capacity of BAL
    (Nadziejko et al., 1992).

    5.4.3  Nitrates

         Only one inhalation study conducted at levels < 1 mg/m3
    NO3- has been reported.  Busch et al. (1986) exposed rats and
    guinea-pigs with either normal lungs or elastase-induced emphysema to
    ammonium nitrate aerosols at 1 mg/m3 for 6 h/day, 5 days/week for
    4 weeks.  Using both light and electron microscopy, the investigators
    concluded that there were no significant effects of exposure on lung
    structure.

    5.5  Summary of studies of the effects of nitrogen compounds on
         experimental animals

         Responses to NO2 exposure have been observed in several
    laboratory animal species, resulting in the conclusion that these
    effects could occur in humans.  In addition, mathematical dosimetry
    models suggest that the greatest dose of NO2 is delivered to the same
    region in both animal and human lungs (i.e. the centriacinar region
    which is the junction of the conducting airway with the gas exchange
    area).  Thus, the responses of laboratory animals can be qualitatively
    extrapolated to humans.

         NO2 exposure causes lung structural alterations.  Exposure to
    3760 g/m3 (2.0 ppm) for 3 days has resulted in centriacinar damage,
    including damaged cilia and alveolar wall oedema.  Prolonged exposures
    produce changes in the cells lining the centriacinar region, and the
    tissue in this region (i.e., alveolar interstitium) becomes thicker. 
    These effects were seen in rats exposed to 940 g/m3 (0.5 ppm)
    baseline with brief peaks of 2800 g/m3 (1.5 ppm) for 6 weeks or
    exposures to 940 g/m3 (0.5 ppm) NO2 for 4 to 6 months.

         Several animal studies clearly demonstrate that chronic exposure
    to concentrations of NO2 > 9400 g/m3 (> 5.0 ppm) can
    cause emphysema of the type seen in human lungs.  Increased lung
    distensibility was reported in mice exposed to 375 g/m3 (0.2 ppm)
    with peaks of 1500 g/m3 (0.8 ppm) after 1 year of exposure.

         NO2 increases susceptibility to bacterial and viral pulmonary
    infections in animals.  Reduced phagocytic activity and reduced
    mobility were observed in AMs from rabbits exposed for 13 days to
    500 g/m3 (0.3 ppm).  The lowest observed concentration that
    increases lung susceptibility to bacterial infections after acute
    exposure is 3750 g/m3 (2.0 ppm) NO2 (a 3-h exposure study in mice). 
    Acute (17 h) exposures to > 4250 g/m3 (> 2.3 ppm) NO2 also
    decrease pulmonary bactericidal activity in mice.  After long-term
    exposures (e.g., 3 to 6 months) to 940 g/m3 (0.5 ppm) NO2, mice
    have decreased resistance to lung bacterial infections. Exposure of
    mice for 1 year to 375 g/m3 (0.2 ppm/week) with 1480 g/m3
    (0.8 ppm) spike followed by infection with streptococcus resulted in

    increased mortality.  NO2 also increases lung susceptibility to viral
    infections in mice.  Subchronic (7-week) exposures to concentrations
    as low as 470 g/m3 (0.25 ppm) NO2 can alter the systemic immune
    system in mice.

         NO2 exposure has been shown to cause a clear dose-related
    decrease in pulmonary antibacterial defences. Decreases in pulmonary
    antibacterial defences occurred at concentrations ranging from
    7520 g/m3 (4 ppm) for  Staphylococcus aureus to 37 500 g/m3
    (20 ppm) for  Proteus mirabilis.  Dose-response increases in
    bacterial-induced mortality in mice was demonstrated with continuous
    exposure to 940 g/m3 (0.5 ppm) after 3 months.

         When the relationship of NO2 exposure concentration and duration
    was studied, concentration had more influence than duration on the
    outcome.  This conclusion is primarily based on investigations of lung
    antibacterial defences of mice, which also indicate that the exposure
    pattern (e.g., baseline level with daily peaks of NO2 or exposure
    24 h/day versus 6 to 7 h/day) has an impact on the study results.

         Structural changes in the lung become more severe as exposure
    progresses from weeks to months at a given NO2 concentration. Longer
    exposures resulted in effects at lower concentrations.

         NO2 showed positive effects in some studies with Salmonella
    strain TA100 and caused DNA strand breaks in a mammalian cell culture. 
    NO seems to be less active.  High concentrations of NO2 have induced
    mutations in lung cells  in vivo, but not in other organs. There are
    no classical chronic bioassays for carcinogenicity. Studies concerning
    enhancement of spontaneous tumours, co-carcinogenic effects, or
    facilitation of the metastases of tumours to the lung are inadequate
    to form conclusions.  Possible secondary effects concern the
     in vivo formation of nitrite and nitrosamines and atmospherically
    formed mutagenic reaction products from NOx and hydrocarbons.

         The effects of exposure to mixtures of NO2 and other pollutants
    are dependent on the exposure regimen, species and end-point measured. 
    Most mixture research involves NO2 and O3 and shows that additivity
    and synergism can occur.  A similar conclusion can be drawn from the
    more limited research with NO2 and sulfuric acid.  Findings of either
    additivity or synergism are of concern because of the ubiquitous
    co-occurrence of NO2 and O3.  Extrapolation of these findings is not
    currently possible.

         NO is a potent vasodilator and effects can be demonstrated with
    inhaled concentrations of approximately 6130 g/m3 (5 ppm) in sheep
    and guinea-pigs.  NO also reduces resistance to bacterial infection
    via the inhalation route in female mice exposed to 2452 g/m3
    (2 ppm).  Morphological alterations in the alveoli and thickening of

    the alveolocapillary membrane are seen in rabbits at 6130 g/m3. 
    Methaemoglobin formation is seen at concentrations above 12 260 g/m3
    (10 ppm).

         NO2 acts as a strong oxidant.  Unsaturated lipids are readily
    oxidized with peroxides as the dominant product.  Both ascorbic acid
    and alpha-tocopherol inhibit the peroxidation of unsaturated lipids. 
    When ascorbic acid is sealed within bi-layer liposomes, NO2 rapidly
    oxidizes the sealed ascorbic acid.  The protective effects of
    alpha-tocophernol (vitamin E) and ascorbic acid (vitamin C) in animals
    and humans are due to the inhibition of NO2 oxidation.  NO2 also
    oxidizes membrane proteins. The oxidation of either membrane lipids or
    proteins results in the loss of cell permeability control.  The lungs
    of NO2-exposed humans and experimental animals have larger amounts of
    protein within the lumen.  The recruitment of inflammatory cells and
    the remodelling of the lung are a consequence of these events.

         The oxidant properties of NO2 also induce the peroxide
    detoxification pathway of glutathione peroxidase, glutathione
    reductase, and glucose-6-phosphate dehydrogenase.  Increases in the
    peroxide detoxification pathway occur in animals in a roughly
    dose-response relationship following NO2 exposure.

         The mechanism of action of NO is less clear.  NO is readily
    oxidized to NO2 and then peroxidation occurs.  Because of concomitant
    exposure to some NO2 in NO exposures, it is difficult to discriminate
    NO effects from those of NO2.  NO is, however, a potent second
    messenger modulating a wide variety of essential cellular functions.

         Peroxyacetyl nitrate (PAN) decomposes in water generating
    hydrogen peroxide.  Little is known of the mechanism of action, but
    oxidative stress is likely for PAN and its congeners.

         Inorganic nitrates may act by alterations in intracellular pH. 
    Nitrate ion is transported into Type 2 cells, acidifying the cell. 
    Nitrate also mobilizes histamine from mast cells.  Nitrous acid could
    also act to alter intracellular pH, but this mechanism is unclear.

         The mechanisms of action of the other nitrogen oxides are unknown
    at present.

    6.  CONTROLLED HUMAN EXPOSURE STUDIES OF NITROGEN OXIDES

    6.1  Introduction

         The effects of nitrogen oxides (NOx) on human volunteers exposed
    under controlled exposure conditions are evaluated in this chapter. 
    Of the NOx species typically found in the ambient air, NO2 has been
    the most extensively studied.  Nitric oxide (NO), nitrates, nitrous
    acid and nitric acid also have been evaluated and are discussed here,
    as are investigations of mixtures of NOy and other co-occurring
    pollutants.  A more extensive detailed review of this literature can
    be found in US EPA (1993).

         Most volunteers for human clinical studies are young, healthy
    adult males, but other potentially susceptible subpopulations,
    especially asthmatics, patients with chronic obstructive pulmonary
    disease (COPD), children and the elderly have also been studied.  Many
    exposures are conducted while the volunteer performs some form of
    controlled exercise.  The exercise increases ventilation, which
    increases the mass of pollutant inhaled per unit time and may alter
    the distribution of the dose within the lung.  More information on
    NO2 dosimetry is presented in chapter 5. Important methodological and
    experimental design considerations for controlled human studies have
    been discussed in greater detail by Folinsbee (1988).

         In many human clinical studies of NO2 exposure, both pulmonary
    function and airway responsiveness to bronchoconstrictors have been
    measured.  Spirometric measurements of lung volume, as well as
    measurements of airway resistance, ventilation volume, breathing
    pattern, and other tests provide information about some of the basic
    physiological functions of the lung.  Dynamic spirometry tests (forced
    expiratory tests such as forced expiratory volume in 1 second (FEV1),
    maximal and partial flow-volume curves (including those using gases of
    different densities such as helium), peak flow measurements, etc.),
    and measurements of specific airway resistance/conductance (SRaw,
    SGaw) are also used.  Most of these tests evaluate large airway
    function.  However, since NO2 deposition occurs primarily around
    the junction of the tracheobronchial and pulmonary regions (section
    5.2.1), many of these tests may not provide the necessary information
    to evaluate fully the effects of NO2.  Other tests that may evaluate
    small airway function (e.g., multiple breath nitrogen washout tests,
    closing volume tests, aerosol deposition/distribution tests, density
    dependence of flow-volume curves, and frequency dependence of dynamic
    compliance) are less frequently used, and the extent to which they
    indicate small airways function is not clearly established.  As
    discussed below, NO2 can increase airway responsiveness to chemicals
    that cause bronchoconstriction, such as histamine or cholinergic
    agonists (i.e., acetylcholine, carbachol or methacholine).  Other
    challenge tests use allergens, exercise, hypertonic saline or cold-dry

    air.  Responses are usually measured by evaluating changes in airway
    resistance (Raw) or spirometry (e.g. FEV1) after each dose of the
    challenge is administered.  Generally, asthmatics are significantly
    more responsive than healthy normal subjects to these types of airway
    challenge (O'Connor et al., 1987).  However, there is some overlap
    between the most responsive healthy subjects and the least responsive
    (to histamine) asthmatics (Pattemore et al., 1990).

         In the following sections, the changes in pulmonary function and
    airway responsiveness after NO2 exposure in healthy subjects  are
    discussed.  Responses of asthmatics and patients with chronic
    obstructive pulmonary disease (COPD) are then evaluated.  A brief note
    regarding age-related susceptibility is followed by a review of the
    effects of NO2 on pulmonary host defences and on biochemical markers
    in lung lavage fluid or in the blood.  The effects of two other
    oxidized nitrogen compounds, NO and nitric acid vapour are also
    discussed.  Finally, the effects of mixtures of oxidized nitrogen
    compounds (NO2, NO, HNO3) with other gaseous or particulate
    pollutants are considered.  An overall summary is presented at the end
    of the chapter.

    6.2  Effects of nitrogen dioxide

    6.2.1  Nitrogen dioxide effects on pulmonary function and airway
           responsiveness to bronchoconstrictive agents

         Much research has focused on NO2-induced changes in pulmonary
    function and airway responsiveness to bronchoconstrictive agents. 
    Healthy adults do not typically respond to low levels of NO2
    (< 1880 g/m3, 1 ppm).  However, asthmatics appear to be the most
    susceptible members of the population (section 6.2.1.2).  Asthmatics
    are generally much more sensitive to inhaled bronchoconstrictors.  The
    potential addition of an NO2-induced increase in airway response to
    the already heightened responsiveness to other substances raises the
    possibility of exacerbation of asthma by NO2.  Another potentially
    susceptible group includes patients with COPD (section 6.2.1.3).  A
    major concern with COPD patients is the absence of an adequate
    pulmonary reserve, so that even a relatively small alteration in lung
    function in these individuals could potentially cause serious
    problems.  In addition, both adolescents and the elderly have
    been evaluated, to determine whether differential age-related
    susceptibility exists (section 6.2.1.4).

        Table 39.  Effects of nitrogen dioxide (NO2) on lung function and airway responsiveness of healthy subjectsa
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristice
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    188          0.1          60                                     15 M           23-29 years,       No symptoms; no odour   Hazucha et al.
                                                                                    NS                 detection; no effect    (1982, 1983)
                                                                                                       on SRaw.

    188          0.1          240                                    6              Normal adults      No effects of NO2       Sackner et al.
    564          0.3                                                                                                           (1980)
    940          0.5
    1880         1.0

    226          0.12         60                                     4 M/6 F        13-18 years        No effects on lung      Koenig et al.
                                                                                                       function.               (1985)

    226          0.12         40         10           32.5           3 M/7 F        14-19 years        No effects on Rtau or   Koenig et al.
    338          0.18         40                                     4 M/6 F        15-19 years        spirometry.             (1987a,b)

    230          0.12         20                                     5 M/4 F        20-36 years,       Suggestion of change    Bylin et al.
    460          0.24                                                               NS                 in SRaw in normals:     (1985)
    910          0.48                                                                                  SRaw tended to increase
                                                                                                       at 476 g/m3 and
                                                                                                       tended to decrease at
                                                                                                       910 g/m3.  Analysis of
                                                                                                       variance indicates no
                                                                                                       significance.  No effects
                                                                                                       on bronchial  reactivity.
                                                                                                       Median odour threshold
                                                                                                       75 g/m3.
                                                                                                                                              

    Table 39  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristice
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    282          0.15         120        60           50 W           6 M            19-24 years        No symptoms; no         Kagawa & Tsuru
                                                                                                       pulmonary function      (1979); Johnson
                                                                                                       effects.  Suggested     et al. (1990)
                                                                                                       individual changes
                                                                                                       in SGaw.

    338          0.18         30         10 (L)       L approx. 25   9 M            18-23 years,       No change in lung       Kim et al.
    564          0.3                     16 (H)       H approx. 72                  "collegiate        function.               (1991)
                                                                                    athletes"

    508          0.27         60                                                    Healthy,           Possible small          Rehn et al.
    1993         1.06                                                               young M            increase in Raw at      (1982)
                                                                                                       508 g/m3 (0.27 ppm).

    564          0.3          120        60           50 W           6              19-25 years        No effect on SGaw.      Kagawa (1986)

    564          0.3          225        30           approx. 40     10 M/10 F      20-48 years        No symptom, lung        Morrow & Utell
                                         (3  10)                                   (FEV1/FVC          function or airway      (1989)
                                                                                    76-95%)            reactivity responses
                                                                                                       to carbachol for either
                                                                                                       of the 20-48 year or
                                                                                                       the 49-69 year age
                                                                                                       groups.

    564          0.3          225        21           30-40          10 M/10 F      49-69 years,
                                         (3  7)                                    (FEV1/FVC
                                                                                    72-84%)
                                                                                                                                              

    Table 39  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristice
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    940          0.5          120        15           Light/         10             Healthy, three     Decreased quasistatic   Kerr et al.
                                                      moderate                      ex-smokers in      compliance. Non-random  (1979)
                                                                                    group              exposure sequence air-
                                                                                                       NO2. No change in
                                                                                                       spirometry or resistance.
                                                                                                       Apparent compliance
                                                                                                       change may be due to
                                                                                                       exposure order.

    940          0.5          120        15                          10             Normal adults      Decreased static lung   Kulle (1982)
                                                                                                       compliance.

    940          0.5          240        30           55             10 M           26.4 years         No significant effects  Stacy et al.
                                                                                                       on spirometry or Raw.   (1983)

    1128         0.6          120        60           25             8 M/8 F        51-76 years        No statistically        Drechsler-Parks
                                                                                                       significant changes     et al. (1987)
                                                                                                       in lung function due
                                                                                                       to NO2 exposure in
                                                                                                       either age group.

                                                                     8 M/8 F        18-26 years,
                                                                                    NS

    1128         0.6          180        60           approx. 40     7 M/2 F        Healthy, NS        No change in            Frampton et al.
                                         (6  10)                                                      spirometry, Raw or      (1989a)
                                                                                                       carbachol reactivity.
                                                                                                                                              

    Table 39  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristice
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    94 with      0.05 with    135        60                          11 M/4 F       Non-reactive
    3760         2.0 spikes   3  15     (6  10)                                   (carbachol)
    spikes

    (1) 1128     (1) 0.6      180        60           39             6 M/2 F        30.3  1.4         There were no changes   Frampton et al.
                                                                                    years, NS          in airway mechanics     (1991)
                                                                                                       (FVC, FEV1, SGaw).
                                                                                                       Responsiveness to
    (2) Var.     (2) Var.     180        60           43             11 M/4 F       25.3  1.2         carbachol was
    (94          (0.05                                                              years, NS          significantly increased
    background   background                                                                            after 2820 g/m3 NO2
    with         with         180        60           approx. 40     5 M/3 F        32.6  1.6         (Group 3) but not after
    3  15 min   3  15                                                             years, NS          the other exposures
    at 3760)     min at                                                                                (Groups 1 and 2). Degree
                 2.0 ppm)                                                                              of baseline
                                                                                                       responsiveness to
                                                                                                       carbachol was not
                                                                                                       related to response after
                                                                                                       2820 g/m3.

    (3) 2820     (3) 1.5      180        60           39             12 M/3 F       23.5  0.7
                                                                                    years, NS
                                                                                                                                              

    Table 39  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristice
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    1128         0.6          120/day    60           approx. 30-40  4 M/1 F        NS, 21-36          No effects of repeated  Boushey et al.
                              for 4 days                                            years, FEV1/       NO2 exposure on         (1988) (Part 2)
                                                                                    FVC% range         respiratory function
                                                                                    73-83%,            (SRaw, FVC, FEV1) or
                                                                                    "normal"           symptoms.
                                                                                    methacholine
                                                                                    responsiveness

    1128         0.6          60         60           70             20 M           Healthy            No effect of NO2 on     Adams et al.
                                                      50             20 F                              spirometry or airway    (1987)
                                                                                                       resistance.

    1166         0.62         120        15           33             5 M            Healthy            No significant          Folinsbee et al.
                                         30           33             5 M                               pulmonary function      (1978)
                                                                                                       responses attributed
                                                                                                       to NO2 exposure.

    1316-3760    0.7-2.0      10                                     10                                Increased resistance    Suzuki &
                                                                                                       10 min after exposure.  Ishikawa (1965)

    1316         0.7          60                                     5              19-22 years,       No effects on airway    Toyama et al.
                                                                                    3 of 5 were        conductance.            (1981)
                                                                                    investigators
                                                                                                                                              

    Table 39  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristice
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    1880         1.0          120 (2     60           Light          16             Healthy            Air-NO2-NO2 fixed       Hackney et al.
                              consecutive                                                              exposure sequence.      (1978)
                              days)                                                                    1.5% decrease in FVC
                                                                                                       after second day of
                                                                                                       NO2. Not clear that
                                                                                                       the decreased FVC is
                                                                                                       an NO2 effect or an
                                                                                                       order effect. No other
                                                                                                       effects.

    1880         1.0          120/day,                               22             Healthy, NS,       Overall trend for a     Goings et al.
    3760         2.0          3 days                                 21, 22         seronegative       slight decrement in     (1989)
    5640         3.0                                                 22                                FEV1 with NO2 exposure
                                                                                                       (< 1%).  No change in
                                                                                                       methacholine
                                                                                                       responsiveness as a
                                                                                                       result of NO2 exposure
                                                                                                       or viral infection
                                                                                                       status.

    1880         1.0          120                                    16             11 S               After 14 100 g/m3      Beil & Ulmer
    4700         2.5          120                                    16             5 NS               (120 min) and           (1976)
    9400         5.0          120                                    16                                9400 g/m3 (14 h),
    14 100       7.5          120                                    16             8 S                responsiveness to
    9400         5.0          840                                    8                                 acetylcholine increased.
                                                                                                       Resistance increased
                                                                                                       after all but the
                                                                                                       1880 g/m3 exposure.
                                                                                                                                              

    Table 39  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristice
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    3760         2.0          60                                     8 M/3 F        18-36 years,       Vitamin C blocked       Mohsenin (1987b)
                                                                                    NS                 NO2-induced increase
                                                                                                       in airway reactivity
                                                                                                       to methacholine.

    3760         2.0          120                                    13 M/5 F       Normal, NS,        No symptoms; no lung    Mohsenin (1988)
                                                                                    18-33 years        function changes.
                                                                                                       Increased methacholine
                                                                                                       reactivity.

    7520-9400    4.0-5.0      10                                                                       Bag exposure            Abe (1967)
                                                                                                       technique. Airway
                                                                                                       resistance increased
                                                                                                       30 min after end of
                                                                                                       exposure. No change in
                                                                                                       spirometry.

    7520         4.0          75         15 (L)       L 20-29        16 M/ 9 F      18-45 years,       No change in SRaw       Linn & Hackney
                                         15 (H)       H 44-57                       NS                 associated with NO2.    (1983); Linn
                                                                                                       Small but significant   et al. (1985b)
                                                                                                       decrease in blood
                                                                                                       pressure; some mild
                                                                                                       increase in symptoms.

    9400         5.0          15                                     16             Healthy            Decreased DLCO 18%.     Von Nieding et
                                                                                                                               al. (1973a)
                                                                                                                                              

    Table 39  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristice
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    9400         5.0          120        Intermittent Light          11 M           Healthy            Increased resistance    Von Nieding et
                                                                                                       60%. Remained elevated  al. (1977)
                                                                                                       for 60 min. Possible
                                                                                                       decrease in PaO2.

