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 Organization or the World Health Organization.

Environmental Health Criteria 229

First draft prepared by Drs J. Kielhorn, U. Wahnschaffe and I. Mangelsdorf, Fraunhofer Institute of Toxicology and Aerosol Research, Hanover, Germany

Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organization 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, 2003

The International Programme on Chemical Safety (IPCS), established in 1980, is a joint venture of the United Nations Environment Programme (UNEP), the International Labour Organization (ILO) and the World Health Organization (WHO). The overall objectives of the IPCS are to establish the scientific basis for assessment of the risk to human health and the environment from exposure to chemicals, through international peer review processes, as a prerequisite for the promotion of chemical safety, and to provide technical assistance in strengthening national capacities for the sound management of chemicals.

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WHO Library Cataloguing-in-Publication Data

Selected nitro- and nitro-oxy-polycyclic aromatic hydrocarbons.

(Environmental health criteria ; 229)

1.Polycyclic hydrocarbons, Aromatic - toxicity 2.Polycyclic hydrocarbons,

Aromatic - adverse effects 3.Environmental exposure 4.Risk assessment

I.International Programme for Chemical Safety II.Series

ISBN 92 4 157229 9        (NLM classification: QD 341.H9)

ISSN 0250-863X

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ENVIRONMENTAL HEALTH CRITERIA FOR
SELECTED NITRO- AND NITRO-OXY-POLYCYCLIC AROMATIC HYDROCARBONS

NOTE TO READERS OF THE CRITERIA MONOGRAPHS

Every effort has been made to present information in the criteria monographs as accurately as possible without unduly delaying their publication. In the interest of all users of the Environmental Health Criteria monographs, readers are requested to communicate any errors that may have occurred to the Director of the International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland, in order that they may be included in corrigenda.

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Environmental Health Criteria

PREAMBLE

Objectives

In 1973, the WHO Environmental Health Criteria Programme was initiated with the following objectives:

1. to assess information on the relationship between exposure to environmental pollutants and human health, and to provide guidelines for setting exposure limits;
2. to identify new or potential pollutants;
3. to identify gaps in knowledge concerning the health effects of pollutants;
4. to promote the harmonization of toxicological and epidemiological methods in order to have internationally comparable results.

The first Environmental Health Criteria (EHC) monograph, on mercury, was published in 1976, and since that time an ever-increasing number of assessments of chemicals and of physical effects have been produced. In addition, many EHC monographs have been devoted to evaluating toxicological methodology, e.g. for genetic, neurotoxic, teratogenic and nephrotoxic effects. Other publications have been concerned with epidemiological guidelines, evaluation of short-term tests for carcinogens, biomarkers, effects on the elderly and so forth.

Since its inauguration, the EHC Programme has widened its scope, and the importance of environmental effects, in addition to health effects, has been increasingly emphasized in the total evaluation of chemicals.

The original impetus for the Programme came from World Health Assembly resolutions and the recommendations of the 1972 UN Conference on the Human Environment. Subsequently the work became an integral part of the International Programme on Chemical Safety (IPCS), a cooperative programme of UNEP, ILO and WHO. In this manner, with the strong support of the new partners, the importance of occupational health and environmental effects was fully recognized. The EHC monographs have become widely established, used and recognized throughout the world.

The recommendations of the 1992 UN Conference on Environment and Development and the subsequent establishment of the Intergovernmental Forum on Chemical Safety with the priorities for action in the six programme areas of Chapter 19, Agenda 21, all lend further weight to the need for EHC assessments of the risks of chemicals.

Scope

The criteria monographs are intended to provide critical reviews on the effects on human health and the environment of chemicals and of combinations of chemicals and physical and biological agents. As such, they include and review studies that are of direct relevance for the evaluation. However, they do not describe every study carried out. Worldwide data are used and are quoted from original studies, not from abstracts or reviews. Both published and unpublished reports are considered, and it is incumbent on the authors to assess all the articles cited in the references. Preference is always given to published data. Unpublished data are used only when relevant published data are absent or when they are pivotal to the risk assessment. A detailed policy statement is available that describes the procedures used for unpublished proprietary data so that this information can be used in the evaluation without compromising its confidential nature (WHO (1990) Revised Guidelines for the Preparation of Environmental Health Criteria Monographs. PCS/90.69, Geneva, World Health Organization).