    9400         5.0          120        60           220            11 M           Healthy            Resistance increased    Von Nieding et
                                         (4  15)                                                      60%. Remained elevated  al. (1979)
                                                                                                       60 min after exposure.
                                                                                                       Possible decrease in
                                                                                                       earlobe PO2.
                                                                                                                                              

    a  Modified from US EPA (1993)
       Abbreviations:
       M = Male; F = Female; S = Active smoker; NS = Non-smoker; FEV1 = Forced expiratory volume in 1 second; FVC = Forced vital capacity;
       SRaw = Specific airway resistance; Var = Variable; Raw = Airway resistance; SGaw = Specific airway conductance; W = Watts; L = Light;
       H = Heavy; RT = Total respiratory resistance; DLCO = Diffusing capacity for carbon monoxide; PaO2 = Arterial partial pressure of oxygen;
       PO2 = Partial pressure of oxygen
        6.2.1.1  Nitrogen dioxide effects in healthy subjects

         The effects of NO2 levels greater than 1880 g/m3 (1.0 ppm) on
    respiratory function in healthy subjects have been examined in several
    studies (Table 39). Early work indicated that NO2 increased Raw
    or total respiratory resistance (RT) at concentrations above
    2820 g/m3 (1.5 ppm) in healthy volunteers (Abe, 1967; Von Nieding et
    al., 1970, 1973a, 1979; Von Nieding & Wagner, 1977).  Although Beil &
    Ulmer (1976) found a small but statistically significant increase in
    RT after a 2-h exposure to > 4700 g/m3 (> 2.5 ppm) NO2, the
    response was not appreciably increased by raising the NO2
    concentration to 9400 or 14 100 g/m3 (5.0 or 7.5 ppm).  Also, airway
    responsiveness to acetylcholine was increased after exposure to
    14 100 g/m3 for 2 h or to 9400 g/m3 for 14 h, but not after the
    2-h exposures to < 9400 g/m3.

         In contrast, some investigators found no effects at high
    concentrations.  For example, a 75-min exposure with light and heavy
    exercise to 7520 g/m3 (4.0 ppm) NO2 did not affect Raw (Linn et
    al., 1985b), and a 1-h resting exposure to 3760 g/m3 (2 ppm) did not
    cause a change in lung volume, flow-volume characteristics on either
    full or partial expiratory flow-volume (PEFV) curves, or SGaw
    (Mohsenin, 1987b, 1988).  However, NO2 did increase airway
    responsiveness to methacholine (Mohsenin, 1987b, 1988).

         Goings et al. (1989) found no effects of exposure to NO2 at
    1880, 3760 or 5640 g/m3 (1, 2 or 3 ppm; for 2 h/day on 3 consecutive
    days) on respiratory symptoms, lung function or airway reactivity to
    methacholine.  Laboratory-induced influenza virus infection did not
    alter airway responsiveness in either sham (clean air) or NO2
    exposure groups.  The infectivity portion of this study is discussed
    in section 6.2.2.

         The influence of exposure pattern was examined by Frampton et al.
    (1991), using healthy subjects exposed for 3 h to either 1128 g/m3
    (0.60 ppm), 2820 g/m3 (1.5 ppm) or a variable concentration protocol
    where three 15 min peaks of 3760 g/m3 (2.0 ppm) were added to a
    background level of 94 g/m3 (0.05 ppm).  Nitrogen dioxide did not
    affect airway mechanics (forced vital capacity (FVC), FEV1, SGaw). 
    However, after exposure to 2820 g/m3, but not to the other
    concentrations, there was a small but statistically significant
    increase in airway responsiveness to carbachol.  This study supported
    the earlier observations by Mohsenin (1987b, 1988) of increased airway
    responsiveness after a 1-h exposure to 3760 g/m3.  Mohsenin (1987b)
    further observed that the NO2-induced increase in airway
    responsiveness could be blocked by elevation of serum ascorbate level
    through pretreatment with the antioxidant ascorbic acid (vitamin C).

         At concentrations below 1880 g/m3 (1.0 ppm) NO2, pulmonary
    function and airway responsiveness have generally not been found to be
    affected in healthy adult subjects (Beil & Ulmer, 1976; Folinsbee et
    al., 1978; Hackney et al., 1978; Kerr et al., 1979; Sackner et al.,
    1980; Toyama et al., 1981; Kulle, 1982; Hazucha et al., 1982, 1983;
    Stacy et al., 1983; Kagawa, 1986; Adams et al., 1987; Drechsler-Parks
    et al., 1987; Drechsler-Parks, 1987; Boushey et al., 1988; Morrow &
    Utell, 1989; Frampton et al., 1989a, 1991; Kim et al., 1991).
    Although some investigators have at times reported statistically
    significant effects, there does not appear to be a consistent pattern
    of acute responses in healthy subjects at these low NO2
    concentrations.

         Kagawa & Tsuru (1979) reported the lowest NO2 exposure
    concentration that appeared to cause an effect.  Healthy men were
    exposed to 282 g/m3 (0.15 ppm) NO2 for 2 h while performing light,
    intermittent exercise.  The authors suggested that NO2 caused some
    statistically significant changes, i.e. a 0.5% decrease in vital
    capacity (VC) and a 16% decrease in an index of small airway function
    (i.e. FEF75HeO2: FEF75AIR; the ratio of forced expiratory flow at 75%
    FVC expired while breathing a helium-oxygen mixture compared to FEF75
    while breathing air).  These findings should be interpreted with the
    consideration that multiple t-tests were used in the statistical
    analysis of these data.  Rehn et al. (1982) reported a small (17%)
    increase in SRaw in men exposed to 500 g/m3 (0.27 ppm) for 1 h, but
    a higher concentration (2000 g/m3, 1.06 ppm) did not cause an
    effect.

         Bylin et al. (1985) reported that the SRaw of normal subjects
    exposed to 230, 460 and 910 g/m3 (0.12, 0.24 and 0.48 ppm) for
    20 min was unaffected. Specific comparisons revealed a significant 11%
    increase in SRaw at 460 g/m3 (0.24 ppm) and a 9% decrease in SRaw
    at 910 g/m3.  Bronchial responsiveness to histamine was increased by
    910 g/m3 NO2.

         Symptomatic responses of subjects exposed to NO2 were evaluated
    in several of the above studies.  None of these studies, including
    exposures for as long as 75 min to 7520 g/m3 (4.0 ppm) NO2 (Linn &
    Hackney, 1983; Linn et al., 1985b), resulted in a significant increase
    in respiratory symptoms.  In studies of sensory effects, subjects were
    unable to detect the odour of 188 g/m3 (0.1 ppm) NO2 (Hazucha et
    al., 1983), but Bylin et al. (1985) observed an odour threshold of
    75 g/m3 (0.04 ppm) for normal subjects and 150 g/m3 (0.08 ppm) for
    asthmatics.

    6.2.1.2  Nitrogen dioxide effects on asthmatics

         Studies of the effects of exposures to NO2 on respiratory
    function and airway responsiveness of asthmatics are summarized in
    Table 40.  Asthmatics are generally more responsive than healthy
    subjects to NO2.  However, as can be seen in Table 40, there is
    substantial variability in observed responses between and even within
    laboratories.  This variability is illustrated in Fig. 22 and 23, in
    which changes in airway resistance and FEV1 are related to the
    "exposure dose" of NO2 (calculated as ppm  litres of air breathed
    over the duration of exposure) (US EPA, 1993).  The individual
    investigations that yielded the data used to develop these
    illustrations will be discussed in more detail below.  Other studies,
    not discussed separately, are also summarized in Table 40.  The review
    by the US EPA (1993) provides more detail on many of these studies. 
    Although differences in exposure protocols may explain some of the
    differences between studies, the explanation most often invoked is
    that there may be differences in the severity of asthma among the
    subject groups tested.  There are numerous definitions of "asthma
    severity" (see, for example, National Institutes of Health, 1991). 
    Those applied to the key asthma studies discussed here (based on the
    data available) are:  (1) mild: controlled by bronchodilators and
    avoidance of known precipitating factors, does not interfere with
    normal activities; and (2) moderate: often requires periodic use of
    inhaled steroids in treatment and may interfere with work or school
    activities.  Those with severe asthma are seldom used as subjects for
    NO2 studies because their disease can include life-threatening
    episodes.  Typical volunteers for the studies described here had mild
    allergic asthma.

         Avol et al. (1988) studied a group of moderate-to-severe
    asthmatics exposed to 564 and 1128 g/m3 (0.3 and 0.6 ppm) NO2 for
    2 h with moderate intermittent exercise. NO2 did not cause
    significant changes in SRaw or FEV1.  Results of tests of airway
    responsiveness to cold air suggested a slightly increased response
    after exposure to 564 g/m3, but not after 1128 g/m3. A post hoc
    analysis of a subgroup of subjects with the most abnormal lung
    function (i.e., FEV1/FVC ratios < 0.65) did not find enhanced
    susceptibility. In a subsequent study using 564 g/m3 NO2, Avol et
    al. (1989) found decreases in FEV1, FVC and peak expiratory flow rate
    (PEFR), but no change in responsiveness to cold air challenge.

        Table 40.  Effects of nitrogen dioxide (NO2) on lung function and airway responsiveness of asthmaticsa
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    188          0.1          60                                     9              20-51 years,       No effect of NO2 on     Ahmed et al.
                                                                                    "history of        FEV1, SGaw or on        (1983a)
                                                                                    bronchial asthma"  ronchial reactivity to
                                                                                                       ragweed antigen, either
                                                                                                       immediately or 24 h
                                                                                                       after exposure.

    188          0.1          60                                     20 M/34 F      18-39 years        No significant effect   Ahmed et al.
                                                                                                       on SGaw, FEV1, VISOV;  (1983b)
                                                                                                       variable effect on
                                                                                                       carbachol reactivity.
                                                                                                       No information on
                                                                                                       controlled exposure.

    188          0.1          60                                     15 M           21-46 years,       No significant          Hazucha et al.
                                                                                    mild or inactive   changes in RT or        (1982, 1983)
                                                                                    disease            responsiveness to
                                                                                                       methacholine associated
                                                                                                       with NO2 exposure.

    207          0.11         60                                     6 M/1 F        1 Smoker,          No change in SRaw or    Orehek et al.
    (132-301)    (0.07-0.16)                                                        3 asthmatic,       in responsiveness to    (1981)
                                                                                    4 allergic         grass pollen in 3
                                                                                                       allergic asthmatics
                                                                                                       and 4 allergic
                                                                                                       subjects.
                                                                                                                                              

    Table 40  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    210          0.11         60                                     13 M/7 F       15-44 years,       13/20 subjects had      Orehek et al.
    (169-244)    (0.09-0.13)                                                        13 mild/7 mod      enhanced responses to   (1976)
                 (n = 20)                                                           asthmatics;        carbachol after
                                                                                                       210 g/m3 NO2. Post
                                                                                                       hoc statistical
                                                                                                       analysis questionable.

    489          0.26                                                               65 years           1/4 subjects had        Orehek et al.
                 (n = 4)                                                                               enhanced responses      (1976)
                                                                                                       to carbachol after
                                                                                                       489 g/m3 NO2.

    226          0.12         60                                     4 M/6 F        12-18 years,       No significant effects  Koenig et al.
                                                                                    asympt.,           on pulmonary function   (1985)
                                                                                    extrinsic          due to NO2. Increased
                                                                                    allergic           symptoms after NO2
                                                                                    asthmatics         exposures.

    226          0.12         60                                     4 M/6 F        12-18 years        No change in FEV1,      Koenig et al.
    226          0.12         40         10           33             4 M/6 F        11-19 years        RT increased 10.4%      (1987a,b)
    338          0.18         40         10           39             7 M/3 F        12-18 years,       (NS), 3% decrease
                                                                                    asympt.,           in FEV1 (p < 0.06).
                                                                                    extrinsic
                                                                                    allergic
                                                                                    asthmatics
                                                                                                                                              

    Table 40  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    230          0.12         20                                     6 M/2 F        17-45 years,       No significant change   Bylin et al.
    460          0.24         20                                                    very mild          in SRaw at any NO2      (1985)
    910          0.48         20                                                    asympt.            levels. Histamine
                                                                                                       reactivity tended to
                                                                                                       increase.

    260          0.14         30                                     8 M/12 F       17-56 years,       Overall trend for SRaw  Bylin et al.
    510          0.27                                                               very mild          to decline during       (1988)
    1000         0.53                                                               asympt.            exposure period, not
                                                                                                       related to NO2
                                                                                                       concentration.
                                                                                                       Histamine bronchial
                                                                                                       reactivity tended to
                                                                                                       increase after 260 and
                                                                                                       510 g/m3 NO2 exposure.

    376          0.2          120        60           approx. 20     12 M/19 F      18-55 years,       No effects on           Kleinman et al.
                                                                                    wide range of      spirometry or airway    (1983)
                                                                                    asthma severity    resistance. Airway
                                                                                                       reactivity to
                                                                                                       methacholine results
                                                                                                       variable-tended to
                                                                                                       increase with exposure.
                                                                                                                                              

    Table 40  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    470          0.25         30         10           30             9 M/2 F        18-55 years,       Mouthpiece exposure     Joerres &
                                                                                    mild asympt.       system. No changes in   Magnussen
                                                                                                       methacholine            (1991)
                                                                                                       responsiveness were
                                                                                                       observed after NO2
                                                                                                       exposure.

    470          0.25         30                                     10 M/4 F       20-55 years,       After NO2 exposure,     Joerres &
                                                                                    mild asthma,       responsiveness to       Magnussen
                                                                                    most asympt.       inhaled SO2 was         (1990)
                                                                                                       increased. No effect
                                                                                                       of NO2 alone on SRaw.

    564          0.3          30         20           approx. 30     5 M/4 F        23-34 years        No changes in SRaw,     Rubinstein et
                                                                                                       FVC, FEV1, SBN2 or      al. (1990)
                                                                                                       symptoms after NO2
                                                                                                       exposure. NO2 exposure
                                                                                                       did not increase airway
                                                                                                       responsiveness to SO2.
                                                                                                                                              

    Table 40  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    564          0.3          30         10           30             15             20-45 years,       Resting 20 min          Bauer et al.
                                                                                    mild asympt.       exposures produced no   (1986)
                                                                                                       effects. Slight excess
                                                                                                       decrease in FEV1 and
                                                                                                       PEFR in NO2 plus
                                                                                                       exercise above that
                                                                                                       caused by exercise
                                                                                                       alone. PEFR, -16% (air).
                                                                                                       -28% (NO2); FEV1 -5.5%
                                                                                                       (air), -9.3% (NO2).
                                                                                                       Significantly increased
                                                                                                       response to cold air
                                                                                                       after NO2 exposure.

    564          0.3          225        30           30-40          10 M/10 F      19-54 years        Group findings          Morrow & Utell
                                         (3  10)                                                      indicated no            (1989)
                                                                                                       significant responses.
                                                                                                       No change in lung
                                                                                                       function, symptoms,
                                                                                                       carbachol reactivity.
                                                                                                       Subjects studied
                                                                                                       previously (Bauer et
                                                                                                       al., 1986) showed
                                                                                                       possible responses to
                                                                                                       NO2. New subject
                                                                                                       subgroup showed
                                                                                                       significantly greater
                                                                                                       response in air
                                                                                                       exposures.
                                                                                                                                              

    Table 40  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    564          A. 03        110        60           42             A. 13 M        19-35 years,       FEV1 decreased 11%      Roger et al.
                                                                                    mild asthmatics    in NO2 but only 7% in   (1990)
                                                                                                       air, after first 10 min
                                                                                                       of exercise. Smaller
                                                                                                       changes later in
                                                                                                       exposure.

    282          B. 0.15      75         30           42             B. 21                             No increase in airway
    564          0.3                                                                                   reactivity to
    1128         0.6                                                                                   methacholine 2 h after
                                                                                                       exposure. Nochange in
                                                                                                       FEV1 or SRaw as a
                                                                                                       result at NO2
                                                                                                       exposure.

    564          0.3          180        90           30             24 M/10 F      10-16 years        After 60 min of         Avol et al.
                                                                                                       exposure, FEV1, FVC     (1989)
                                                                                                       and PEFR (-3.4, -4.0
                                                                                                       and -5.6%,
                                                                                                       respectively) were
                                                                                                       significantly reduced.
                                                                                                       No change in airways
                                                                                                       responsiveness to cold
                                                                                                       air challenge. SRaw
                                                                                                       increased 17% after NO2
                                                                                                       exposure. After 180 min
                                                                                                       of exposure, the
                                                                                                       responses had returned
                                                                                                       to baseline levels.
                                                                                                                                              

    Table 40  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    564          0.3          120        60           40             27 M/32 F      18-50 years,       Exercise-related        Avol et al.
                                                                                    some moderate      increases in symptoms.  (1988)
                                                                                    asthmatics         Possible NO2-related
                                                                                                       decrease in FEV1,
                                                                                                       PEFR. Increased cold
                                                                                                       air response after
                                                                                                       564 g/m3.

    1128         0.6          120        60           41                                               More consistent
                                                                                                       increases in SRaw at
                                                                                                       1128 g/m3 but not
                                                                                                       significantly different
                                                                                                       from air and 564 g/m3.

    564          0.3          60         30           41             15 M/6 F       20-34 years,       No effect of NO2.       Linn et al.
    1880         1.0          60         30           41                            mild asthmatics    Exercise-related        (1986)
    5640         3.0          60         30           41                                               increase in SRaw under
                                                                                                       all conditions.

    940          0.5          120        15                          9 M/4 F        19-50 years,       Increased respiratory   Kulle (1982)
                                                                                    3 Smokers          symptoms in 4/13
                                                                                                       subjects. Also,
                                                                                                       increased static lung
                                                                                                       compliance. Impossible
                                                                                                       to determine amount of
                                                                                                       effect due to NO2.
                                                                                                                                              

    Table 40  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    940          0.5          60                                     10             22-44 years,       No change in symptoms.  Mohsenin (1987b)
                                                                                    mild asthmatics    Significant group mean
                                                                                                       increase in
                                                                                                       responsiveness to
                                                                                                       methacholine after
                                                                                                       NO2 exposure. No other
                                                                                                       function changes.

    940 +        0.5 +        120        60           approx. 20     6 M/12 F       33 years,          No significant effect   Linn et al.
    857          0.3 ppm                                             6 ex-smokers   physician-         on spirometry, RT.      (1980a)
    SO2          SO2                                                 asthma         diagnosed

    7520         4.0          75         a. 15        a. 25          12 M/11 F      18-34 years,       No NO2 effects on       Linn & Hackney
                                         b. 15        b. 49                         physician-         SRaw, symptoms, heart   (1984); Linn et
                                                                                    diagnosed          rate, skin              al. (1985b)
                                                                                    asthma             conductance. Small
                                                                                                       decrease in
                                                                                                       systolic blood
                                                                                                       pressure.
                                                                                                                                              

    a  Modified from US EPA (1993)
       M = male; F = female; SGaw = specific airway conductance; FEV1 = forced expiratory volume in 1 second; VISOV = volume of isoflow;
       PEFR = peak expiratory flow; SRaw = specific airway resistance; FVC = forced vital capacity; Asympt. = asymptomatic;
       RT = total respiratory resistance; NS = not significant; SO2 = sulfur dioxide; SBN2 = single breath nitrogen washout

    FIGURE 22

    FIGURE 23

         Roger et al. (1990) reported the effects of NO2 exposure on mild
    asthmatics.  Their first study was a pilot study of 12 mild asthmatics
    exposed to 564 g/m3 (0.3 ppm) for 110 min, including three 10-min
    periods of exercise.  After the first 10 min of exercise in NO2,
    there was a decrease in FEV1 that persisted for the remainder of the
    exposure period, although the overall responses were progressively
    less with successive periods of exercise, as is common with
    exercise-induced asthma when the exercise is intermittent.  Their
    subsequent concentration-response study of twenty-one subjects 
    included six responsive subjects from the pilot study; volunteers were
    exposed to 282, 564 and 1128 g/m3 (0.15, 0.30 and 0.60 ppm) NO2 for
    75 min, with three 10-min exercise periods.  In contrast to the pilot
    study, there were no effects of NO2 on pulmonary function or airway
    responsiveness to methacholine, tested 2 h after exposure ceased.  The
    authors suggested that the differences between the pilot and the main
    study may have been due to more reactive airways in the pilot study
    asthmatics.  Because the studies were conducted during different
    seasons, seasonal differences in temperature, air pollution, ambient
    aeroallergens or other factors may have contributed to some of the
    variability in response.

         Asthmatics exposed to 230, 460 and 910 g/m3 (0.12, 0.24 and
    0.48 ppm) NO2 for 20 min were studied by Bylin et al. (1985). 
    Changes in SRaw during the four exposures averaged +3% after air and
    +9%, -2% and -14% after the three levels of NO2, respectively; these
    changes were not significantly different.  There was a tendency for an
    increase in thoracic gas volume (TGV) after NO2 exposures (9 to
    10%), but differences in pre-exposure values for TGV were probably
    responsible, rather than NO2.  There were no significant changes in
    tidal volume or respiratory rate.  At the highest concentration tested
    (910 g/m3, 0.48 ppm), histamine bronchial responsiveness was
    increased.

         In mild asthmatics exposed for 30 min to 260, 510 and 1000 g/m3
    (0.14, 0.27 and 0.53 ppm), there were no significant changes in SRaw,
    although there was a general trend for SRaw to fall throughout the
    period of exposure at all NO2 concentrations (Bylin et al., 1988). 
    There was, however, a significant increase (p = 0.03) in airway
    responsiveness to histamine after 30 min of exposure to 510 g/m3
    (0.27 ppm) only. The absence of a concentration-related increase in
    responsiveness is not inconsistent with other studies.  This
    observation contrasts with earlier results (Bylin et al., 1985) that

    suggested a possible increased responsiveness after exposure to
    910 g/m3 (0.48 ppm). Because of the use of a non-parametric pair
    comparison test that was not adjusted for multiple comparisons, the
    raw data presented in the paper were subjected to reanalysis (US EPA,
    1993) using a Friedman non-parametric analogue of an F test, which
    is probably more appropriate for these data than a series of
    Wilcoxon matched pairs signed rank tests.  This analysis showed no
    statistically significant change in histamine responsiveness due to
    NO2 exposure.

         Asthmatics exposed to 564 g/m3 (0.3 ppm) NO2 by mouthpiece
    for 20 min at rest followed by 10 min of exercise (30 litres/min)
    experienced a statistically significant spirometric response to NO2
    (Bauer et al., 1986).  After NO2 exposure, 9 out of 15 asthmatics had
    a decrease in FEV1; both the pre-post exposure difference on the NO2
    day (10.1%) and the pre-post NO2 minus the pre-post air (i.e.,
    delta-delta) differences (6%) were significant using a paired t-test. 
    Maximum expiratory flow at 60% total lung capacity (PEFV curve) was
    also decreased, but FVC and SGaw were not altered.  Nine out of
    twelve subjects experienced an increase in airway responsiveness to
    cold air.  The mouthpiece exposure system used in this study contained
    relatively dry air (relative humidity, RH, of 9 to 14% at 20C) and
    airway drying may have interacted with NO2 to cause greater
    responses.  However, Bauer et al. (1986) controlled for the airway
    drying effect by exposing subjects to clean air at the same
    temperature and RH.  Nevertheless, air temperature and humidity
    effects may be an important consideration for NO2 effects in winter
    in the temperate regions of the world.
 
        Linn et al. (1985b) and Linn & Hackney (1984) exposed mild
    asthmatics to 7520 g/m3 (4.0 ppm) NO2 for 75 min, with two 15-min
    exercise periods.  There was no significant difference in lung
    function that could be attributed to NO2; if anything, SRaw tended
    to be slightly lower with the NO2 exposures.

         The reasons for the differences between the group of asthmatics
    exposed to 7520 g/m3 (4 ppm) for 75 min (with exercise) (Linn et
    al., 1985b) and the group exposed to 564 g/m3 (0.30 ppm) for 30 min
    with exercise studied by Bauer et al. (1986) are not clear.  The
    subjects of Bauer et al. were exposed to NO2 in dry air through a
    mouthpiece which could have caused some drying of the upper airways;
    Linn et al. (1985b) used a chamber exposure.  Second, the subjects in
    the Linn et al. (1985b) study tended to have milder asthma than the
    subjects in the Bauer et al. (1986) study.  There were differences in
    the season in which the two studies were conducted, and there may have
    been a difference in background exposure to NO2 (outdoors and/or
    indoors).  In addition, increased bronchial reactivity to cold air was
    an important finding in the Bauer et al. (1986) study, but it was not
    measured by Linn et al. (1985b).

 

         Further research was conducted by Linn et al. (1986) on mild
    asthmatics exposed to 564, 1880 and 5640 g/m3 (0.30, 1.0 and
    3.0 ppm) NO2 for 1 h.  The exposures included intermittent, moderate
    exercise.  As in the previous study with 7520 g/m3 (4.0 ppm) NO2,
    there were no significant effects of NO2 on spirometry, SRaw or
    symptoms.  Furthermore, there was no significant effect on airway
    responsiveness to cold air.  In order to examine the suggestion that
    the severity of response to NO2 may be related to the clinical
    severity of asthma, the authors selected three subjects characterized
    as having more severe illness.  Although they experienced markedly
    larger changes in resistance than other milder asthmatics under all
    exposure conditions, there was no indication that the responses of
    these subjects were related to NO2 exposure.

         Mohsenin (1987a) found no changes in symptoms, spirometry, or
    plethysmography in mild asthmatics exposed to 940 g/m3 (0.5 ppm)
    NO2 for 1 h at rest.  However, airway responsiveness to methacholine
    increased after the NO2 exposure.

         The effects of previous NO2 exposure on SO2-induced
    bronchoconstriction has been  examined by Joerres & Magnussen (1990)
    and Rubinstein et al. (1990).  Neither study found changes in
    pulmonary function after NO2 exposure.  Joerres & Magnussen (1990)
    exposed mild-to-moderate asthmatic subjects to 470 g/m3 (0.25 ppm)
    NO2 for 30 min while breathing through a mouthpiece at rest.  After
    the NO2 exposure, airway responsiveness to 1965 g/m3 (0.75 ppm)
    SO2 was increased.  Rubinstein et al. (1990) exposed asthmatics to
    564 g/m3 (0.30 ppm) NO2 for 30 min (including 20 min light
    exercise).  No mean change in responsiveness to SO2 occurred, but one
    subject showed a tendency toward increased responsiveness.  The
    reasons for the different findings in these two studies is not clear,
    especially as the subjects of Rubinstein et al. (1990) were exposed to
    a higher NO2 concentration and exercised during exposure.  However,
    Joerres & Magnussen's subjects appeared to have had slightly more
    severe asthma and were somewhat older.  The modest increase in SRaw
    caused by exercise in the Rubinstein et al. (1990) study may have
    induced a refractory state to SO2.  Finally, the different method of
    administering the SO2 bronchoprovocation test may have had an
    influence.  Joerres & Magnussen (1990) increased minute ventilation
    (V.E) at a constant SO2 concentration, whereas Rubinstein et al.
    (1990) increased SO2 concentration at constant VE.

         A number of studies of the effects of NO2 exposure in asthmatics
    on changes in airway responsiveness to bronchoconstrictors have been
    presented in Table 40, but not evaluated in the text.  Various types
    of inhalation challenge tests have been used (methacholine, histamine,
    cold air, etc.).  Some exposures were conducted at rest and others
    while performing some exercise.  For twenty studies for which
    individual data were available, a meta analysis (Folinsbee, 1992) was
    performed to assess the changes in airway responsiveness in asthmatics
    exposed to NO2.  The aim of the meta analysis was to examine the
    diversity of response seen in the various studies and to examine
    factors such as NO2 concentration, exercise, and airway challenge
    method that could help explain some of the variability in response. 
    Such questions could not be adequately addressed using individual
    studies.  The analysis provides only a qualitative examination of
    concentration-response relationships.  For this analysis, the
    directional change (i.e., increased or decreased) in airway
    responsiveness after NO2 exposure was determined for each subject. 
    The data were then organized by exposure concentration range and
    whether or not exposures included exercise.  Within each exposure
    category the fraction of subjects with increased airway responsiveness
    was determined (see Table 41).  For the total of 355 individual NO2
    exposures, 59% of the asthmatics had increased responsiveness.  If the
    response was not associated with NO2 exposure, the fraction would be
    expected to approach 50%.  The excess increase in responsiveness can
    be attributed primarily to the NO2 exposures conducted at rest
    (fraction was 69%).  There was a larger fraction of increased
    responsiveness during the resting exposures in all three concentration
    ranges (see Table 41).  In the exercising studies, however, there was
    no effect because only 51% had an increase in airway responsiveness. 
    There was a trend for a slightly larger percentage (approx. 75%) of
    subjects to have increased airway responsiveness after NO2 exposures
    above 376 g/m3 (0.20 ppm) and under resting conditions.  Of those
    six studies independently reporting a statistically significant
    response (Kleinman et al., 1983; Bylin et al., 1985, 1988; Bauer et
    al., 1986; Mohsenin, 1987a; Joerres & Magnussen, 1990), four were
    resting exposures, and in four the exposure duration was 30 min or
    less.  Although the authors offered various hypotheses for this

    apparent effect of low-level NO2 resting exposures, the mechanisms
    are unknown.  Changes in responsiveness were seen with relatively
    brief exposures.  One possible explanation for the absence of
    response in the exercising exposures is that exercise-induced
    bronchoconstriction may interfere with the NO2-induced response or
    that prior exercise may cause the airways to become refractory to the
    effects of NO2.  Possible confounding influences of nitric oxide, not
    measured in most studies, cannot be determined.

    Table 41.  Fraction of nitrogen dioxide-exposed subjects with
               increased airway responsivenessa
                                                                        

    Nitrogen dioxide   All             Exposures      Exposure
    concentration      exposures       with exercise  at rest
    (ppm)
                                                                        

    Asthmatics

    0.05-0.20          0.64 (105)b     0.59 (17)      0.65 (88)b
    0.20-0.30          0.57 (169)      0.52 (136)     0.76 (33)b
    > 0.30             0.59 (81)       0.49 (48)      0.73 (33)c
    All NO2            0.59 (355)b     0.51 (202)     0.69 (154)b
      concentrations

    Healthy

    < 1.0              0.47 (36)                      0.47 (36)
    < 1.0              0.79 (29)b    0.73 (15)        0.86 (14)c
                                                                        

    a  Data are fraction of subjects with an increase in airways
       responsiveness above the value for clean air. Numbers in
       parenthesis indicate actual number of subjects in each category.
       Total number = 355.  Ties (i.e. no change) were excluded.
    b  p < 0.01 two-tailed sign test
    c  p < 0.05 two-tailed sign test

         A similar meta analysis for healthy subjects indicated increased
    airway responsiveness after exposure to NO2 concentrations greater
    than 1880 g/m3 (1 ppm). Exercise during exposure did not appear to
    influence the responses as much in the healthy subjects as in the
    asthmatics, but a similar trend was evident.

    6.2.1.3  Nitrogen dioxide effects on patients with chronic obstructive
             pulmonary disease

         Patients with COPD represent an important potentially sensitive
    population group. Studies evaluating NO2 effects on respiratory
    function in COPD subjects are summarized in Table 42.  The results of
    two NO2 exposure studies (9400 to 15 040 g/m3, 5 to 8 ppm NO2 for
    up to 5 min) were discussed by Von Nieding et al. (1980), who found
    that the responses of bronchitics were generally similar to those of
    healthy subjects.  There was a tendency for the response to NO2 to be
    greater in the subjects with the highest baseline Raw.  Percentage
    changes ranged from approximately 25 to 50%.  In a review of their
    studies, Von Nieding & Wagner (1979) showed that Raw increased in
    chronic bronchitics exposed to > 3760 g/m3 (2.0 ppm) NO2.

         The responses of COPD patients were affected by exposure (with
    mild exercise) to 564 g/m3 (0.3 ppm) NO2 for 3.75 h (Morrow &
    Utell, 1989).  Forced vital capacity showed progressive and
    significant decreases during and following NO2 exposure, the largest
    change of -9.6% occurring after 3.75 h of exposure.  Smaller
    decrements in FEV1 (-5.2%) occurred at the end of exposure. There was
    no effect of NO2 on SGaw or diffusing capacity.  The severity of
    disease (based on impairment of lung function: FEV1 < 60% predicted
    vs. > 60% predicted) generally did not influence the magnitude of
    response to NO2.  The COPD patients showed a decrement in FEV1
    compared to the healthy, elderly non-smokers who experienced an
    improvement in FEV1.  In contrast, Linn et al. (1985a) found no
    effects from a 1-h exposure (with exercise) to 940, 1880 and
    3760 g/m3 (0.5, 1.0 and 2.0 ppm) NO2 in a diverse group of COPD
    patients.  The reasons for the marked difference in responses between
    the two studies are not known.  Ambient exposure to air pollution in
    general and NO2 in particular was probably much higher for the
    subjects in the Linn et al. (1985a) study.  Thus, attenuation of
    physiological responses may have been a factor.

         Hackney et al. (1992) studied effects of field exposure to
    ambient air and chamber exposure to 564 g/m3 (0.3 ppm) NO2 in       
    older adults with evidence of COPD and a history of heavy smoking. 
    They reported only slight adverse effects of NO2.  The study did not
    strongly confirm the findings  of Morrow & Utell (1989) and Morrow et
    al. (1992), and the authors speculated that ambient exposure history
    may have been responsible for differences between these studies.

    6.2.1.4  Age-related differential susceptibility

         Studies evaluating possible age-related differences in
    susceptibility to NO2 effects on respiratory function in healthy
    subjects are summarized in Table 39.

         Research on asthmatics is summarized in Table 40.  Spirometry
    measurements of young (18 to 26 years old) and older (51 to 76 years
    old) men and women were not affected by exposure to 1128 g/m3
    (0.6 ppm) NO2 with light intermittent exercise (Drechsler-Parks et
    al., 1987; Drechsler-Parks, 1987).  In addition, Morrow & Utell (1989)
    did not observe any pulmonary function or airway responsiveness
    effects due to a lower level of NO2 (564 g/m3, 0.3 ppm) in young or
    elderly healthy subjects.

         Koenig et al. (1985) found no "consistent significant changes in
    pulmonary functional parameters" after 1-h resting exposures of
    asthmatic adolescents to 226 g/m3 (0.12 ppm) NO2. Subsequent
    mouthpiece exposures to 226 g/m3 NO2, with exercise, caused
    increases in RT and decreases in FEV1 after both air and NO2
    exposure, which were apparently due to exercise alone (Koenig et al.,
    1987a,b).  When subjects were exposed to a higher level of NO2
    (338 g/m3, 0.18 ppm), no differences in RT occurred.  Decreases in
    FEV1 were -1.3 and -3.3% for air and NO2, respectively; this
    difference (p = 0.06) may indicate a possible response trend.

    6.2.2  Nitrogen dioxide effects on pulmonary host defences and
           bronchoalveolar lavage fluid biomarkers

         Nitrogen dioxide can enhance susceptibility to infectious
    pulmonary disease, as clearly demonstrated in the animal toxicological
    literature (chapter 5).  Epidemiological studies (chapter 7) suggest
    similar effects.  Human clinical studies of NO2 effects on host
    defences are summarized in Table 43.

         Kulle & Clements (1988) and Goings et al. (1989) (two reports of
    the same study) examined the effect of NO2 exposure on susceptibility
    to attenuated influenza virus.  Healthy adults were exposed for
    2 h/day for 3 days to either clean air or 1880, 3760 or 5640 g/m3
    (0, 1.0, 2.0 or 3.0 ppm) NO2.  The virus was administered
    intranasally after the second day of exposure, and infectivity was
    defined as the presence of virus in nasal washes, a rise in either
    nasal wash or serum antibody titres to the virus, or both.  Although
    the rates of infection were elevated after NO2 exposure in some of
    the NO2-exposed groups (91% of subjects exposed to 1880 or
    3760 g/m3 (1 or 2 ppm) infected vs. 71% of controls), the changes
    were not significant.  The investigators concluded that the results of
    the study were inconclusive, rather than negative, because the
    experimental design had a low power to detect a 20% difference in
    infection rate, decreasing the possibility of statistical
    significance.

        Table 42.  Effects of nitrogen dioxide on lung function and airway responsiveness of chronic obstructive pulmonary disease patientsa
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    564          0.3          225        21           25             13 M/7 F       47-70 years,       Total NO2 inhaled       Morrow &
                                         (3  7)                                    8 mild,            dose 1.215 mg.          Utell (1989)
                                                                                    12 moderate        Decrease in FVC after
                                                                                                       exposure-9.6%. 5.2%
                                                                                                       decline in FEV1
                                                                                                       significant after
                                                                                                       approx. 4-h exposure.

    564          0.3          240        28           25             15 M/11 F      47-69              No significant change   Hackney et al.
                                         (4  7)                                                       in FVC or FEV1 with     (1992)
                                                                                                       NO2 exposure

    940          0.5          120        15           25             7              24-53 years,       No effects in           Kerr et al.
                                                                                    daily cough        bronchitics alone.      (1979)
                                                                                    for 3 months       Possible decrease in
                                                                                                       quasistatic compliance.

    940          0.5          60         30           16             13 M/9 F       48-69 years,       No change in FVC,       Linn et al.
                                                                                    some with          FEV1, etc. at any NO2   (1985a)
    1880         1.0                                                                emphysema,         level. SRaw tended to
                                                                                    some with          increase after first
    3760         2.0                                                                chronic            exercise period.
                                                                                    bronchitis         Possible decrease in
                                                                                                       peak flow at
                                                                                                       3760 g/m3. No symptom
                                                                                                       changes. No change
                                                                                                       in SaO2.
                                                                                                                                              

    Table 42  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    940-9400     0.5-5        15                                     88                                Decrease in earlobe     Von Nieding et
                                                                                                       blood PO2 at            al. (1971, 1970)
                                                                                                       > 7520 g/m3.
                                                                                                       Increased Raw at
                                                                                                       > 3008 g/m3.

    1880-9400    1-5          30 breaths                             84 M           30-72 years,       Increase in Raw         Von Nieding et
                              (15 min)                                              chronic non-       related to NO2          al. (1973a)
                                                                                    specific disease   concentration. No
                                                                                                       effect on Raw below
                                                                                                       2820 g/m3.

    9400         5            60                                                                       Changes in PO2 of
                                                                                                       earlobe capillary
                                                                                                       blood. Change occurred
                                                                                                       in first 15 min,
                                                                                                       effect did not
                                                                                                       increase with further
                                                                                                       exposure.
                                                                                                                                              

    Table 42  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    1880-15 040  1-8 ppm      5-60                                   116            25-74 years        At 7520-9400 g/m3      Von Nieding &
                                                                                                       for 15 min, PaO2        Wagner (1979)
                                                                                                       decreased
                                                                                                       (arterialized capillary
                                                                                                       blood). Raw increased
                                                                                                       with exposure to
                                                                                                       > 3008 g/m3.
                                                                                                                                              

    a  Modified from US EPA (1993)
       Abbreviations: FVC = Forced vital capacity; FEV1 = Forced expiratory volume in 1 second; PaO2 = Arterial partial pressure of oxygen;
       PO2 = Partial pressure of oxygen; Raw = Airway resistance; SRaw = Specific airway resistance; SaO2 = Arterial oxygen saturation

    Table 43.  Effects of nitrogen dioxide on host defences of humansa
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    508          0.27         60                                     M              Healthy, young     No change in nasal or   Rehn et al.
    1993         1.06                                                                                  tracheobronchial        (1982)
                                                                                                       clearance.

    (1) 1128     (1) 0.6      180        60           39             6 M/2 F        30.3  1.4         Total NO2 uptake (1)    Frampton et al.
                                                                                    years, healthy,    3.4 mg (2) 5.6 mg, (3)  (1989b)
                                                                                    NS                 approx.3.3 mg (4)
                                                                                                       8.1 mg. BAL fluid
                                                                                                       analysis showed no
                                                                                                       significant effect on
                                                                                                       total protein or
                                                                                                       albumin
    (2) Var      (2) Var      180        60           43             11 M/4 F       25.3  1.2         Apparent increase in
    (94          (0.05                                                              years, healthy,    alpha-2-macro-globulin
    background   background                                                         NS                 3.5 h after exposure
    with 3      with 3  15                                                                           to 0.6 ppm (Group 1)
    15 min at    min at                                                                                but not after the
    3760)        2.0 ppm)                                                                              other protocols. No
                                                                                                       changes in percentage
                                                                                                       of lymphocytes or
                                                                                                       neutrophils. Concluded
                                                                                                       that NO2 at these
                                                                                                       concentrations neither
    (3) 1128     (3) 0.6      180        60           approx. 40     5 M/3 F        32.6  1.6         altered epithelial
                                                                                    years, healthy,    permeability nor
                                                                                    NS                 caused inflammatory
                                                                                                       cell influx.
                                                                                                                                              

    Table 43  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    (4) 2820     (4) 1.5      180        60           39             12 M/3 F       23.5  0.7
                                                                                    years, healthy,
                                                                                    NS

    1128         0.6          120/day    60           approx. 30-40  4 M/1 F        21-36 years,       Slight increase in      Boushey et al.
                              for 4 days                                            Healthy, NS.       circulating (venous)    (1988) (Part 2)
                                                                                    FEV1/FVC%          lymphocytes:
                                                                                    range 73-83%,      1792  544 per mm3
                                                                                    "normal"           (post-NO2) vs.
                                                                                    methacholine       1598  549 per mm3
                                                                                    responsiveness     (baseline). No change
                                                                                                       in BAL lymphocytes
                                                                                                       except an increase in
                                                                                                       natural killer cells:
                                                                                                       7.2  3.1% (post-NO2)
                                                                                                       vs. 4.2  2.4%
                                                                                                       (baseline). No change
                                                                                                       observed in IL-1
                                                                                                       or TNF.

    1128         0.6          180        60           approx. 40     7 M/2 F        Healthy, NS        No change in cell       Frampton et al.
                                         (6  10)                                                      recovery or             (1989a)
                                                                                                       differential counts.
                                                                                                       Possible decrease in
                                                                                                       macrophage
                                                                                                       inactivation of virus
                                                                                                                                              

    Table 43  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    94 with      0.05 with    135        60                          11 M/4 F       Nonreactive        in vitro. Possible
    3760         2.0 spikes   3  15     (6  10)                                   (carbachol), no    sensitive subgroup.
    spikes                                                                          recent upper
                                                                                    resp. infection

    1880         1.0          180        Intermittent                3 M/5 F        Healthy            No responses.           Jorres et al.
                                                                                                                               (1992)

    1880         1.0          120/day                                22             Healthy, NS,       Study conducted over    Goings et al.
    3760         2.0          3 days                                 21, 22         seronegative       3-year period. NO2 did  (1989)
    5640         3.0                                                 22                                not significantly
                                                                                                       increase viral
                                                                                                       infectivity, although
                                                                                                       a trend was observed.
                                                                                                       This study had a low
                                                                                                       power to detect small
                                                                                                       differences in
                                                                                                       infection rate.

    3760         2.0          240        120          50             10             Healthy, NS        Increased bronchial     Devlin et al.
                                                                                                       PMN's and decreased     (1992); Becker
                                                                                                       macrophage phagocytosiset al. (1993)

    3760         2.0          360        Intermittent                12             Healthy, NS        Immediate and 18-h      Frampton et al.
                                                                                                       post-BAL increase       (1992)
                                                                                                       in PMN.
                                                                                                                                              

    Table 43  (Con't)
                                                                                                                                              

    NO2 concentration         Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    4230         2.25         20         20           approx. 35     8              Healthy, NS        Increased levels of     Sandstroem et
    7520         4.0                                                 8                                 mast cells in BAL       al. (1989)
    10 340       5.5                                                 8                                 fluid at all
                                                                     Total n = 18                      concentrations.
                                                                                                       Increased numbers of
                                                                                                       lymphocytes at
                                                                                                       > 7520 g/m3
                                                                                                       (BAL 24-h
                                                                                                       post-exposure).