In the evaluation of human health risks, sound human data, whenever available, are preferred to animal data. Animal and in vitro studies provide support and are used mainly to supply evidence missing from human studies. It is mandatory that research on human subjects is conducted in full accord with ethical principles, including the provisions of the Helsinki Declaration.

The EHC monographs are intended to assist national and international authorities in making risk assessments and subsequent risk management decisions. They represent a thorough evaluation of risks and are not, in any sense, recommendations for regulation or standard setting. These latter are the exclusive purview of national and regional governments.

Content

The layout of EHC monographs for chemicals is outlined below.

• Summary — a review of the salient facts and the risk evaluation of the chemical
• Identity — physical and chemical properties, analytical methods
• Sources of exposure
• Environmental transport, distribution and transformation
• Environmental levels and human exposure
• Kinetics and metabolism in laboratory animals and humans
• Effects on laboratory mammals and in vitro test systems
• Effects on humans
• Effects on other organisms in the laboratory and field
• Evaluation of human health risks and effects on the environment
• Conclusions and recommendations for protection of human health and the environment
• Further research
• Previous evaluations by international bodies, e.g., IARC, JECFA, JMPR

Selection of chemicals

Since the inception of the EHC Programme, the IPCS has organized meetings of scientists to establish lists of priority chemicals for subsequent evaluation. Such meetings have been held in Ispra, Italy, 1980; Oxford, United Kingdom, 1984; Berlin, Germany, 1987; and North Carolina, USA, 1995. The selection of chemicals has been based on the following criteria: the existence of scientific evidence that the substance presents a hazard to human health and/or the environment; the possible use, persistence, accumulation or degradation of the substance shows that there may be significant human or environmental exposure; the size and nature of populations at risk (both human and other species) and risks for environment; international concern, i.e., the substance is of major interest to several countries; adequate data on the hazards are available.

If an EHC monograph is proposed for a chemical not on the priority list, the IPCS Secretariat consults with the Cooperating Organizations and all the Participating Institutions before embarking on the preparation of the monograph.

Procedures

The order of procedures that result in the publication of an EHC monograph is shown in the flow chart. A designated staff member of IPCS, responsible for the scientific quality of the document, serves as Responsible Officer (RO). The IPCS Editor is responsible for layout and language. The first draft, prepared by consultants or, more usually, staff from an IPCS Participating Institution, is based on extensive literature searches from reference databases such as Medline and Toxline.

EHC PREPARATION FLOW CHART

The draft document, when received by the RO, may require an initial review by a small panel of experts to determine its scientific quality and objectivity. Once the RO finds the document acceptable as a first draft, it is distributed, in its unedited form, to well over 150 EHC contact points throughout the world who are asked to comment on its completeness and accuracy and, where necessary, provide additional material. The contact points, usually designated by governments, may be Participating Institutions, IPCS Focal Points or individual scientists known for their particular expertise. Generally some four months are allowed before the comments are considered by the RO and author(s). A second draft incorporating comments received and approved by the Director, IPCS, is then distributed to Task Group members, who carry out the peer review, at least six weeks before their meeting.

The Task Group members serve as individual scientists, not as representatives of any organization, government or industry. Their function is to evaluate the accuracy, significance and relevance of the information in the document and to assess the health and environmental risks from exposure to the chemical. A summary and recommendations for further research and improved safety aspects are also required. The composition of the Task Group is dictated by the range of expertise required for the subject of the meeting and by the need for a balanced geographical distribution.The three cooperating organizations of the IPCS recognize the important role played by nongovernmental organizations. Representatives from relevant national and international associations may be invited to join the Task Group as observers. Although observers may provide a valuable contribution to the process, they can speak only at the invitation of the Chairperson. Observers do not participate in the final evaluation of the chemical; this is the sole responsibility of the Task Group members. When the Task Group considers it to be appropriate, it may meet in camera.