    7520         4.0          20 min-    20           approx. 35     8              Healthy, NS        Total cell counts       Sandstroem et
                              alternate                                                                were reduced. Alveolar  al. (1990a)
                              days for                                                                 macrophages had
                              12 days                                                                  enhanced phagocytic
                                                                                                       activity but fewer
                                                                                                       were present.
                                                                                                       Decreased numbers
                                                                                                       of mast cells, T and
                                                                                                       B lymphocytes, and
                                                                                                       natural killer cells
                                                                                                       (BAL 24-h
                                                                                                       post-exposure).
                                                                                                                                              

    a  Modified from US EPA (1991)

       Abbreviations: M = Male; F = Female; NS = Non-smoker; FEV1 = Forced expiratory volume in 1 second; FVC = Forced vital capacity;
       BAL = Bronchoalveolar lavage; IL-1 = Interleukin-1; TNF = Tumour necrosis factor; VAR = Variable
             Others investigated the effects of NO2 on cells and fluids in
    bronchoalveolar lavage (BAL) of healthy adults.  Frampton et al.
    (1989a) used two different exposure protocols that had the same
    concentration  time product.  One group was exposed for 3 h to
    1128 g/m3 (0.6 ppm), whereas the other was exposed to a background
    level of 94 g/m3 (0.05 ppm) with three 15-min spikes of 3760 g/m3
    (2.0 ppm).  Both exposures included exercise.  Pulmonary function and
    airway responsiveness were not affected.  Alveolar macrophages (AM)
    obtained by BAL after exposure to 1128 g/m3 NO2 tended to
    inactivate virus less effectively than AM collected after air
    exposure.  The AMs that showed the impairment of virus inactivation
    also showed an increase in interleukin-1 production, not seen in the
    AMs from other subjects.  Interleukin-1 is a proinflammatory protein
    produced by AMs, which performs a number of immunoregulatory
    functions, including induction of fibroblast proliferation, activation
    of lymphocytes, and chemotaxis for monocytes.  The study had
    relatively low statistical power to detect an effect.  Becker et al.
    (1993) reported no change in virus inactivation properties of alveolar
    macrophages lavaged from subjects exposed to 3760 g/m3 (2 ppm) for
    4 h.

         Using exposures similar to the above, with the addition of two
    groups exposed to 2820 g/m3 (1.5 ppm) NO2 for 3 h, one with BAL at
    3.5 h post-exposure and the other with BAL at 18 h post-exposure,
    Frampton et al. (1989b) examined changes in protein in BAL fluid.  The
    total protein and albumin content of BAL fluid obtained at either 3.5-
    or 18-h post-exposure was not changed.  In BAL fluid obtained 3.5 h
    after exposure to 1128 g/m3 (0.60 ppm) there was an increase in
    alpha-2-macroglobulin, a regulatory protein that has antiprotease
    activity and immunoregulatory effects.  This response was not seen in
    the group lavaged at 18 h post-exposure and no such effect occurred at
    a higher NO2 concentration (2820 g/m3).

         Sandstroem et al. (1989) exposed healthy subjects to 4230, 7520
    and 10 340 g/m3 (2.25, 4.0 and 5.5 ppm) for 20 min (with moderate
    exercise) and performed BAL 24 h after exposure.  Increased numbers of
    mast cells were observed at all NO2 concentrations; numbers of
    lymphocytes were increased only at > 7520 g/m3.  In order to
    determine the time course of this response, Sandstroem et al. (1990a)
    exposed four groups of healthy subjects to 7520 g/m3 NO2 for 20 min
    (mild exercise) and then performed BAL 4, 8, 24 or 72 h after
    exposure. Increased numbers of mast cells and lymphocytes were
    observed at 4, 8 and 24 h but not at 72 h.  There was no change in the
    numbers of AMs, eosinophils, polymorphonuclear leukocytes, T cells or
    epithelial cells, or in the albumin concentration of lavage fluid. 
    The authors interpreted the increased numbers of mast cells and
    lymphocytes as a nonspecific inflammatory response.

         Sandstroem et al. (1990b) also evaluated responses to repeated
    NO2 exposures.  Healthy subjects were exposed to 7520 g/m3
    (4.0 ppm) NO2 for 20 min/day (with moderate exercise) on alternate
    days over a 12-day period (seven exposures in all); BAL was performed

    24 h after the last exposure.  The first 20 ml of BAL fluid was
    treated separately and presumed to represent primarily bronchial cells
    and secretions; subsequent fractions presumably were from the alveolar
    region.  In the first fraction, there was a reduction in the numbers
    of mast cells and AMs; AM phagocytic activity (on a per cell
    basis) was increased.  In addition, there were reduced numbers of
    T-suppressor cells, B cells and natural killer (NK) cells in the
    alveolar portion of the BAL.  This pattern of cellular response
    contrasts with that after single NO2 exposure (Sandstroem et al.,
    1990a).

         Rubinstein et al. (1991) studied five healthy volunteers exposed
    for 2 h/day for 4 days to 1128 g/m3 (0.60 ppm) NO2 with
    intermittent exercise.  A slight increase in circulating (venous
    blood) lymphocytes was observed.  The only change observed in BAL
    cells was a modest increase in the percentage of NK cells, suggesting
    a possible increase in immune surveillance.

         Three recent studies examined the effects of longer exposures to
    1880 or 3760 g/m3 (1.0 to 2.0 ppm) NO2 on lavaged cells and
    mediators.  Devlin et al. (1992) (also Becker et al., 1993) studied
    healthy subjects exposed to 3760 g/m3 NO2 for 4 h with alternating
    15-min periods of rest and moderate exercise.  One of the main
    findings after NO2 exposure was that there was a three-fold increase
    in PMNs in the first lavage sample, representing predominantly
    bronchial cells and fluid.  In addition, macrophages recovered from
    the predominantly alveolar fraction showed a 42% decrease in ability
    to phagocytose  Candida albicans  and a 72% decrease in release of
    superoxide anion.  In another study, Frampton et al. (1992) exposed
    exercising subjects to 3760 g/m3 NO2 for 6 h.  Bronchoalveolar
    lavage was performed either immediately or 18 h after exposure.
    There was a modest increase in lavage fluid PMN levels (< two-fold
    increase) but no change in lymphocytes.  Alveolar macrophage
    production of superoxide anion was not altered in these subjects. 
    These two studies suggest that NO2 exposure may induce a mild
    bronchial inflammation and may also lead to impaired macrophage
    function.  On the other hand, Joerres et al. (1992) examined both
    healthy and asthmatic subjects exposed to 1880 g/m3 NO2 for 3 h,
    but observed no changes in cells or mediators in BAL fluid or in the
    appearance of bronchial mucosal biopsies after this exposure.  Neither
    macrophage function nor a specific bronchial washing were examined in
    this study.

         Rehn et al. (1982) reported that a 1-h exposure to either 500 or
    2000 g/m3 (0.27 or 1.06 ppm) NO2 did not alter nasal or
    tracheobronchial mucociliary clearance rates.

    6.2.3  Other classes of nitrogen dioxide effects

         There have been isolated reports that higher levels of NO2
    (> 7520 g/m3, 4.0 ppm) can decrease arterial oxygen partial
    pressure (PaO2) (Von Nieding & Wagner, 1977; Von Nieding et al.,
    1979) and cause a small decrease in systemic blood pressure (Linn et
    al., 1985b).  However, the impact of such changes is not clear,
    especially considering the high concentrations of NO2 required.

         The effects of NO2 on the constituents of BAL fluid, blood and
    urine have been examined in very few studies and are reviewed in more
    detail elsewhere (US EPA, 1993).  The general purpose of this research
    was to examine mechanisms of pulmonary effects or to determine whether
    NO2 exposure could result in systemic effects.  Investigations of the
    effects of NO2 on levels of serum enzymes and antioxidants have been
    conducted, but few effects were found and they cannot be interpreted
    (Posin et al., 1978; Chaney et al., 1981).  For example, Chaney et al.
    (1981) found an increase in glutathione levels, but Posin et al.
    (1978), using a higher NO2 concentration, did not find such an
    effect.  Studies of exposure to NO2 concentrations between 2820 and
    7520 g/m3 (1.5 and 4.0 ppm) found either slight or no changes in BAL
    levels of alpha-1-antitrypsin, which inhibits protease activity
    (Mohsenin & Gee, 1987; Johnson et al., 1990; Mohsenin, 1991). Healthy
    subjects exposed to 7520 g/m3 NO2 (Mohsenin, 1991) at rest for 3 h
    showed increased lipid peroxidation products in BAL fluid obtained
    immediately after exposure. In addition, the activity or the elastase
    inhibitory capacity (EIC) of alpha-1-protease inhibitor (alpha-1-PI)
    was decreased after NO2 exposure.  However, vitamin C supplementation
    for 4 weeks prior to NO2 exposure markedly attenuated the EIC
    response and resulted in a lower level of lipid peroxidation products. 
    The author suggested that the reduced activity of alpha-1-PI may have
    implications for the pathogenesis of emphysema, especially in smokers. 
    At a lower NO2 concentration (3760 g/m3, 2.0 ppm, for 4 h), Becker
    et al. (1993) reported no change in alpha-1-antitrypsin.  Potential
    effects of NO2 on collagen metabolism have been investigated by
    examining urinary excretion of collagen metabolites after a 3-day
    (4 h/day) exposure to 1128 g/m3 (0.6 ppm) NO2, but no effects were
    found (Muelenaer et al., 1987).

    6.3  Effects of other nitrogen oxide compounds

         Relatively few controlled human exposure studies have been
    conducted that evaluate NOx species other than NO2.  Such studies
    are summarized in Table 44 and concisely discussed here.

        Table 44.  Effects of other nitrogen oxide (NOx) compounds on humansa
                                                                                                                                              

    Concentrations            Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    HNO2         0.004        210                                    15             Healthy            A dose-dependent        Kjaergaard et
                 0.077                                               (11 M/4 F)     22-57 years        vasodilation in         al. (1993)
                 0.395                                                                                 bulbar conjunctiva.
                                                                                                       Significant increase
                                                                                                       of polymorphonuclear
                                                                                                       neutrophils, cuboidal
                                                                                                       and squamous epithelium
                                                                                                       cell counts in the
                                                                                                       tear fluid

    HNO3

    129          0.050        40         10           approx. 25-30  5 M/4 F        12-17 years,       FEV1 decreased -4.4%    Koenig et al.
                                                                                    asthmatic          after HNO3 and -1.7%    (1989a)
                                                                                                       after HNO3 plus air
                                                                                                       exposure. RT increased
                                                                                                       +22.5% after HNO3 and
                                                                                                       +7.4% after air
                                                                                                       exposure.

    200          0.078        120        100          Mod.           4 M/1 F        Healthy            In BAL, increase in     Becker et al.
                                                                                                       AM phagocytosis and     (1991)
                                                                                                       AM infection
                                                                                                       resistance.

    500          0.194        240        240          40             10             Healthy            No effect on FEV1,      Aris et al.
                                                                                                       FVC, SRaw or BAL        (1991)
                                                                                                       cells.
                                                                                                                                              

    Table 44  (Con't)
                                                                                                                                              

    Concentrations            Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    NO

    1230         1.0          120        60           50 W           8 M            19-24 years        Suggested change in     Kagawa (1982)
                                                                                                       density dependance of
                                                                                                       expired flow.

    12 300-      10-39        15                                     191            Healthy,           Increase in total       Von Nieding et
    47 970                                                                          20-50 years        respiratory resistance  al. (1973b)
                                                                                                       at > 24 600 g/m3 and
                                                                                                       a decrease in PaO2 at
                                                                                                       > 18 450 g/m3.

    NH4NO3

    200          (1.1 MMAD)   120        60           approx. 20     20             Normal             No significant changes  Kleinman et al.
                                                                     19             Asthmatic          due to NH4NO3 in        (1980)
                                                                                                       normals or asthmatics
                                                                                                       except possible
                                                                                                       decrease in RT.
                                                                                                       No symptoms and
                                                                                                       effects.

    80 + 940     (0.55 MMAD)  240        30           55             12             Normal             No effects.             Stacy et al.
    g/m3        +0.5 ppm                                                                                                      (1983)
    NO2          NO2
                                                                                                                                              

    Table 44  (Con't)
                                                                                                                                              

    Concentrations            Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    NaNO3

    10, 100,     (0.2 MMAD)   10                                     5              Normal             No effects.             Sackner et al.
    1000                                                             5              Asthmatic                                  (1979)

    1000                                                             6              Normal
                                                                     6              Asthmatic

    7000         (0.46        16 ( 2)                               10             Normal             No effects.             Utell et al.
                 MMAD)        32 (total)                             11             Mild asthmatics                            (1979)

    7000         (0.49        16 ( 2)                               11             Influenza          No symptoms. SGaw       Utell et al.
                 MMAD)        32 (total)                                            patients           decrease 17% and VE    (1980)
                                                                                                       max 40% TLC decreased
                                                                                                       by 12% after nitrate,
                                                                                                       within 2 days of onset
                                                                                                       of illness. Similar
                                                                                                       effect 1 week later
                                                                                                       but not 3 weeks later.
                                                                                                                                              

    a  Modified from US EPA (1993)
       Abbreviations:

       W = Watt; M = Male; PaO2 = Arterial partial pressure of oxygen; HNO3 = Nitric acid; FEV1 = Forced expiratory volume in 1 second;
       FVC = Forced vital capacity; SRaw = Specific airway resistance; BAL = Bronchoalveolar lavage; AM = Alveolar macrophage; F = Female;
       RT = Total respiratory resistance; NS = Not significant; MMAD = Mass median aerodynamic diameter; SGaw = Specific airway conductance;
       VE max 40% TLC = Maximum expiratory flow at 40% of total lung capacity on a partial expiratory flow-volume curve
             Von Nieding et al. (1973b) exposed healthy subjects and smokers
    to 12 300 to 47 970 g/m3 (10 to 39 ppm) NO for 15 min.  Total
    respiratory resistance increased significantly (approx. 10-12%) after
    exposure to > 24 600 g/m3 (> 20 ppm) NO.  Diffusing capacity
    was not changed, but a small decrease (7 to 8 torr) in PaO2 was noted
    between 18 450 and 36 900 g/m3 (15 to 30 ppm).  Kagawa (1982)
    examined the effects of a 1230 g/m3 (1 ppm) NO exposure for 2 h in
    normal subjects.  A few individuals had increases in SGaw, and a few
    had decreases.  Analysis of the group mean data produced only one
    apparently statistically significant change: an 11% decrease in flow
    at 50% FVC in a helium-air mixture compared to this flow in air. 
    However, because the data were analysed by multiple t-tests the
    results should be interpreted with this in mind.

         NO is naturally formed in the body from the amino acid L-arginine
    and performs a second messenger function in several organ systems.  It
    has been measured in expired air (Gustafsson et al., 1991) and causes
    vasodilation in the pulmonary circulation.  Recently, NO has been used
    clinically to treat pulmonary hypertension in COPD patients and in
    infants with persistent pulmonary hypertension of the newborn (Zapol
    et al., 1994).

         In healthy volunteers made hypoxic by breathing 12% oxygen in
    nitrogen, the inhalation of 49 403 g/m3 (40 ppm) NO prevented the
    hypoxia-induced increase in pulmonary artery pressure (Frostell et
    al., 1993).  Systemic arterial pressure was not changed.  No
    evaluation of effects on lung function were performed.  Adnot et al.
    (1993) studied a group of COPD patients who had pulmonary artery
    pressures averaging 32 mmHg.  They breathed 6130 to 49 403 g/m3
    (5 to 40 ppm) NO for successive 10-min periods.  There was a
    dose-dependant decrease in pulmonary artery pressure during NO
    inhalation and no alteration of systemic arterial pressure.  Moinard
    et al. (1994) observed a 20% drop in pulmonary artery pressure in COPD
    patients after breathing 18 391 g/m3 (15 ppm) NO for 10 min.  Based
    on an improvement in alveolar ventilation in some segments of the
    lung, the authors postulated that NO may also act as a bronchodilator. 
    Hoegman et al. (1993) suggested a modest bronchodilator effect of
    98 080 g/m3 (80 ppm) NO.  Based on findings in animals, which are
    summarized in chapter 5, NO does cause bronchodilation at similar
    concentrations (Barnes, 1993).

         Nitrous acid and nitric acid may be formed from the reaction of
    NO2 with water.  Nitrous acid may also be produced directly in the
    combustion process.

         Koenig et al. (1989a) examined the responses of adolescent
    asthmatics to a 40-min exposure to 129 g/m3 (0.05 ppm) HNO3 vapour
    via a mouthpiece exposure system.  After 30 min of rest and 10 min of
    exercise while breathing HNO3, there was a 4.4% decrease in FEV1
    compared to a 1.7% decrease after air  breathing.  A 22.5% increase in
    total respiratory resistance was also observed after HNO3 exposure,
    compared to a 7.4% increase after air breathing.

         The effects of HNO3 on BAL endpoints have been reported.  Becker
    et al. (1992) exposed healthy subjects to 200 g/m3 (0.078 ppm) HNO3
    for 120 min, including 100 min of moderate exercise.  Bronchoalveolar
    lavage performed 18-h after exposure indicated increased phagocytic
    activity of AMs and increased resistance to respiratory syncytial
    virus infection.  There were no changes in markers of tissue damage. 
    Aris et al. (1991) exposed healthy subjects to 500 g/m3 (0.194 ppm)
    HNO3 for 4 h, including moderate exercise.  No change in lactate
    dehydrogenase levels, lavage fluid protein or differential cell counts
    in the BAL were observed.  Pulmonary function (FEV1, FVC and SRaw)
    was not significantly affected.

         Kjaergaard et al. (1993) studied the effects of nitrous acid on
    the eyes of 15 healthy non-smokers exposed to 8, 148 or 758 g/m3
    (4, 77 or 395 ppb) for 3.5 h.  There was an increase in trigeminal
    sensitivity (CO2 induced eye irritation) related to the concentration
    of nitrous acid.  Eye inflammation was increased, as indicated by
    increased PMNs and epithelial cells in tear fluid.

         Neither sodium nitrate (NaNO3) nor ammonium nitrate caused
    effects on pulmonary function of normal or asthmatic subjects (Sackner
    et al., 1979; Utell et al., 1979; Kleinman et al., 1980; Stacy et al.,
    1983).  However, there was a decrease in airway conductance and in
    PEFV curves in normal subjects with acute influenza exposed to
    7 mg/m3 of NaNO3 aerosol (Utell et al., 1980).  This is several
    orders of magnitude above the nitrate concentrations found in most
    ambient air.

    6.4  Effects of nitrogen dioxide/gas or gas/aerosol mixtures on lung
         function

         Table 45 summarizes studies of human subjects exposed to
    NO2-containing pollutant mixtures.  Most of the studies have been
    limited primarily to spirometry and plethysmography.  More extensive
    discussion can be found in US EPA (1993).

         With a few exceptions (to be discussed below), most research on
    interactions either showed no effects of the individual pollutants or
    the mixture, or it indicated that NO2 did not enhance the effects of
    the other pollutant(s) in the mixture (Table 45).  Most attention has
    focussed on NO2 mixtures with ozone (O3), although combinations with
    SO2, NO, particles, and a mixture of SO2 plus O3 have also been
    tested.  Due to the varied exposure protocols in the database, no
    consistent physiological trends are evident.  The generally negative
    responses could either reflect a true lack of interaction or other
    important design considerations.  For example, asthmatics were not
    studied.  Because pulmonary function studies of NO2 alone  cause
    variable effects with no clear concentration-responses, detecting
    interactions would be expected to be difficult unless there was
    significant synergism.

        Table 45.  Effects of nitrogen dioxide mixtures on healthy subjectsa
                                                                                                                                              

    Concentrations            Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    75 NO2       0.04 NO2     60         60           56             42 M/8 F       Healthy            No apparent effect      Avol et al.
                 (Amb)                                                                                 over and above that     (1983)
                                                                                                       of O3 alone.

    75 NO2       0.04 NO2     60         60           22.4           33 M/33 F      Children,          No effects of ambient   Avol et al.
                 (Amb)                                                              8-11 years         air exposures.          (1985a, 1987)

    103 NO2      0.055 NO2    60         60           32             46 M/13 F      Adolescents,       Ambient air exposures   Avol et al.
                 (Amb)                                                              12-15 years        effect  attributed      (1985b)
                                                                                                       to O3.

    132 NO2      0.07 NO2     120        60           approx. 20     14 M/20 F      29 years           Small decreases in      Linn et al.
                 (Amb)                                                                                 FVC, FEV1, in ambient   (1980b)
                                                                                                       air mostly attributable
                                                                                                       to O3. No association
                                                                                                       of NO2 levels with lung
                                                                                                       function change.

    545 NO2      (a) 0.29 NO2 240 (2     120          approx. 20     4              Healthy            With each group,        Hackney et al.
    +980 O3      +0.50 O3     consecutive                                                              minimal alterations     (1975b)
                              days of                                                                  in pulmonary function
    545 NO2      (b) 0.29 NO2 exposure                                                                 caused by O3 exposure.
    +980 O3      +0.50 O3     to each                                                                  Effects were not
    +34 350      +30.0 CO     mixture)                                                                 increased by addition
    CO                                                                                                 of NO2 or NO2 plus CO
                                                                                                       to test atmospheres.
                                                                                                                                              

    Table 45  (Con't)
                                                                                                                                              

    Concentrations            Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    545 NO2      (a) 0.29 NO2 120 (2     60           approx. 20     7              Healthy            Little or no change     Hackney et al.
    +490 O3      +0.25 O3     consecutive                                                              in pulmonary function   (1975b)
                              days of                                                                  found with O3 alone.
    545 NO2      (b) 0.29 NO2 exposure)                                                                Addition of NO2 or of
    +490 O3      +0.25 O3                                                                              NO2 plus CO did not
    +34 350      +30.0 CO                                                                              noticeably increase
    CO                                                                                                 the effect. Seven
                                                                                                       subjects included;
                                                                                                       some believed to be
                                                                                                       unusually reactive
                                                                                                       to respiratory
                                                                                                       irritants.

    940 NO2      0.50 NO2     120        30           40             10 M           Young adults,      FEV1, decreased 8-14%.  Folinsbee
    +980 O3      +0.5 O3                                                            NS                 No differences between  et al. (1981)
                                                                                                       O3 plus NO2 and O3
                                                                                                       alone.

    1128 NO2     0.60 NO2     120        60           25             8 M/8 F        18-26 years,       No significant          Drechsler-Parks
    +882 O3      +0.45 O3                                                           NS                 changes attributable    (1987)
                                                                                                       to NO2.

                                                                     8 M/8 F        51-76 years        Tendency (p > 0.05)
                                                                     8 M/8 F        51-76 years        for NO2 plus O3 to be
                                                                                                       greater than O3 alone.

    1128 NO2     0.60 NO2     60         60           70             20 M           Healthy            No additional effect    Adams et al.
    +588 O3      + 0.30 O3                            50             20 F                              of NO2 over and above   (1987)
                                                                                                       effect of O3.
                                                                                                                                              

    Table 45  (Con't)
                                                                                                                                              

    Concentrations            Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    1128         0.60 ppm NO2 120        60           40             21 F           Healthy, NS        NO2 exposure increased  Hazucha et al.
    NO2                                                                                                airway responses to     (1994)
                 0.3 ppm O3   120        60           40                                               methacholine after a
                 (3 h later)                                                                           subsequent O3 exposure.

    282 NO2      0.15 NO2     120        60           approx. 25     6 M            Some               Possible small          Kagawa (1986)
    +294 O3      + 0.15 O3                                                          smokers            decrease in SGaw.
    +200         + H2SO4
    H2SO4

    282 NO2      0.15 NO2     120        60           approx. 25     3 M            Some               Possible small
    +294 O3      + 0.15 O3                                                          smokers            decrease in FEV1.
    +393 SO2     + 0.15 SO2
    +200         + H2SO4
    H2SO4

    564 NO2      0.30 NO2     120        20           approx. 25     6 M            Some               Possible small
    +588 O3      +0.30 O3                                                           smokers            decrease in SGaw.
    +200         + H2SO4
    H2SO4

    282 NO2      0.15 NO2     120        60           approx. 25     7 M            19-23 years        No significant          Kagawa
    +294 O3      +0.15 O3                                                                              enhancement of the      (1983a,b)
    +393 SO2     +0.15 SO2                                                                             effects of O3 and/or
                                                                                                       SO2 by presence of
                                                                                                       NO2.
                                                                                                                                              

    Table 45  (Con't)
                                                                                                                                              

    Concentrations            Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    301 NO2      0.16 NO2     480        0                           15             16-26 years        No change in FVC,       Islam &
    +157 O3      +0.08 O3                                                                              acetylcholine airway    Ulmer (1979b)
    +891 SO2     +0.34 SO2                                                                             reactivity.

    564 NO2      0.3 NO2      120        60                          6 F            19-25 years        No significant effects  Kagawa (1990)
    +738 NO      +0.6 NO                                                            NS                 on pulmonary function
                                                                                                       or airway
                                                                                                       responsiveness to
                                                                                                       acetylcholine.

    940 NO2      0.50 NO2     135        60           approx. 20     11 M/9 F       20-53 years        No effects on           Kleinman
    1310 SO2     + 0.5 SO2                                                                             function; possible      et al. (1985)
    +26          +                                                                                     symptom responses.
    Zn(NH4)2     Zn(NH4)2                                                                              NO2 effects not
    (SO4)2       (SO4)2                                                                                discernible from
    +330 NaCl    + NaCl                                                                                mixture.

    940 NO2      0.50 NO2     120        60           approx. 20     10 M/14 F      26  4             No significant effect   Linn et al.
    1310 SO2     + 0.50 SO2                                                         years, 21 NS,      on lung function in     (1980a)
                                                                                    3 S                normals. Trend for a
                                                                                                       slight decrease in
                                                                                                       FVC after combined
                                                                                                       exposure.
                                                                                                                                              

    Table 45  (Con't)
                                                                                                                                              

    Concentrations            Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    7520-9400    4-5 NO2      10                                     5 M            21-40 years,       Time course of          Abe (1967)
    NO2          +4-5 SO2                                                           4 NS, 1 S          response different.
    +4920-6150                                                                                         SO2 alone had immediate
    SO2                                                                                                increase in resistance;
                                                                                                       NO2 had delayed
                                                                                                       increase. Mixture had
                                                                                                       intermediate effects
                                                                                                       on resistance.

    9400 NO2     5.0 NO2      120        60           ?              8 M            < 30 years         FVC (-5%), FEV1.0       Islam &
    +1960 O3     +0.1 O3                                             8 M            30-40 years        (-11.7%), decreased     Ulmer (1979a)
    +13 100 SO2  +5.0 SO2                                            8 M            > 49 years         with exercise exposure
                                                                                                       to this mixture
                                                                                                       in < 30 years group.

    9400 NO2     5.0 NO2      120        intermittent                9 M            Healthy,           No interaction on PaO2  Von Nieding
    +196 O3      +0.1 O3                                                            20-38 years        or RT                   et al. (1977)
    +13 100 SO2  +5.0 SO2

    9400 NO2     5.0 NO2      120        intermittent                11 M           Healthy,           No interaction on PaO2
    +196 O3      +0.1 O3                                                            20-38 years,       or Rt
                                                                                    25 S, 9 NS

    9400 NO2     5.0 NO2      120        60           approx. 20                    23-38 years,       RT increased from 1.5   Von Nieding
    +196 O3      + 0.1 O3                             (70 W)                        two atopic         to 2.4 (p < 0.01);      et al. (1979)
    +13 100      + 5.0 SO2                                                                             questionable decrease
    SO2                                                                                                in PaO2 (8 torr).
                                                                                                                                              

    Table 45  (Con't)
                                                                                                                                              

    Concentrations            Exposure   Exercise     Exercise       Number of      Subject            Effects                 Reference
                              duration   duration     ventilation    subjects/      characteristics
                              (min)      (min)        (litres/min)   gender
    g/m3        ppm
                                                                                                                                              

    188 NO2      0.1 NO2      120        60           approx. 20                    23-38 years,       No effects.
    +786 SO2     +0.3 SO2                                                           two atopic
                                                                                                                                              

    a  Modified from US EPA (1993)
       Abbreviations:

       Amb = Ambient air; CO = Carbon monoxide; F = Female; FEV1 = Forced expiratory volume in 1 second; FEV1.0 = Forced expiratory volume
       in 1 second; FVC = Forced vital capacity; H2SO4 = Sulfuric acid; M = Male; NaCl = Sodium chloride; (NH4)2SO4 = Ammonium sulfate;
       NO = Nitric oxide; NS = Non-smoker; O3 = Ozone; PaO2 = Arterial partial pressure of oxygen; RT = Total respiratory resistance;
       S = Active smoker; SGaw = Specific airway conductance; SO2 = Sulfur dioxide; W = Watts; ZnSO4 = Zinc sulfate
             Abe (1967) studied brief exposures to NO2-SO2 mixtures.  Both
    gases were at 4 to 5 ppm (i.e., 7520 to 9400 g/m3 NO2 and 4920 to
    6150 g/m3 SO2).  The effects were additive, with both gases causing
    bronchoconstriction.  Independently, the effect of SO2 was immediate
    and short-lasting, whereas the effect of NO2 was delayed and more
    persistent.  The effect of the mixed gases was intermediate between
    the two independent responses.  Kagawa (1983a,b) reported that the
    interaction of 282 g/m3 (0.15 ppm) NO2 plus 393 g/m3 (0.15 ppm)
    SO2 in normal  subjects exposed for 2 h with light intermittent
    exercise caused an increase in SGaw.  However, because a large number
    of repeated t-tests with an alpha level of 0.05 were used, it is
    possible that the responses were due to chance.

         The Rancho Los Amigos group (Linn et al., 1980b; Linn & Hackney,
    1983; Avol et al., 1983, 1985a, 1987) conducted several studies of
    NO2-containing ambient air mixtures.  The mean NO2 level in the
    ambient air (from the Los Angeles Air Basin) ranged from 75 to
    132 g/m3 (0.04 to 0.07 ppm).  Normal and  asthmatic adults,
    adolescents and children were exposed for approximately 2 h during the
    summer smog seasons of 1978 to 1984.  The various pulmonary function
    effects observed (see Table 45) were attributed to O3.  However, in
    another study, Hazucha et al. (1994) found that ozone-induced
    increases in airway responsiveness to methacholine were enhanced by
    prior (3 h earlier) exposure to 1128 g/m3 (0.60 ppm) NO2.  There
    was also a slightly greater FEV1 decrement after the NO2-O3
    sequence.

         There has been one study on the effects of HNO3 vapour in
    combination with O3 (Aris et al., 1991).  Ten healthy men were
    exposed (with moderate exercise) to 430 g/m3 HNO3 for 2 h and then,
    after 1 h, to 392 g/m3 (0.20 ppm) O3 for 3 h.  No changes were
    observed in FVC, FEV1 or SRaw after HNO3 exposure.  Ozone exposure
    caused increased SRaw and decreased FVC and FEV1.  Prior exposure to
    HNO3 vapour rather than air resulted in somewhat smaller changes in
    lung function after ozone exposure.  Clearly HNO3 did not potentiate
    responses to ozone.

    6.5  Summary of controlled human exposure studies of oxides of
         nitrogen

         Human responses to a variety of oxidized nitrogen compounds have
    been evaluated.  By far, the largest database and the one most
    suitable for risk assessment is that available for controlled
    exposures to NO2.  The database on human responses to NO, nitric acid
    vapour, nitrous acid vapour and inorganic nitrate aerosols is not as
    extensive.  A number of sensitive or potentially sensitive subgroups
    have been examined, including adolescent and adult asthmatics, older
    adults, and patients with chronic obstructive pulmonary disease and
    pulmonary hypertension.  Exercise increases the total uptake and

    alters the distribution of the inhaled material within the lung.  The
    proportion of NO2 deposited in the lower respiratory tract is also
    increased by exercise.  This may increase the effects of the above
    compounds in people who exercise during exposure.

         As is typical with human biological response to inhaled particles
    and gases, there is variability in the biological response to NO2. 
    Healthy individuals tend to be less responsive to the effects of NO2
    than  individuals with lung disease.  Asthmatics are clearly the most
    responsive group to NO2 that has been studied to date.  Individuals
    with chronic obstructive pulmonary disease may be more responsive than
    healthy individuals, but they have limited capacity to respond to NO2
    and thus quantitative differences between COPD patients and others are
    difficult to assess.  There is not sufficient information available at
    present to evaluate whether age or gender should be considered in the
    risk evaluation.

         NO2 causes decrements in lung function, particularly increased
    airway resistance in resting healthy subjects at 2-h concentrations as
    low as 4700 g/m3 (approx. 2.5 ppm).  Available data are insufficient
    to determine the nature of the concentration-response relationship.

         NO2 exposure results in increased airway responsiveness to
    broncoconstrictive agents in exercising healthy, non-smoking subjects
    exposed to concentrations as low as 2800 g/m3 (approx. 1.5 ppm) for
    exposure durations of 1 h or longer.

         Exposure of asthmatics to NO2 causes, in some subjects,
    increased airway responsiveness to a variety of provocative mediators,
    including cholinergic and histaminergic chemicals, SO2 and cold air. 
    The presence of these responses appears to be influenced by the
    exposure protocol, particularly whether or not the exposure includes
    exercise.  These responses may begin at concentrations as low as
    380 g/m3 (0.20 ppm).  A meta analysis suggests that effects may
    occur at even lower concentrations.  However, no concentration-
    response relationship is observed between 350 and 1150 g/m3
    (approx. 0.2 and 0.6 ppm).

         Modest increases in airway resistance may occur in patients with
    COPD from brief exposure (15-60 min) to concentrations of NO2 as low
    as 2800 g/m3 (approx. 1.5 ppm) and decrements in spirometric
    measures of lung function (3 to 8%) change in FEV1 may also be
    observed with longer exposures (3 h) to concentrations as low as
    600 g/m3 (approx. 0.3 ppm).

         Exposure to NO2 at levels above 2800 g/m3 (approx. 1.5 ppm)
    may alter numbers and types of inflammatory cells in the distal
    airways or alveoli.  NO2 may alter the function of cells within the
    lung and production of mediators that may be important in lung host

    defences. The constellation of changes in host defences, alterations
    in lung cells and their activities, and changes in biochemical
    mediators is consistent with the epidemiological findings of increased
    host susceptibility associated with NO2 exposure.

         In studies of mixtures of NO2 with other pollutants, NO2 has
    not been observed to increase responses to other co-occurring
    pollutant(s) beyond what would be observed for the other pollutant(s)
    alone.  A notable exception is the observation that pre-exposure to
    NO2 enhances the ozone-induced change in airway-responsiveness in
    healthy, exercising subjects during a subsequent ozone exposure.  This
    observation suggests the possibility of delayed or persistent
    responses to NO2.

         Within an NO2 concentration range that may be of interest with
    regard to risk evaluation (i.e., 100-600 g/m3), the characteristics
    of the concentration-response relationship for acute changes in lung
    function, airway responsiveness to bronchoconstricting agents, or
    symptoms cannot be determined from the available data.

         NO is acknowledged as an important endogenous second messenger
    within several organ systems.  Inhaled NO concentrations above
    6000 g/m3 (approx. 5 ppm) can cause vasodilation in the pulmonary
    circulation without affecting the systemic circulation.  The lowest
    effective concentration is not established.  Information on pulmonary
    function and lung host defences consequent to NO exposure are too
    limited for any conclusions to be drawn at this time.  Relatively high
    concentrations (> 40 000 g/m3) have been used in clinical
    applications for brief periods (< 1 h) without reported adverse
    reactions.

         Nitric acid levels in the range of 250-500 g/m3 (100-200 ppb)
    may cause some pulmonary function responses in adolescent asthmatics,
    but not in healthy adults.

         Limited information on nitrous acid suggests that it may cause
    eye inflammation at 760 g/m3 (0.40 ppm).  There are currently no
    published data on human pulmonary responses to nitrous acid.

         Limited data on inorganic nitrates suggest that there are no
    lung function effects of nitrate aerosols with concentrations of
    7000 g/m3 or less.

    7.  EPIDEMIOLOGICAL STUDIES OF NITROGEN OXIDES

    7.1  Introduction

         This chapter discusses epidemiological evidence regarding effects
    of NOx on human health.  Primary emphasis is placed on assessment of
    the effects of NO2 because it is the oxide of nitrogen measured in
    most epidemiological studies and the one of greatest concern from a
    public health perspective.  Human health effects associated with
    exposure to NO2 have been the subject of several literature reviews
    since 1970 (National Research Council, 1971, 1977; US EPA, 1982a,
    1993; Samet et al., 1987, 1988).  Oxides of nitrogen have also been
    reviewed previously by the World Health Organization (WHO, 1977),
    which presented a comprehensive review of studies conducted up to
    1977.  This chapter focuses on studies conducted since 1977, while
    also using some key information from earlier literature, as reviewed
    in more detail by US EPA (1993).

         The studies discussed in this chapter are those that provide
    useful quantitative information on exposure-effect relationships for
    health effects associated with levels of NO2 likely to be encountered
    in the ambient air.  In addition, some studies that do not provide
    quantitative information are briefly discussed in the text in order to
    help elucidate particular points concerning the health effects of
    NO2.

    7.2  Methodological considerations

         Key epidemiological studies on NO2 health effects are evaluated
    below for several factors of importance for interpreting their results
    (US EPA, 1982a,c). Such factors include: (1) exposure measurement
    error; (2) misclassification of the health outcome; (3) adjustment
    for covariates; (4) selection bias; (5) internal consistency; and
    (6) plausibility of the effect based on other evidence.

    7.2.1  Measurement error

         Measurement error regarding exposure may be a major problem in
    epidemiological studies of NO2.  Ideally, personal monitors should be
    placed on all subjects for the entire period of a study, but this is
    often not feasible.  Moreover, personal monitoring may not overcome
    measurement error altogether.  For example, the monitors that are
    presently available do not accurately measure short-term peaks or
    long-term chronic exposures.  Other means of estimating NO2 exposure
    include source description, in-home monitors and fixed-site outdoor
    monitors.  These approaches are generally cheaper than personal
    monitors but may be subject to greater measurement error, both random
    (non-systematic) and systematic.

         In general, a measurement error in estimation of exposure that is
    independent of the health outcome will result in underestimation of
    associations between exposure and dichotomous health outcomes (Samet &
    Utell, 1990).  Whittlemore & Keller (1988) examined the data of Melia
    et al. (1980) and showed that a 20% misclassification rate of the
    exposure category could result in an underestimate of the logistic
    regression coefficient by as much as 50%.  Even when exposure
    measurement error is not independent of the outcome, measures of
    association are biased towards the null, unless the probability of the
    health outcome is very close to 0 or 1 (Stefanski & Carroll, 1985).

         At present, there is little information on the relative
    importance of peak and average NO2 levels as causes of respiratory
    effects in humans. In most homes and outdoor settings, peak values may
    be related to average values, and reduction of peaks may lower
    time-weighted averages.  However, if health effects are largely
    associated with the peak levels of NO2, then the use of averages as
    the sole guide to exposures will increase measurement error.

         NO2 may act as a precursor for other biologically active
    substances (such as nitrous acid).  If these agents are responsible
    for some or all of the observed respiratory effects, then measurement
    of NO2 will provide an imprecise estimate of the effective dose.

    7.2.2  Misclassification of the health outcome

         Misclassification of the health outcome can occur whether the
    outcome is continuous, (such as a measure of pulmonary function) or
    dichotomous (such as the presence or absence of respiratory symptoms). 
    Lung function is typically measured with spirometry, a well-
    standardized technique (Ferris, 1978).  The measurement errors of the
    instruments collecting the data have also been carefully estimated,
    and random errors will simply add to the error variance.  On the other
    hand, respiratory symptoms and health status are usually measured by a
    questionnaire.  Responses to symptom questions will be correlated and
    will depend on the interpretation of the respondent.  As noted below,
    a specific respiratory disease is likely to be reflected by a
    constellation of symptoms.  Therefore, it is appropriate to consider
    aggregate, as well as single, specific symptom reports.  Obviously,
    questionnaire measurements involving recent recall are better than
    those based on recall of events occurring several years earlier. 
    Questionnaires for cough and phlegm production have been standardized,
    e.g., the British Medical Research Council (BMRC) questionnaire
    (American Thoracic Society, 1969) and revisions of that questionnaire
    (Ferris, 1978; Samet, 1978).  These questionnaires and modifications
    of them have been used extensively.

    7.2.3  Adjustment for covariates

         It is common when analysing a data set to discover that one or
    more key covariates for the analysis were not measured.  Schenker et
    al. (1983) discussed socioeconomic status, passive smoking and gender
    as important covariates in childhood respiratory disease studies. 
    Other covariates often of importance are age, humidity and other
    co-occurring pollutants (e.g., particulate matter).  The concern is
    that, had missing covariates been measured, the estimate of the
    regression coefficient of a variable of interest would have been
    significantly different.  Although the problem is faced by most
    investigators, literature on the subject is sparse.  For example,
    Kupper (1984) showed that high correlations between the variables just
    described will result in "unreliable parameter estimates with large
    variances".  Gail (1986) considered the special case of omitting a
    balanced covariate from the analysis of a cohort study and concluded
    that: "In principle, the bias may be either toward or away from zero,
    though in more important examples - the bias is toward zero.  In
    important applications with additive or multiplicative regression,
    there is no bias".  Neither report provided information on how to
    attempt to correct for the bias or on approaches for investigating the
    possible bias in a given situation.

         Most studies of respiratory disease and NO2 exposure discussed
    here measured important covariates such as age, socioeconomic level of
    the parents, gender and parental smoking habits.  The estimated effect
    (regression coefficient of disease on NO2 exposure) will be
    overestimated if a missing covariate is positively or negatively
    correlated with both exposure and health outcome.  The estimated
    effect will be underestimated if positively correlated with exposure
    or outcome and negatively correlated with the other.  Ware et al.
    (1984) found that parents with some college education were more likely
    to report respiratory symptoms and less likely to use a gas stove,
    leading to an underestimate of the health effect, if education were
    omitted from the analysis.

    7.2.4  Selection bias

         The possibility of selection bias, although a concern of every
    study, seems very low for NO2 epidemiological studies.  Selection
    bias would require selection of participants based on exposure
    (e.g., use of gas stove) and also health outcome.  Because most
    epidemiological studies of these exposures are population based, there
    is little possibility of selection based on health end-points. 
    Nevertheless, the loss of subjects by attrition associated with both
    exposure and health studies must be considered.

    7.2.5  Internal consistency

         Internal consistency is also a useful check on the validity of a
    study, but authors often do not report sufficient detail to check for
    such consistency.  For example, in the case of known risk factors for
    respiratory effects, a study should find the anticipated associations
    (e.g., passive smoking with increased respiratory illness or with more
    wheeze in asthmatic children), and certain patterns of age or gender
    effects should be observed.  Consistency between studies also provides
    an indication of the overall strength of the database.

    7.2.6  Plausibility of the effect

         Health outcomes should be ones for which there are plausible
    bases to suspect that NO2 exposure could contribute to such effects. 
    Two health outcome measures have been most extensively considered
    in the epidemiological studies: lung function measurements and
    respiratory illness occurrence.  Human clinical and animal
    toxicological studies have not indicated a demonstrated effect on lung
    function at ambient levels in normal subjects.  On the other hand,
    human clinical and animal toxicological studies have shown that NO2
    exposure can impair components of the respiratory host defence system,
    resulting in increased susceptibility of the host to respiratory
    infection.  Thus, reported increases in respiratory symptoms and
    disease among children in epidemiological studies of NO2 exposure can
    be plausibly hypothesized to reflect an increase in respiratory
    infection.

         Each study is subsequently reviewed with special attention given
    to the above factors.  Those studies that address these factors most
    appropriately provide a stronger basis for the conclusions that they
    draw.  Consistency between studies indicates the level of the strength
    of the whole database.

    7.3  Studies of respiratory illness

         Respiratory illness and factors determining its occurrence and
    severity are important public health concerns.  The possible
    association of NO2 exposure with respiratory illness is of public
    health importance because both the potential for exposure to NO2 and
    childhood respiratory illness are common (Samet et al., 1983; Samet &
    Utell, 1990).  This takes on added importance because recurrent
    childhood respiratory illness (independent of NO2) may be a risk
    factor for later susceptibility to lung damage (Samet et al., 1983;
    Glezen, 1989; Gold et al., 1989).  The epidemiological studies
    relating NO2 exposure to respiratory illness are discussed in
    sections 7.3.1 and 7.3.2.

    7.3.1  Indoor air studies

         In this section, studies that meet criteria for use in a
    quantitative analysis are presented.  Firstly, studies conducted by
    Melia and colleagues in the United Kingdom are discussed.  This is
    followed by an evaluation of two large studies conducted in six cities
    in the USA.  Several other quantitative studies conducted by different
    authors in various countries and cities are then presented.  These are
    followed by discussion of some additional recent large-scale studies
    that yield useful quantitative information, e.g., a study of NO2
    relationship to respiratory disease in young children in Albuquerque,
    New Mexico, USA.  Lastly, other studies that provide information
    concerning respiratory illness are also discussed.

    7.3.1.1  St Thomas' Hospital Medical School Studies (United Kingdom)

         Results of several British studies have been reported by Melia
    et al. (1977, 1978, 1979, 1980, 1982a,b, 1985, 1988), Goldstein et al.
    (1979, 1981), and Florey et al. (1979, 1982).  Parts of these studies
    were reviewed previously (US EPA, 1982a), but their importance
    requires a more complete discussion of them.

         The initial study (Melia et al., 1977) was based on a survey of
    5658 children (excluding asthmatics, thus 100 less than the number
    reported), aged 6 to 11 years, with sufficient questionnaire
    information in 28 randomly selected areas of England and Scotland.  A
    self-administered questionnaire was completed by a parent to obtain
    information on the presence of morning cough, day or night cough,
    colds going to chest, chest sounds of wheezing or whistling, and
    attacks of bronchitis.  The questionnaire, distributed in 1973, asked
    about symptoms during the previous 12 months.  Colds going to the
    chest accounted for the majority of symptoms reported.  Information
    about cooking fuel (gas or electric), age, gender and social class
    (manual versus non-manual labour) was obtained, but there were no
    questions about parental smoking.  Melia et al. (1977) noted that
    although they could not include family smoking habits in the analysis,
    the known relation between smoking and social class (Tobacco Research
    Council, 1976) allowed them to avoid at least some of the potential
    bias from this source.  It seemed unlikely that, within the social
    class groups studied, there was a higher prevalence of smoking in
    homes where gas was used for cooking.  No measurements of NO2, either
    indoors or outdoors, were given.

         The authors presented their results in the form of a contingency
    table for non-asthmatics with complete covariate information. Table 46
    is a summary of that data for non-asthmatic children. The authors
    indicated that there was a trend for increased symptoms in homes with
    gas stoves, but the increase was only significant for girls in urban
    areas.  The authors gave no measures of increased risk. The data in

    Table 46 have been reanalysed using a multiple logistic model as shown
    in Table 47.  Because it had been suggested that gender had an effect
    on the relationship with "gas cooker", interaction terms for gender
    were included in the original model.  None of these proved to be
    significant, and they were subsequently dropped from the model.  When
    separate terms for each gender were used for the effect of "gas
    cooker", an estimated odds ratio of 1.25 was obtained for boys and an
    odds ratio of 1.39 was obtained for girls.  The combined odds ratio
    for both genders was 1.31 (95% confidence limits of 1.16 and 1.48) and
    was statistically significant (p < 0.0001).  The other main effects
    of gender, SES and age were all statistically significant.  This
    reanalysis suggests that gas stove use was associated with an
    estimated 31% increase in the odds of children having respiratory
    illness symptoms.

         Melia et al. (1979) reported further results of a national survey
    covering a new cohort of 4827 boys and girls, aged 5 to 10 years, from
    27 randomly selected areas that were examined in 1977.  The study
    collected information on the number of smokers in the home.  In the
    1977 cross-sectional study, only prevalence of day or night cough in
    boys (p approx. or equal 0.02) and colds going to the chest in girls
    (p < 0.05) were found to be significantly higher in children from
    homes where gas was used for cooking compared with children from homes
    where electricity was used.  As shown in Table 48, grouping responses
    according to the six respiratory questions into (1) none or (2) one or
    more symptoms or diseases yielded a prevalence higher in children
    from homes where gas was used for cooking than in those from homes
    where electricity was used (p approx. or equal 0.01 in boys,
    p = 0.07 in girls).  The effects of gender, social class, use of pilot
    lights and number of smokers in the house were examined.

         The reanalysis of the data in Table 48, applying a multiple
    logistic model, is given in Table 49. This model contained the same
    terms as the analysis in Table 47. As in the previous analysis, none
    of the interaction terms proved to be significant, and they were
    subsequently dropped from the model.  When separate terms for each
    gender were used for the effect of "gas cooker", an estimated odds
    ratio of 1.29 was obtained for boys and an odds ratio of 1.19 was
    obtained for girls.  The combined odds ratio for both genders was
    1.24 (95% confidence limits of 1.09 and 1.42).  This effect was
    statistically significant (p < 0.0002).  The other main effects of
    gender, SES and age were all statistically significant.  This
    reanalysis suggests that gas stove use in this study is associated
    with an estimated 24% increase in the odds of having symptoms.

    Table 46.  Symptom rates of United Kingdom children by age, gender,
               social class and type of cookera
                                                                        

                       Social classes I-IIIa     Social classes IIIb-V
                         (non-manual)                  (manual)
                       Electric   Gas            Electric       Gas
                                                                        

    Age < 8 years

    Boys               25.6%      26.1%          29.9%          37.5%
                       (203)      (88)           (375)          (309)

    Girls              22.2%      30.4%          31.8%          33.5%
                       (171)      (112)          (393)          (337)

    Age 8 to 11 years

    Boys               20.8%      23.3%          25.0%          29.0%
                       (365)      (189)          (675)          (654)

    Girls              18.1%      19.2%          17.8%          27.8%
                       (303)      (187)          (674)          (623)
                                                                        
    a  Numbers in parentheses refer to number of subjects; source:
       Melia et al. (1977)

    Table 47.  Multiple logistic analysis of data from the study of Melia
               et al. (1977)
                                                                        

    Factora                      Odds ratio   95% Confidence    p value
                                              interval
                                                                        

    SES and age by gender
      interactions (2 d.f.)                                     0.2922

    Gas by gender
      interaction (1 d.f.)                                      0.3953

    Gas cooker                   1.31         1.16-1.48         < 0.0001

    Gender (female)              0.86         0.76-0.97         0.0121

    SES (manual)                 1.31         1.14-1.51         0.0001

    Age (< 8 years)              1.47         1.30-1.66         < 0.0001
                                                                        
    a  SES = Socioeconomic status; d.f. = Degrees of freedom

    Table 48.  Unadjusted rates of one or more symptoms among United
               Kingdom children by age, gender, social class and type of
               cookera
                                                                        

                       Social classes I-IIIa     Social classes IIIb-V
                           (non-manual)                (manual)
                       Electric   Gas            Electric       Gas
                                                                        

    Age < 8 years
    Boys               27.4%      31.7%          32.8%          36.7%
                       (277)      (145)          (485)          (313)

    Girls              24.4%      27.6%          27.8%          36.3%
                       (291)      (134)          (497)          (336)

    Age 8 to 11 years
    Boys               19.2%      28.3%          23.6%          26.9%
                       (286)      (113)          (501)          (338)

    Girls              14.8%      18.6%          21.5%          18.5%
                       (243)      (118)          (437)          (313)
                                                                        

    a  Numbers in parentheses refer to number of subjects; source:
       Melia et al. (1979)

    Table 49.  Multiple logistic analysis of data from the study of Melia
               et al. (1979)
                                                                        

    Factora                      Odds ratio   95% Confidence    p value
                                              interval
                                                                        

    SES and age by gender
      interactions (2 d.f.)                                     0.5749

    Gas by gender
      interaction (1 d.f.)                                      0.5566

    Gas cooker                   1.24         1.09-1.42         < 0.0001

    Gender (female)              0.82         0.72-0.94         0.0030

    SES (manual)                 1.25         1.08-1.45         0.0034

    Age (< 8 years)              1.69         1.48-1.93         < 0.0001
                                                                        
    a  SES = Socioeconomic status; d.f. = Degrees of freedom

         In 1978, 808 schoolchildren (Melia et al., 1980), aged 6 to
    7 years, were studied in Middlesborough, an urban area of northern
    England.  Respiratory illness was defined as in the previous study. 
    Weekly indoor NO2 measurements were collected from 66% of the homes,
    the remaining 34% refusing to participate.  NO2 was measured weekly
    by triethanolamine diffusion tubes (Palmes tubes) attached to walls in
    the kitchen area and in the children's bedrooms.  In homes with gas
    stoves, weekly levels of NO2 in kitchens ranged from 10 to 596 g/m3
    (0.005 to 0.317 ppm) with a mean of 211 g/m3 (0.112 ppm) and levels
    in bedrooms ranged from 8 to 318 g/m3 (0.004 to 0.169 ppm) with a
    mean of 56 g/m3 (0.031 ppm). In homes with electric stoves, weekly
    levels of NO2 in kitchens ranged from 11 to 353 g/m3 (0.006 to
    0.188 ppm) with a mean of 34 g/m3 (0.018 ppm), and levels in
    bedrooms ranged from 6 to 70 g/m3 (0.003 to 0.037 ppm) with a mean
    of 26 g/m3 (0.014 ppm).  Outdoor levels of NO2 were determined
    using diffusion tubes systematically located throughout the area; the
    weekly average ranged from 26 to 45 g/m3 (0.014 to 0.024 ppm).  One
    analysis by the authors was restricted to those 103 children in homes
    where gas stoves were present and where bedroom NO2 exposure was
    measured; the data are shown in Table 50.  A linear regression model
    was fit to the logistic transformation of the rates.  Cooking fuel was
    found to be associated with respiratory illness, independent of social
    class, age, gender or presence of a smoker in the house (p = 0.06). 
    However, when social class was excluded from the regression, the
    association was weaker (p = 0.11).  For the 6- and 7-year-old children
    living in homes with gas stoves, there appeared to be an increase in
    respiratory illness with increasing levels of NO2 in their bedrooms
    (p = 0.10), but no significant relationship was found between
    respiratory symptoms in those children, their siblings or parents and
    levels of NO2 in kitchens.

         Because no concentration-response estimates were given by the
    authors, a multiple logistic model was fitted to the data in Table 50
    with a linear slope for NO2 and separate intercepts for boys and
    girls.  NO2 levels for the groups were estimated by fitting a
    log-normal distribution to the grouped NO2 data, and the average
    exposures within each interval were estimated (see Hasselblad et al.,
    1980).  The estimated logistic regression coefficient for NO2
    (in g/m3) was 0.015 with a standard error of 0.007.  The likelihood
    ratio test for NO2 gave a chi-square of 4.94 with one degree of
    freedom, with a corresponding p value of 0.03.

         The study was repeated in January to March of 1980 by Melia et
    al. (1982a,b).  This time, children aged 5 to 6 years were sampled
    from the same neighbourhood as the previous study, but only families
    with gas stoves were recruited.  Environmental measurements were made
    and covariate data were collected in a manner similar to the previous
    study (Melia et al., 1980).  Measurements of NO2 were available for
    54% of the homes.  The unadjusted rates of one or more symptoms by

    Table 50.  Unadjusted rates of one or more symptoms among United
               Kingdom boys and girls according to bedroom levels of
               nitrogen dioxidea
                                                                        

                                Bedroom levels of NO2 (ppm)
                                                                        

                   < 0.020      0.020-0.039      > 0.039      Total
                                                                        

    Boys           43.5%        57.9%            69.2%        54.5%
                   (23)         (19)             (13)         (55)

    Girls          44.0%        60.0%            75.0%        54.2%
                   (25)         (15)             (8)          (48)

    TOTAL          43.7%        58.8%            71.4%        54.4%
                   (48)         (34)             (21)         (103)
                                                                        

    a  Numbers in parentheses refer to number of subjects
       (from: Melia et al., 1980)

    gender and exposure level are shown in Table 51.  The authors
    concluded that "... no relation was found between the prevalence of
    respiratory illness and levels of NO2".  A reanalysis by Hasselblad
    et al. (1992) of the data in Table 51 was made using a multiple
    logistic model similar to the one used for the previous study (Melia
    et al., 1980).  The model included a linear slope for NO2 and
    separate intercepts for boys and girls.  Nitrogen dioxide levels for
    the groups were estimated by fitting a log-normal distribution to the
    grouped bedroom NO2 data.  The estimated logistic regression
    coefficient for NO2 (in g/m3) was 0.0037 with a standard error of
    0.0052.  The likelihood ratio test for the effect of NO2 gave a
    chi-square of 0.51 with one degree of freedom (p = 0.48).

         Melia et al. (1983) investigated the association between gas
    cooking in the home and respiratory illness in a study of 390 infants
    born between 1975 and 1978.  When the child reached 1 year of age, the
    mother was interviewed by a trained field worker to complete a
    questionnaire.  The mother was asked whether the child usually
    experienced morning cough, day or night cough, wheeze or colds going
    to the chest, and whether the child had experienced bronchitis, asthma
    or pneumonia during the past 12 months.  No relation was found between
    type of fuel used for cooking at home and the prevalence of
    respiratory symptoms and diseases recalled by the mother after
    allowing for the effects of gender, social class and  parental
    smoking.  The authors gave prevalence rates of children having at

    least one symptom, according to gas stove use and gender.  The
    combined odds ratio for presence of symptoms according to gas stove
    use was 0.63 with 95% confidence interval of 0.36 to 1.10.

    Table 51.  Unadjusted rates of one or more symptoms among United
               Kingdom boys and girls according to bedroom levels of
               nitrogen dioxidea
                                                                         

                                 Bedroom levels of NO2 (ppm)
                                                                        

                   < 0.020       0.020-0.039       > 0.039      Total
                                                                        

    Boys             56.4%          67.6%          72.0%        64.4%
                     (39)           (37)           (25)         (101)

    Girls            60.0%          41.0%          52.2%        49.4%
                     (25)           (39)           (23)         (87)

    Total            57.8%          53.9%          62.5%        57.5%
                     (64)           (76)           (48)         (188)
                                                                        

    a  Numbers in parentheses refer to number of subjects; source:
       Melia et al. (1982a,b)

         Melia et al. (1988) studied factors affecting respiratory
    morbidity in 1964 primary school children living in 20 inner city
    areas of England in 1983 as part of a national study of health and
    growth.  Data on age, gender, respiratory illness, cooking fuels,
    mother's education and size of family were obtained by questionnaire.
    Smoking was not studied. The same respiratory questions were asked as
    in previous studies.  Melia et al. (1990) reported indoor levels of
    NO2 associated with gas stoves in inner city areas of England in
    1987.  The mean weekly NO2 level measured in 22 bedrooms of homes
    with gas stoves was 45  25 g/m3 (24.1  13.2 ppb).  The mean weekly
    NO2 level measured in four bedrooms of homes without gas stoves was
    40  22 g/m3 (20.7  11.8 ppb).  Melia et al. (1988) reported a
    relative risk of 1.06 (95% confidence interval of 0.94 to 1.17) for
    one or more respiratory conditions associated with exposure to gas or
    kerosene fuel used in the home after adjustment for ethnic group,
    gender, age group, mother's education, family size and single parent
    family status.

    7.3.1.2  Harvard University - Six Cities Studies (USA)

         Several authors (Spengler et al., 1979, 1986; Speizer et al.,
    1980; Ferris et al., 1983; Ware et al., 1984; Berkey et al., 1986;
    Quackenboss et al., 1986; Dockery et al., 1989a; Neas et al., 1990,
    1991) have reported on two cohorts of children studied in six
    different cities in the USA.  The six cities were selected to
    represent a range of air quality based on their historic levels of
    outdoor pollution.  They included: Watertown, Massachusetts; Kingston
    and Harriman, Tennessee; southeast St. Louis, Missouri; Steubenville,
    Ohio; Portage, Wisconsin; and Topeka, Kansas.  In each community
    during 1974-1977, approximately 1000 first- and second-grade
    schoolchildren were enrolled in the first year and an additional
    500 first-graders were enrolled in the next year (Ferris et al.,
    1979).  Families reported the number of people living in the home and
    their smoking habits, parental occupation and educational background,
    and fuels used for cooking and heating.  Outdoor pollution was
    measured at fixed sites in the communities as well as at selected
    households.  Indoor pollution including NO2 was measured in several
    rooms of selected households.

         Speizer et al. (1980) reported results from the six cities
    studies based on 8120 children, aged 6 to 10 years, who had been
    followed for 1 to 3 years.  Health end-points were measured by a
    standard respiratory questionnaire completed by the parents of the
    children.  The authors used log-linear models to estimate the effect
    of current use of gas stoves versus electric stoves on the rates of
    serious respiratory illness before age 2, yielding an odds ratio of
    1.12 (95% confidence limits of 1.00 and 1.26) for gas stove use.  The
    results were adjusted for presence of adult smokers, presence of air
    conditioning, and family SES.

         Ware et al. (1984) reported results for a larger cohort of
    10 160 white children, aged 6 to 9 years, in the same six cities over
    a longer period (1974-1979).  Directly standardized rates of reported
    illnesses and symptoms did not show any consistent pattern of
    increased risk for children from homes with gas stoves.  Logistic
    regression analyses controlling for age, gender, city and maternal
    smoking level gave estimated odds ratios for the effect of gas stoves
    ranging from 0.93 to 1.07 for bronchitis, chronic cough, persistent
    wheeze, lower respiratory illness index, and illness for the last
    year.  The lower respiratory illness index indicated the presence of
    bronchitis, restriction of activity due to lower respiratory illness,
    or chronic cough during the past year.  The 95% confidence bounds
    around all of these symptom-specific odds ratios included 1.  Only two
    odds ratios approached statistical significance: (1) history of
    bronchitis (odds ratio = 0.86, 95% confidence interval 0.74 to 1.00)
    and (2) respiratory illness before age 2 (odds ratio = 1.13, 95%
    confidence interval 0.99 to 1.28).  When the odds ratio for

    respiratory illness before age 2 was adjusted for parental education,
    the odds ratio was 1.11 with 95% confidence limits of 0.97 and 1.27
    (p = 0.14).  Thus, the study suggests an increase of about 11% in
    respiratory illness before the age of 2 years, which is about the same
    as that reported by Speizer et al. (1980), although the increase was
    not statistically significant at the 0.05 level.  The end-point in the
    Ware et al. (1984) study most similar to that of the Melia studies was
    the lower respiratory illness index.  The authors gave the unadjusted
    prevalence, and from those data, an estimated odds ratio of 1.08 with
    95% confidence limits of 0.97 and 1.19 was calculated.  Although this
    odds ratio was not adjusted for other covariates, such adjustments
    minimally affected other end-points in this study.  Analyses by Ware
    et al. (1984) on the other end-points found that effects of adjustment
    for covariates was minimal.

         During the period from 1983 to 1986, a new cohort of about
    1000 second- to fifth-grade schoolchildren in each community was
    enrolled and given an initial symptom questionnaire (Dockery et al.,
    1989a).  The authors studied reported respiratory symptoms on a
    subsequent symptom questionnaire (second annual) for 5338 white
    children aged 7 to 11 years at the time of enrolment.  The end-points
    of chronic cough, bronchitis, restriction of activity due to chest
    illness, and persistent wheeze were not associated with gas stove use
    in the home, but the health end-point of doctor-diagnosed respiratory
    illness prior to age 2 yielded an odds ratio of 1.15 with 95%
    confidence limits of 0.96 to 1.37.  The odds ratio for chronic cough
    was 1.15 with 95% confidence limits of 0.89 and 1.91.  The odds ratio
    was adjusted for age, sex, parental education, city of residence, and
    use of unvented kerosene heaters.

         Neas et al. (1990, 1991) studied the effects of measured NO2
    among a stratified one-third random sample of the children that were
    part of the Dockery et al. (1989a) analysis.  The sample was
    restricted to 1286 white children 7 to 11 years of age at enrolment
    with complete covariate information and at least one valid indoor
    measurement of both NO2 and respirable particles.  Methods for
    measuring indoor pollutants were described by Spengler et al. (1986). 
    Indoor pollutants were measured in each child's home for 2 weeks
    during the heating season and 2 weeks during the cooling season.  The
    two 2-week measurements were averaged to estimate each child's annual
    average NO2 exposure.  NO2 was measured by Palmes passive diffusion
    tubes at three locations: kitchen, activity room and the child's
    bedroom.  The three locations were averaged to create a household
    annual average NO2 exposure.

         The analysis of the Neas et al. (1990, 1991) study was based on
    the final symptom questionnaire (third annual), completed by parents
    following the indoor measurements.  The questionnaire reported
    symptoms during the previous year, including attacks of shortness of
    breath with wheeze, persistent wheeze, chronic cough, chronic phlegm

    and bronchitis.  The authors used a multiple logistic model with
    separate city intercepts, indicator variables for gender and age,
    parental history of chronic obstructive pulmonary disease, parental
    history of asthma, parental education and single parent family status. 
    Increases in symptoms were estimated for an additional NO2 exposure
    of 28.3 g/m3 (0.015 ppm).  Table 52 shows the odds ratios for the
    five separate symptoms associated with the increase in NO2 exposure.

    Table 52.  Odds ratios and 95% confidence intervals for the effect of
               an additional load of 0.015 ppm NO2 on the symptom
               prevalence (from: Neas et al., 1991)
                                                                        

    Symptom              Odds ratio   95% Confidence interval
                                                                        

    Shortness of breath    1.23           0.93 to 1.61
    Persistent wheeze      1.16           0.89 to 1.52
    Chronic cough          1.18           0.87 to 1.60
    Chronic phlegm         1.25           0.94 to 1.66
    Bronchitis             1.05           0.75 to 1.47
                                                                        

         Neas et al. (1990, 1991) defined a combined symptom as the
    presence of any of the symptoms just reported.  A multiple logistic
    regression of this combined lower respiratory symptom, equivalent to
    the single response regression, gave an estimated odds ratio of 1.40
    with a 95% confidence interval of 1.14 to 1.72.  The odds ratio for
    the combined symptom score was slightly higher than in other studies,
    but was not inconsistent with those results.  The reference category
    for each of the symptom-specific odds ratios included some children
    with the other lower respiratory symptoms, whereas the children in the
    reference category for combined lower respiratory symptoms were free
    of any of these symptoms.  When split by gender, the odds ratio was
    higher in girls, a result similar to the gender modification reported
    by Melia et al. (1979).  When separate logistic analyses were
    performed for each community, the adjusted odds ratios ranged from
    1.26 for Topeka, Kansas, to 1.86 for Portage, Wisconsin.  When the
    cohort was restricted to the 495 children in homes with a gas stove,
    the adjusted odds ratio was 1.37 with a 95% confidence interval of
    1.02 to 1.84.  Table 53 provides the adjusted odds ratios for combined
    lower respiratory symptoms across ordered NO2 exposure categories. 
    The association is statistically significant for the upper exposure
    category and the overall results are consistent with a linear
    dose-response relationship between NO2 and lower respiratory symptoms
    in children.

    Table 53.  Odds ratios and 95% confidence intervals for the effect of
               ordered NO2 exposures on the prevalence of lower
               respiratory symptoms (from: Neas et al., 1991)
                                                                        

    NO2 level (ppm)                Number of     Odds     95% Confidence
                                   children      ratio    interval

    Range              Mean
                                                                        

    0 to 0.0049        0.0037        263          1.00
    0.005 to 0.0099    0.0073        360          1.06     0.71 to 1.58
    0.010 to 0.0199    0.0144        317          1.36     0.89 to 2.08
    0.020 to 0.0782    0.0310        346          1.65     1.03 to 2.63
                                                                        

         Neas et al. (1992) reported that the estimated effect of an
    additional load of 28.3 g NO2/m3 (0.015 ppm) on lower respiratory
    symptoms was consistent across the seasons and sampling locations. 
    Table 54 provides the odds ratios and 95% confidence intervals for
    this association by season and sampler location.  The NO2 levels
    measured by the activity room and bedroom sampler were more strongly
    associated with lower respiratory symptoms than those in the kitchen. 
    The NO2 measurements in the kitchen were influenced more by transient
    peak levels associated with meal preparation on gas stoves, whereas
    the other sampling locations were more reflective of the child's
    long-term average exposures to NO2 in the home.  Spengler et al.
    (1992) suggested that children spend relatively little time (0.5 h per
    day) in the kitchen when the range is operating.

    7.3.1.3  University of Iowa Study (USA)

         Ekwo et al. (1983) surveyed 1355 children 6 to 12 years of age
    for respiratory symptoms and lung function in the Iowa City School
    District.  Parents of the children completed a questionnaire that was
    a modification of one developed by the American Thoracic Society.  The
    children were a random sample from those families whose parents had
    completed the questionnaire.  Eight measures of respiratory illness
    were reported by the authors, but only two were similar to the
    end-points used in the United Kingdom studies (section 7.3.1.1) and
    the Harvard Six City studies (section 7.3.1.2).  Parental smoking was
    also measured and used as a covariate in the analyses.  Results of the
    analyses, based on 1138 children, are presented in Table 55.  No
    measurements of NO2 exposure, either inside or outside the homes,
    were reported.

    Table 54.  Odds ratios and 95% confidence intervals for the effect of
               an additional 0.015 ppm NO2 on the prevalence of lower
               respiratory symptoms according to sampling location and
               season (from: Neas et al., 1992)
                                                                        

    Sampler location and        Mean difference    Odds    95% Confidence
    season                      gas vs. electric   ratio   interval
                                    (ppm)
                                                                        

    Household annual average        0.016          1.40    1.14 to 1.72
    Household winter average        0.018          1.16    1.04 to 1.29
    Household summer average        0.014          1.46    1.13 to 1.89
    Kitchen annual average          0.022          1.23    1.05 to 1.44
    Activity room annual
      average                       0.014          1.50    1.20 to 1.87
    Bedroom annual average          0.013          1.47    1.17 to 1.85
                                                                        


    Table 55.  Analysis of Iowa city school children respiratory symptoms
               according to gas stove type and parental smoking
               (from: Ekwo et al., 1983)
                                                                        

    Factor                   Hospitalization for     Chest congestion and
                             chest illness           phlegm with colds
                             before age two
                                                                      

                             Odds ratio     SEa      Odds ratio     SEa
                                                                        

    Gas stove use              2.4b        0.684        1.1        0.188
    Smoking effects
      Father alone smokes      2.3b        0.856        1.0        0.213
      Mother alone smokes      2.9b        1.239        1.3        0.363
      Both smoke               1.6         0.859        1.2        0.383
                                                                         

    a  SE = Standard error of the odds ratio
    b  Indicates statistical significance at the 0.05 probability level

    7.3.1.4  Agricultural University of Wageningen (The Netherlands)

         Houthuijs et al. (1987), Brunekreef et al. (1987), and Dijkstra
    et al. (1990) studied the effect of indoor factors on respiratory
    health in 6- to 9-year-old children from 10 primary schools in five

    non-industrial communities in the southeast region of the Netherlands. 
    Personal exposure to NO2 and home concentrations were measured.  An
    important NO2 emission and exposure source in these homes are
    geysers, which are unvented, gas-fired, hot water sources at the water
    tap.  Exposure to tobacco smoke was assessed by a questionnaire that
    also reported symptom information.  The study used Palmes diffusion
    tubes to measure a single weekly average personal NO2 exposure.  In
    January and February 1985, NO2 in the homes of 593 children who had
    not moved in the last 4 years was measured for 1 week.  Personal
    exposure was also estimated from time budgets and room monitoring. 
    Estimated and measured exposures to NO2 are given in Table 56.

    Table 56.  Estimated and measured personal NO2 exposure (g/m3)
               for a single weekly average (from: Houthuijs et al., 1987)
                                                                         

    NO2 Source                     Estimated              Measured
                                                                         

                     Number  Arithmetic  Standard   Arithmetic  Standard
                                mean     deviation     mean     deviation
                                                                         

    No geyser        370        22          7           22          9
    Vented geyser    112        29          9           31          12
    Unvented geyser  111        40          9           42          11
                                                                         


         Three health measures were obtained from the questionnaire, a
    modified form of the WHO questionnaire.  The different items were
    combined to create three categories: cough, wheeze and asthma.  Asthma
    was defined as attacks of shortness of breath with wheezing in the
    past year.  The presence of any of the three symptoms was used as a
    combination variable.  The results are presented in Table 57.  A
    logistic regression model was used to fit the combination variable.
    Exposure was estimated by fitting a log-normal distribution to the
    grouped data, and the mean exposure values for each group were
    estimated by a maximum likelihood technique (Hasselblad et al.,
    1980).  The estimated logistic regression coefficient was 0.002,
    corresponding to an odds ratio of 0.94 for an increase of 28.3 g/m3
    (0.015 ppm) in NO2, with 95% confidence interval of 0.70 to 1.27. 
    Thus, these studies did not demonstrate an increase in respiratory
    disease with increasing NO2 exposure, but the range of uncertainty is
    quite large and the rates were not adjusted for covariates such as
    parental smoking and age of the child.  One potential explanation
    offered by the authors for the negative findings with respect to NO2
    exposure was the smaller sample size of the measured NO2 data
    compared to the categorical data (i.e., gas stove versus electric

    stove use).  They could not estimate whether more precision was gained
    by use of measured NO2 than was lost by the reduction in the sample
    size.  Houthuijs et al. (1987) reported earlier that the presence of
    an unvented geyser in the kitchen is associated with a higher
    prevalence of respiratory symptoms and that the NO2 difference
    between no geyser present and an unvented geyser is about 0.01 ppm.

    7.3.1.5  Ohio State University Study (USA)

         Mitchell et al. (1975) and Keller et al. (1979a) conducted a
    12-month study of respiratory illness and pulmonary function in
    families in Columbus, Ohio, prior to 1978.  The sample included 441
    families divided into two groups using either gas or electric cooking. 
    Participating households were given diaries to record respiratory
    illnesses for 2-week periods.  Respiratory illnesses included colds,
    sore throat, hoarseness, earache, phlegm and cough.  Only one incident
    of illness per person per 2-week period was recorded.  The study
    measured NO2 exposure, by both the Jacobs-Hochheiser and continuous
    chemiluminescence methods.  The electric stove users averaged
    38 g/m3 (0.02 ppm) NO2 exposure, whereas the gas stove users
    averaged 94 g/m3 (0.05 ppm).  The report did not indicate which
    rooms were measured in order to obtain this average.

         No differences were found in any of the illness rates for
    fathers, mothers or children.  No analyses were carried out using
    multiple logistic regression or Poisson regression (these methods were
    relatively new at the time).  No estimates were made that can be
    considered comparable to the odds ratios reported in the other
    studies.  However, the authors did show a bar graph of all respiratory
    illness for children under 12.  The rates were 389 (per 100 person-
    years) for electric stove use and 377 for gas stove use.  These rates
    were not significantly different even after adjustment for covariates,
    including family size, age, gender, length of residence and father's
    education.  No mention was made of adjustments for smoking status or
    smoking exposure for the children.

         In a second, related study (Keller et al., 1979b), 580 people
    drawn from households that participated in the earlier study were
    examined to confirm the reports and to determine the frequency
    distribution of reported symptoms among parents and children in gas or
    electric cooking homes.  A nurse-epidemiologist examined selected
    subjects who reported ill and obtained throat cultures.  The
    percentage of children having respiratory illnesses in homes with a
    gas stove was 85.1% (n = 87) versus 88.8% (n = 89) in homes with
    electric stoves.  The unadjusted proportions permit the calculation of
    an estimated odds ratio of 0.71 with 95% confidence interval of 0.30
    to 1.74.  Unfortunately the adjusted rates were not reported.

         Neas et al. (1991) commented that Keller's model controls for a
    series of variables that specify the child's prior illness history and
    that if chronic exposure to NO2 is a risk factor for prior illnesses,
    controlling for the child's illness history would substantially reduce
    the estimated effect of current NO2 exposure.

    7.3.1.6  University of Dundee (United Kingdom)

         Ogston et al. (1985) studied infant mortality and morbidity in
    the Tayside region of northern Scotland.  The subjects were 1565
    infants born to mothers who were living in Tayside in 1980.  Episodes
    of respiratory illness were recorded during the first year of life. 
    The information was supplemented by observations made by a health
    visitor and scrutinized by a paediatrician who checked diagnostic
    criteria and validity.  One health end-point assessed was defined as
    the presence of any respiratory disease during the year.  The use of
    gas cooking fuel was associated with increase respiratory illness
    (odds ratio = 1.14, 95% confidence interval 0.86 to 1.50) after
    adjustment for parental smoking, mother's age and type of home heating
    (Table 58).  The study did not give measured NO2 exposure values, but
    referenced the other studies conducted elsewhere in the United Kingdom
    for exposure estimates.

    7.3.1.7  Harvard University - Chestnut Ridge Study (USA)

         Schenker et al. (1983) reported a large respiratory disease study
    of 4071 children aged 5 to 14 in the Chestnut Ridge region of western
    Pennsylvania.  The region is predominately rural, with numerous
    underground coal mines and four large coal-fired electricity-
    generating plants in the area.  A standardized children's
    questionnaire (Ferris, 1978) was sent to parents of all children in
    grades 1 to 6 in targeted schools.  An SES scale derived from the
    parent's occupation and education was divided into quintiles to
    provide SES strata.  Important confounding factors considered in the
    analysis were gender, SES and maternal smoking.  In the multiple
    logistic model, no significant association was found between gas stove
    use and any of the respiratory or illness variables after adjusting
    for SES.  No odds ratios or other numerical data were reported.

        Table 57.  Frequency and prevalence of reported respiratory symptoms with respect to different
               categories of mean indoor NO2 concentrations in a population of 775 children
               aged 6 to 12 old (from: Dijkstra et al., 1990)
                                                                                                       

                                       Frequency and prevalence in category of indoor NO2
                                                                                                       

    Symptom                    0-20 g/m3          21-40 g/m3         41-60 g/m3        > 60 g/m3
                               (n = 336)            (n = 267)            (n = 93)          (n = 79)
                                                                                                       

    Cough                    16       4.8%       12       4.5%       7        7.5%       3        3.8%

    Wheeze                   30       8.9%       18       6.7%       3        3.2%       7        8.9%

    Asthma                   22       6.6%       12       4.5%       2        2.2%       3        3.8%

    One or more symptoms     36      10.7%       24       9.0%       8        8.6%       8       10.1%
                                                                                                       
    
    Table 58.  Regression coefficients for multiple logistic analyses of   
               respiratory illness in Tayside children (from: Ogston et
               al., 1985)
                                                                        

    Factor                   Regression    Odds ratio      95% Confidence
                             coefficient                   limits
                                                                        

    Parental smoking         0.429         1.54

    Age of mother            -0.094        not available
    (in 5-year groups)

    Presence of gas stove    0.130         1.14            0.86, 1.50
                                                                        


    7.3.1.8  University of New Mexico Study (USA)

         Samet et al. (1993) conducted a prospective cohort study between
    January 1988 and June 1990 to test the hypothesis that exposure to
    NO2 increases the incidence and severity of respiratory illness
    during the first 18 months of life.  A total of 1315 infants were
    enrolled into the study at birth in Albuquerque, New Mexico.  The
    subjects were healthy infants from homes without smokers and who spent
    less than 20 h/week in day care.  Illness experience was monitored by
    a daily diary of symptoms completed by the mother and a telephone
    interview conducted every two weeks.  For a sample of the ill
    children, a nurse practitioner made a home visit to conduct a
    standardized history and physical assessment.  Exposure to NO2 was
    estimated by a 2-week average concentration measured in the subjects'
    bedrooms with passive samplers.  Estimates of exposure based on
    bedroom concentration were tightly correlated with estimates of
    exposures calculated as time-weighted averages of the concentrations
    in the kitchen, bedroom and activity room.  The authors defined
    illness events as the occurrence on at least two consecutive days of
    any of the following: runny or stuffy nose, wet cough, dry cough,
    wheezing or trouble with breathing.  Wheezing was defined as a


    high-pitched musical sound audible during breathing, and trouble with
    breathing as the parent's perception of rapid or laboured breathing. 
    Illness events ended with two consecutive symptom-free days.

         The analysis was limited to the 1205 subjects completing at least
    1 month of observation; of these, 823 completed the full protocol. 
    Multivariate methods were used to control for potential confounding
    factors and to test for effect modification.  In analyses of
    determinants of incident illnesses, the outcome variable was the
    occurrence of illness during 2-week intervals of days at risk.  The
    independent variables considered in the multivariate analyses included
    the fixed factors of birth order, gender, ethnicity, parental asthma
    and atopic status, household income, and maternal education.  Other
    variables considered were the temporally varying factors of age,
    calendar month, day-care attendance and breast-feeding. Potential
    confounding and effect modification by cigarette smoking was
    controlled by excluding subjects from households with smokers.

         Lambert et al. (1993) reported that in this prospective cohort
    study during the winter, bedroom concentrations in homes with gas
    stoves averaged 0.021 ppm (SD = 0.022 ppm).  In bedrooms of homes with
    electric stoves, concentrations averaged 0.007 ppm (SD = 0.006 ppm). 
    Approximately 77% of the bedroom NO2 observations were less than
    0.02 ppm; only 5% were greater than 0.04 ppm.  The 90th percentile of
    the weekly measured concentrations was 0.05 ppm NO2 in bedrooms.

         Samet et al. (1993) performed the analysis using the generalized
    estimated equations described by Zeger & Liang (1986). This takes into
    account the correlation structure when estimating regression
    coefficients and their standard errors.  The multivariate models
    examined the effects of the unlagged NO2 exposures, lagged NO2
    exposures and stove type (Table 59).  None of the odds ratios was
    significantly different from unity, the value for the reference
    category of 0 to 0.02 ppm.  Additionally, the odds ratios did not tend
    to increase consistently from the middle category of exposure to the
    highest category.  Furthermore, exposure to NO2 and the durations of
    the four illness categories were not associated.  The authors added
    NO2 exposure to the model as a continuous variable, while controlling

    for the same covariates included in Table 59.  For each of the five
    illness variables, the estimated multiplier of the odds ratio per
    0.001 ppm increment of NO2 was 0.999, with confidence limits
    extending from approximately 0.995 to 1.002.

    7.3.1.9  University of Basel Study (Switzerland)

         Braun-Fahrlaender et al. (1989, 1992) and Rutishauser et al.
    (1990a,b) studied the incidence and duration of common airway symptoms
    in children up to 5 years old over a 1-year period in a rural, a
    suburban and two urban areas of Switzerland.  Parents were asked to
    record daily their child's respiratory symptoms (from a list) over a
    6-week period.  Additionally, covariates, including family size,
    parental education, living conditions, health status of the child,
    parents' respiratory health, and smoking habits of the family, were
    assessed by questionnaire.  During the same 6-week period NO2 was
    measured weekly using Palmes tubes, both inside and outside the home
    of the participants.  Meteorological data were obtained from local
    monitoring stations, but additional air quality data from fixed
    monitoring sites were only available for the two urban study areas. 
    NO2 concentrations inside the home were on average lower than in the
    outside air (Fig. 24). Indoor levels for Basel, Zurich, Wetzikon and
    Rufzerfeld were 33.8, 28.4, 20.5 and 11.2 g/m3 (0.018, 0.015, 0.011
    and 0.006 ppm), respectively.  The indoor NO2 concentration depended
    to some extent on the concentration of the outside air.

         The analysis was restricted to 1063 Swiss nationals (from a total
    of 1225 participating families).  For all four study areas, regional
    mean incidence rates of upper respiratory illness, cough, breathing
    difficulties and total respiratory illness, adjusted for individual
    covariates and weather data, were regressed (using Poisson regression)
    against regional differences in annual mean NO2 concentrations.  All
    the relative risks were computed for a 20-g/m3 (0.011-ppm) increase
    in pollution concentration.  The NO2 concentration measured by indoor
    passive sampler was associated with the duration of any episode
    (relative duration of 1.16, 95% confidence interval of 1.12 to 1.21),
    upper respiratory episodes (relative duration of 1.18, 95% confidence
    interval of 1.01 to 1.38), and coughing episodes (relative duration of
    1.15, 95% confidence interval of 1.03 to 1.29).  A discussion of
    associations with outdoor levels is presented in section 7.3.2.

    FIGURE 24

        Table 59.  Odds ratiosa for effect of nitrogen dioxide exposure on incidence of respiratory illness
               (from: Samet et al., 1993)
                                                                                                                                          

    NO2 exposure              All illnesses                  All lower                    Lower, with                   Lower, with
                                                                                           wet cough                      wheezing
                                                                                                                                    

                        Odds ratio     95% CIb        Odds ratio     95% CIb        Odds ratio     95% CIb        Odds ratio     95% CIb
                                                                                                                                          

    Unlaggedc              1.04       0.96-1.12          0.98       0.89-1.09          1.00       0.89-1.12          0.92       0.73-1.15
    0.02-0.06 ppm          0.94       0.81-1.08          0.93       0.76-1.13          0.94       0.77-1.16          0.88       0.56-1.37
    > 0.04 ppm

    Laggedc                1.01       0.93-1.10          0.97       0.87-1.08          0.97       0.87-1.09          0.95       0.75-1.19
    0.02-0.06 ppm          0.92       0.77-1.10          0.91       0.72-1.15          0.89       0.68-1.16          0.98       0.66-1.48
    > 0.04 ppm

    Gas Stoved             0.98       0.90-1.07          0.91       0.81-1.04          0.94       0.82-1.07          0.84       0.64-1.09
                                                                                                                                          

    a  Obtained by generalized estimating equation method. Adjusted for season, age, gender, ethnicity, birth order, day care, income,
       maternal education, breast feeding, parental atopy and asthma, and maternal history of respiratory symptoms.
    b  CI = Confidence interval
    c  Reference category is 0-0.02 ppm NO2
    d  Reference category is electric stove
        7.3.1.10  Yale University Study (USA)

         Berwick et al. (1984, 1987, 1989), Leaderer et al. (1986) and
    Berwick (1987) reported on a 12-week study (six 2-week time periods)
    of lower and upper respiratory symptoms in 159 women and 121 children
    (aged 12 or less) living in Connecticut.  Levels of NO2 were measured
    in 91% of the homes, 57 of which had kerosene heaters and 62 of which
    did not.  Ambient NO2 levels ranged from 9 to 19 g/m3 (0.005 to
    0.01 ppm) for the six 2-week time periods.  Two-week average indoor
    NO2 levels in homes of monitored children were highest for homes with
    kerosene heaters  and  gas stoves (91 g/m3, 0.05 ppm; n = 8),
    second highest for kerosene only (36 g/m3, 0.02 ppm; n = 45), third
    highest for gas stoves only (32 g/m3, 0.02 ppm; n = 13), and lowest
    for no sources (6 g/m3, 0.003 ppm; n = 43).  Indoor levels did not
    fluctuate greatly over time, as indicated by the 2-week averages.  A
    comparison of personal NO2 exposures, as measured by Palmes diffusion
    tubes, and NO2 exposures measured in residences had a correlation of
    0.94 for a subsample of 23 individuals.  Results of this comparison
    show an excellent correlation between average household exposure and
    measured personal exposure (see section 3.6 and Fig. 13).

         The study defined lower respiratory illness as the presence of at
    least two of the following: fever, chest pain, productive cough,
    wheeze, chest cold, physician-diagnosed bronchitis, physician-
    diagnosed pneumonia and asthma.  Information on many potential
    covariates (e.g., SES, age, gender and exposure to environmental
    tobacco smoke) was obtained.  The covariates having the largest effect
    were age of child, family SES and history of respiratory illness, as
    shown by multiple logistic analysis.  When controlling for SES and
    history of respiratory illness, children under 7 years of age exposed
    to 30 g NO2/m3 (0.016 ppm) or more were found to have a risk of
    lower respiratory symptoms 2.25 times higher than that of unexposed
    children (95% confidence limits of 1.69 and 4.79).  Older children and
    adults showed no increased risk.

         Although the Berwick study had relatively extensive information
    on exposure, several problems are evident.  Unvented kerosene
    space-heaters also release volatile organic compounds and combustion
    particles.  The 4-year age-specific relative risks for lower
    respiratory disease are very variable, and it is not clear why these
    3-year strata were collapsed into 2 strata at 7 years of age.  The

    analyses may be sensitive to the adjustment for SES, which can be
    correlated with exposure.  This is less of a problem in studies with
    larger sample sizes (e.g., Melia et al. 1977, 1979), but may be
    critical in the Berwick study.  Furthermore, Neas et al. (1991) noted
    that the Berwick study controlled for prior illnesses, as did the
    Keller study, which would reduce the estimated effect of current NO2
    exposure.

    7.3.1.11  Freiburg University Study (Germany)

         Kuehr et al. (1991) conducted a cross-sectional study on the
    prevalence of asthma in childhood in relation to NO2 levels in the
    city of Freiburg and two Black Forest communities. A study group of
    704 children (with 41 asthmatic) aged 7 to 16 years took part in a
    standardized interview and medical examination. Indoor and outdoor
    exposure information was taken into account.  Passive smoking
    exposures were assessed.  Stoves used as heating devices carried a
    4.8-fold relative risk for asthma compared to other types of heating
    (95% CI 1.95-11.8).

    7.3.1.12  McGill University Study (Canada)

         In a case-control study carried out in Montreal, Quebec, Canada,
    between 1988 and 1990, NO2 levels measured by passive NO2 monitoring
    badge were studied in relation to the incidence of asthma among 3- and
    4-year-old children (Infante-Rivard, 1993).  Multivariate
    unconditional logistic regression was carried out for the 140 subjects
    who had NO2 measurements; the analysis included NO2 and the
    variables retained in the final conditional model that includes SES
    and parental smoking.  The author reported an increase in asthma
    incidence associated with NO2 exposure levels.  However, the Task
    Group noted the exceptionally large effect estimates given the
    exposure levels.

    7.3.1.13  Health and Welfare Canada Study (Canada)

         Dekker et al. (1991) studied asthma and wheezing syndromes as
    part of a questionnaire-based study of 17 962 Canadian school
    children.  The questionnaire was developed from the 1978 American
    Thoracic Society questionnaire, which was the same as that used in the
    Harvard Six Cities Study.  For analysis, the sample was restricted to
    children aged 5 to 8 years and excluded those children with cystic
    fibrosis as well as those living in mobile homes, tents, vans,
    trailers and boats.  The authors calculated odds ratios adjusted for

    age, race, gender, parental education, gender of the respondent,
    region of residence, crowding, dampness and environmental tobacco
    smoke.  The adjusted odds ratio of asthma as a function of gas cooking
    was 1.95 with 95% confidence limits of 1.41 and 2.68.  The adjusted
    odds ratio of wheezing as a function of gas cooking was 1.04 with 95%
    confidence limits of 0.77 and 1.42.  The authors noted that this
    finding needed to be treated with caution, however, because of the few
    subjects with asthma in the study who were exposed to gas cooking
    (n = 60).

    7.3.1.14  University of North Carolina Study (USA)

         Margolis et al. (1992) studied the prevalence of persistent
    respiratory symptoms in 393 infants of different SES by analysing data
    from a community-based cohort study of respiratory illness in the
    first year of life in central North Carolina between 1986 and 1988. 
    Infants were limited to those weighing more than 2000 g and who did
    not require neonatal care outside the normal newborn nursery.  Of
    those eligible, 47% were enrolled and, of these, 77% completed the
    study and were included in the analysis.  Compared with the 1241
    infants from families refusing enrolment, the 1091 eligible study
    infants were more likely to be of high SES and were more often black. 
    Study infants were less likely to have mothers who smoked.

         The presence of persistent respiratory symptoms was measured at
    the 12-month home interview using an American Thoracic Society
    children questionnaire (modified for infants) for studies of
    respiratory illness.  Infants who were reported to "usually cough" or
    "occasionally wheeze" were classified as having persistent respiratory
    symptoms.

         Of the 393 infants that Margolis et al. (1992) included in their
    study, approximately 41 lived in homes with gas cooking.  The relative
    risk of persistent respiratory symptoms among infants exposed to gas
    cooking unadjusted for any covariates was 1.12 (95% confidence
    interval of 0.63 to 2.04).

    7.3.1.15  University of Tucson Study (USA)

         The study by Dodge (1982) was based on a cohort of 676 children
    in the third and fourth grades (about 90% aged 8-10 years) of schools
    in three Arizona communities.  Gas cooking stoves were associated with
    increased symptoms: asthma odds ratio = 1.47, wheeze odds ratio 
    = 1.24, sputum odds ratio = 2.28, and cough odds ratio = 2.21. 
    However, only 79 children (19%) had electric heat, so the numbers were
    small and only cough was significant at the 0.05 level.  After
    controlling for height and age, gas stoves were not associated with a
    decline in the growth of FEV1.

    7.3.1.16  Hong Kong Anti-Cancer Society Study (Hong Kong)

         In 1985, 362 primary school children (age 7-13 years) were
    included in a study of NO2 exposure and respiratory illness in Hong
    Kong (Koo et al., 1990).  Exposures to NO2 were estimated by use of
    personal badge monitors, worn for a single period of 24 h, and
    supplemented by monitors placed in classrooms.  NO2 exposures were
    estimated in the same manner for the mothers of the study children. 
    Mothers and children completed respiratory illness questionnaires.  No
    association was found between respiratory symptoms and NO2 exposures
    for children (mean 19 ppb).  Among the mothers (mean exposure 19 ppb)
    allergic rhinitis and chronic cough were associated with NO2.

    7.3.1.17  Recent studies

         This section includes studies that have reported preliminary
    results only or have appeared recently in the scientific literature.

         Spengler et al. (1993) reported results for evaluation of more
    than 15 000 schoolchildren in various sites in the USA and Canada, but
    found no statistically significant increases in respiratory symptoms
    to be associated with use of gas heaters or cookers.

         Goren et al. (1993) reported no association between gas heating
    and respiratory health effects among 8000 schoolchildren in Israel.

         Preliminary results reported by Peat et al. (1990) indicated no
    relationship between relatively high NO2 in Australian homes with
    gas use in Sydney and respiratory symptoms or bronchial hyper-
    responsiveness.

         Pilotto (1994) reported a prospective study of health effects of
    unflued gas heater emissions on 425 Australian schoolchildren aged
    6-11 years.  Short-term indoor monitoring by means of passive
    diffusion badge monitors placed in classrooms or worn at home was
    carried out to determine daily 6-h averages.  Children exposed to a
    level of 0.08 ppm or more, compared with a background level of
    0.02 ppm, had increased rates of respiratory illnesses and school
    absences.

    7.3.2  Outdoor studies

         Several studies have examined the relationship of estimated
    ambient NO2 levels to respiratory health outcome measures, including
    various respiratory symptomatologies.  Those that provide a
    quantitative estimate of effect are indicated in Table 60.

        Table 60.  Effects of outdoor NO2 exposure on respiratory disease
                                                                                                                                          

    Study                              Health end-point              NO2 levels (ppm)/period            Odds ratio or       95% CI
                                                                                                           estimate
                                                                                                                                          

    Dockery et al. (1989b)             Bronchitis                    0.007-0.023 annual average              1.7            0.5 to 5.5
                                       Chronic cough                                                         1.6            0.3 to 10.5
                                       Chest illness                                                         1.2            0.3 to 4.8
                                       Wheeze                                                                0.8            0.4 to 1.6
                                       Asthma                                                                0.6            0.3 to 0.9

    Braun-Fahrlaender et al. (1992)    Duration of respiratory       Change of 0.011 6-week                  1.11           1.07 to 1.16
                                       episodes                      average

    Schwartz et al. (1991)             Croup                         0.005-0.037 daily                       1.28           1.07 to 1.54

    Jaakkola et al. (1991)             Upper respiratory             Contrasted polluted versus              1.6            1.1 to 2.1
                                       infection                     less polluted areas by
                                                                     comparison of annual levels
                                                                                                                                          
        7.3.2.1  Harvard University - Six City Studies (USA)

         As part of the US Six City Studies, Dockery et al. (1989b)
    obtained respiratory illness and symptom data from questionnaires
    distributed from September 1980 to April 1981.  Indoor air aspects
    of this study (Dockery et al., 1989a) were described in the section
    on indoor studies.  The questionnaires obtained information on
    bronchitis, cough, chest illness, wheeze and asthma.  A centrally
    located air monitoring station was established in 1974 where ambient
    sulfur dioxide, NO2, ozone, total suspended particulate matter and
    meteorological variables were measured.  The authors used multiple
    logistic regression analysis in order to adjust for covariates of
    gender, age, maternal smoking, gas stove use and separate intercepts
    for each city.  Although the strongest associations were found between
    respiratory symptoms and particulate matter, there were increased odds
    ratios of respiratory symptoms with ambient NO2.  These were not
    statistically significant, but the direction for bronchitis, chronic
    cough and chest illness was consistent with the studies of indoor
    exposure.  The odds ratios for various health end-points for an
    increase in NO2 from the lowest-exposure city to the highest-exposure
    city 12 to 43 g/m3 (0.0065 to 0.0226 ppm) are shown in Table 60.

    7.3.2.2  University of Basel Study (Switzerland)

         Braun-Fahrlaender et al. (1992) studied the incidence and
    duration of common airway symptoms in children up to 5 years old. 
    This study, also discussed in section 7.3.1.9, was conducted over
    a 1-year period in a rural, a suburban and two urban areas of
    Switzerland.  Parents were asked to record their child's respiratory
    symptoms (from a list) daily over a 6-week period.  Additionally,
    covariates including family size, parental education, living
    conditions, health status of the child, parents' respiratory health
    and smoking habits of the family were assessed by questionnaire. 
    Weekly NO2 measurements were made during the same 6-week period using
    Palmes tubes, both inside and outside the home of the participants. 
    Meteorological data were obtained from local monitoring stations, but
    additional air quality data from fixed monitoring sites were only
    available for the two urban study areas.  The analysis was restricted
    to 1063 Swiss nationals (from a total of 1225 participating families). 
    For all four study areas, regional mean incidence rates of upper
    respiratory illness, cough, breathing difficulties and total
    respiratory illness, adjusted for individual covariates and weather
    data, were regressed (using Poisson regression) against regional
    differences in annual mean NO2 concentrations.  There was no
    association between long-term differences in NO2 levels by region and
    mean annual rates of respiratory incidence.

         The adjusted annual mean symptom duration by region and the
    corresponding NO2 levels (measured by passive samplers) are shown in
    Table 61.  A second-stage regression of the adjusted natural logarithm
    of regional mean duration on NO2 levels yields significant
    associations between outdoor NO2 levels and the average duration of
    any respiratory episode (relative duration of 1.11, 95% confidence
    interval of 1.07 to 1.16) and upper respiratory episodes (relative
    duration of 1.14, 95% confidence interval of 1.03 to 1.25).  A
    positive trend for the duration of coughing episodes was also seen
    (relative duration of 1.09, 95% confidence interval of 0.97 to 1.22). 
    No association was seen with the duration of breathing difficulties. 
    All the relative risks are computed for a 20-g/m3 (0.011-ppm)
    increase in pollution concentration.  In the suburban and rural areas,
    NO2 was the only air pollutant measured.  Correlation between
    outdoor passive NO2 sampler and total suspended particulate (TSP)
    measurements in the two urban study areas was quite high (0.52).  The
    high correlation between NO2 and TSP suggests that this NO2
    association may reflect confounding with TSP.  The lack of TSP data
    for the other two regions precludes eliminating TSP as a possible
    confounder in this analysis.  But the consistency of the NO2 findings
    are evident and, although the association with symptom duration in
    Zurich and Basel may well be due to confounding with TSP, the
    cross-sectional association across the four regions supports a
    possible NO2 role.

    7.3.2.3  University of Wuppertal Studies (Germany)

         Schwartz et al. (1991) evaluated respiratory illness in five
    German communities.  Children's hospitals, paediatric departments of
    general hospitals, and paediatricians reported daily the numbers of
    cases of croup.  A diagnosis of croup was based on symptoms of
    hoarseness and barking cough, inspiratory stridor, dyspnoea, and a
    sudden onset.  The counts were modelled using Poisson regression
    with adjustments for weather, season, temperature, humidity and
    autoregressive errors.  Statistically significant effects of both
    ambient particulate matter and NO2 were found on the counts of
    respiratory illnesses.  A relationship between short-term fluctuations
    in air pollution and short-term fluctuations in medical visits for
    croup symptoms was found in this study.  The estimated relative risk
    was 1.28 with 95% confidence limits of 1.07 and 1.54 for an increase
    from 10 to 70 g NO2/m3 (0.005 to 0.037 ppm).

    7.3.2.4  University of Tubigen (Germany)

         Rebmann et al. (1991) studied 875 cases of croup in Baden-
    Wrttemberg in relation to ambient NO2 levels over a 2-year period. 
    Monthly NO2 means varied from 23 to 78 g/m3.  Statistical
    regression methods indicated weak but statistically significant
    influences of the daily ambient NO2 mean on the occurrence of croup.

        Table 61.  Adjusted annual symptom duration (days) and NO2 levels in four regions of Switzerland
               (from: Braun-Fahrlaender et al., 1992)
                                                                                                                           

    Region         Any symptom     URI durationa      Cough       Breathing difficulty     Indoor NO2         Outdoor NO2
                     duration                        duration             duration        concentration      concentration
                                                                                               (ppm)             (ppm)
                                                                                                                           

    Basel             4.50             1.99            2.32                 1.55              0.0166             0.0272

    Zurich            4.21             1.85            2.01                 1.72              0.0118             0.0248

    Wetzikon          4.00             1.62            2.10                 3.47              0.0103             0.0173

    Rafzerfeld        3.88             1.72            2.02                 1.25              0.0059             0.0133
                                                                                                                           

    a  URI = Upper respiratory illness
        7.3.2.5  Harvard University - Chestnut Ridge Study (USA)

         In the autumn of 1980, Vedal et al. (1987) conducted a panel
    study on 351 children selected from the 1979 Chestnut Ridge study. 
    Parents and children were instructed at the beginning of the school
    year in completing daily diaries of respiratory symptoms.  Lower
    respiratory illness was defined as wheeze, pain on breathing, or
    phlegm production.  Of the 351 subjects selected for the 8 month of
    follow-up, 128 participated in the completion of diaries.  Three
    subgroups were established: one without respiratory symptoms, one with
    symptoms of persistent wheeze, and one with cough or phlegm production
    but without persistent wheeze.  Maximum hourly NO2 levels, measured
    at a single monitoring site in the study region, for each 24-h period
    were used to reflect the daily pollutant level.  During September 1980
    to April 1981, the mean NO2 maximum daily level was 40.5 g/m3
    (0.021 ppm) with a range of 12 to 79 g/m3 (0.006 to 0.042 ppm). 
    Regression models could not be fit for asymptomatic subjects; thus 55
    subjects were included in the analysis of lower respiratory illness,
    but NO2 levels were not predictive of any symptom outcome.

    7.3.2.6  University of Helsinki Studies (Finland)

         Jaakkola et al. (1991) studied the effects of low-level air
    pollution in three cities by comparing the frequency of upper
    respiratory infections over a 12-month period in 1982 as reported by
    parents of children aged 14 to 18 months (n = 679) and 6 years
    (n = 759).  Pollutants studied included ambient levels of NO2, the
    annual mean of which was 15 g/m3 (0.008 ppm).  Other pollutants
    monitored were sulfur dioxide, hydrogen sulfide and particles. 
    Passive smoking and SES were taken into account.  The authors reported
    a significant association between the occurrence of upper respiratory
    infections and living in an air-polluted area for both age groups
    studied, both between and within cities.  The adjusted odds ratio was
    1.6 (95% confidence interval of 1.1 to 2.1) in the 6-year-old age
    group.  The authors concluded that the combined effect of sulfur
    dioxide, particulates, NO2, hydrogen sulfide and other pollutants may
    be a contributing factor in the study results.

    7.3.2.7  Helsinki City Health Department Study (Finland)

         Pnk (1991) studied effects of ambient air pollution and minimum
    temperature on the number of patients admitted to hospital for asthma
    attacks in Helsinki from 1987 to 1989.  During the 3-year period,
    4209 hospitalizations for asthma occurred. The temperature ranged from
    37 to +26C, with a 3-year mean of 5C, and the number of admissions
    increased during cold weather.  After standardization for minimum
    temperature, the multiple-regression analysis indicated that NO2 and
    carbon monoxide levels were significantly related to asthma admission. 
    The NO2 levels averaged 38.6 g/m3 (0.02 ppm) for the 3-year period,
    ranging from 4.0 to 169.6 g/m3 (0.002 to 0.09 ppm).  During the

    period of high NO2 (mean 45.8 g/m3, 0.024 ppm) levels, the mean
    number of all admissions was 29% greater than during the lower
    pollution period (28.1 g/m3, 0.015 ppm).  Indoor NO2 levels and
    cooking fuel use were not reported.

    7.3.2.8  Oulu University Study (Finland)

         The number of daily attendances for asthma at the emergency room
    of the Oulu University Central Hospital, Finland, was recorded for one
    year, along with daily measures of air pollutants at four points
    around the city (Rossi et al., 1993).  Daily mean levels of NO2
    ranged up to 69 g/m3 (maxima 0-154 g/m3).  Asthma visits were
    reported to be significantly associated with NO2, SO2, H2S and TSP
    levels.  After adjustment for daily temperature, only NO2 was
    significantly correlated with attendances.  The association of NO2
    and asthma attacks was stronger in winter months than during the
    summer.

    7.3.2.9  Seth GS Medical College Study (India)

         A survey of air pollution and health was carried out in Bombay,
    India, in 1978 (Kamat et al., 1980).  The study included 4129 adults
    in three urban areas and one rural area.  A single monthly mean NO2
    level was reported for each study area - annual averages were 4 g/m3
    in the rural area, and 14-16 g/m3 in the city.  Winter levels in
    the city study were higher than at other times of the year (up to
    40 g/m3).  It was reported that chronic cough with sputum, frequent
    colds and exertional dyspnoea were significantly associated with NO2
    levels.  These symptoms were also associated with atmospheric levels
    of SO2 and suspended particulate matter, and it was not possible to
    identify a separate influence of NO2 alone.

    7.4  Pulmonary function studies

         Pulmonary function studies are part of any comprehensive
    investigation of the possible effects of any air pollutant. 
    Measurements can be made in the field, they are non-invasive, and
    their reproducibility has been well documented.  Age, height, gender
    and presence of respiratory symptoms are important determinants of
    lung function. Furthermore, changes in pulmonary function have been
    associated with exposure to tobacco smoke, particulate matter and
    other factors.  The studies reviewed below evaluate pulmonary function
    changes in relation to indoor or outdoor NO2 exposures.  Several of
    the respiratory disease studies described earlier also included
    information on pulmonary function.

    7.4.1  Harvard University - Six City Studies (USA)

         Ware et al. (1984) described analysis of lung function values
    using multiple linear regression on the logarithm of the lung function
    measures.  Covariates included sex, height, age, weight, smoking
    status of each parent, and educational attainment of the parents. 
    Exposure to gas stoves was associated with reductions of 0.7% in mean
    FEV1 (forced expiratory volume in 1 second) and 0.6% in mean forced
    vital capacity (FVC) at the first examination (p < 0.01), and
    reductions of 0.3% at the second examination (not significant). 
    The estimated effect of exposure to gas stoves was reduced by
    approximately 30% after adjustment for parental education.  The
    authors stated that the adjustment for parental education may be an
    over-adjustment, and may partially represent gas stove use because of
    association between parental education and type of stove.

         Berkey et al. (1986) used the data from children seen at two to
    five annual visits to study factors affecting pulmonary function
    growth.  Children whose mothers smoked one pack of cigarettes per day
    had FEV1 growth rates approximately 0.17% per year lower (p = 0.05). 
    The same data provided no evidence for an effect of gas stove exposure
    on growth rate.

         Dockery et al. (1989b) obtained pulmonary function data
    during the 1980 and 1981 school year.  Only TSP concentration was
    consistently associated with estimated lower levels of pulmonary
    function.  There was little evidence for an association between lower
    pulmonary function levels and the annual mean concentration of NO2 or
    any other pollutant.

         Neas et al. (1991) also reported that indoor NO2 levels were not
    significantly associated with a deficit in children's pulmonary
    function levels in either of two examinations (FEV1 and FVC).

    7.4.2  National Health and Nutrition Examination Survey Study (USA)

         Schwartz (1989) studied air pollution effects on lung function in
    children and youths aged 6 to 24 years.  FVC, FEV1, and peak flow
    measurements taken as part of the National Health and Nutrition
    Examination Survey II (NHANES II) were examined after controlling
    for age, height, race, gender, body mass, cigarette smoking and
    respiratory symptoms.  Air pollution measurements were taken from all
    popul