All individuals who as authors, consultants or advisers participate in the preparation of the EHC monograph must, in addition to serving in their personal capacity as scientists, inform the RO if at any time a conflict of interest, whether actual or potential, could be perceived in their work. They are required to sign a conflict of interest statement. Such a procedure ensures the transparency and probity of the process.

When the Task Group has completed its review and the RO is satisfied as to the scientific correctness and completeness of the document, it then goes for language editing, reference checking and preparation of camera-ready copy. After approval by the Director, IPCS, the monograph is submitted to the WHO Office of Publications for printing. At this time, a copy of the final draft is sent to the Chairperson and Rapporteur of the Task Group to check for any errors.

It is accepted that the following criteria should initiate the updating of an EHC monograph: new data are available that would substantially change the evaluation; there is public concern for health or environmental effects of the agent because of greater exposure; an appreciable time period has elapsed since the last evaluation.

All Participating Institutions are informed, through the EHC progress report, of the authors and institutions proposed for the drafting of the documents. A comprehensive file of all comments received on drafts of each EHC monograph is maintained and is available on request. The Chairpersons of Task Groups are briefed before each meeting on their role and responsibility in ensuring that these rules are followed.

WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR SELECTED NITRO- AND NITRO-OXY-POLYCYCLIC AROMATIC HYDROCARBONS

Members

Professor D. Anderson, Department of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire, United Kingdom (Chairperson)

Professor J. Arey, Air Pollution Research Center, University of California, Riverside, California, USA

Dr R.P. Bos, Department of Pharmacology & Toxicology, UMC St. Radboud, University of Nijmegen, Nijmegen, The Netherlands

Dr A. Cecinato, Istituto sull’Inquinamento Atmosferico-CNR, CP10 Monterotondo Stazione, Rome, Italy

Dr K. El-Bayoumy, Division of Cancer Etiology & Prevention, American Health Foundation, Valhalla, New York, USA (Vice-Chairperson)

Dr P.C. Howard, Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas, USA (Co-Rapporteur)

Dr J. Kielhorn, Chemical Risk Assessment, Fraunhofer Institute of Toxicology and Aerosol Research, Hanover, Germany (Co-Rapporteur)

Professor M. Kirsch-Volders, Laboratory of Cell Genetics, Free University of Brussels, Brussels, Belgium

Dr I. Mangelsdorf, Chemical Risk Assessment, Fraunhofer Institute of Toxicology and Aerosol Research, Hanover, Germany

Dr S. Pavittranon, Toxicology and Environmental Laboratory, National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nontaburi, Thailand

Dr H. Tokiwa, Department of Environmental Health Science, Kyushu Women’s University, Kitakyushu, Japan

Dr U. Wahnschaffe, Consultant, Uetze, Germany

Professor Z. Yuxin, Institute of Occupational Medicine, Chinese Academy of Preventive Medicine, Beijing, People’s Republic of China

Secretariat

Mr T. Ehara, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland

Mrs P. Harlley, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland

WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR SELECTED NITRO- AND NITRO-OXY-POLYCYCLIC AROMATIC HYDROCARBONS

The first and second drafts of this monograph were prepared by the authors, Drs J. Kielhorn, U. Wahnschaffe and I. Mangelsdorf.

A WHO Task Group on Environmental Health Criteria for Selected Nitro- and Nitro-oxy-Polycyclic Aromatic Hydrocarbons met at the Fraunhofer Institute of Toxicology and Aerosol Research, in Hanover, Germany, on 26–30 November 2001. The group reviewed the draft and the peer review comments, revised the draft and made an evaluation of the risks for human health and environment from exposure to selected nitro- and nitro-oxy-polycyclic aromatic hydrocarbons.

Dr P. Jenkins and Mr T. Ehara of the IPCS central unit were responsible for the scientific aspects of the monograph, and Ms. Marla Sheffer was responsible for the technical editing.

The efforts of all, especially the Fraunhofer Institute of Toxicology and Aerosol Research, which helped in the preparation and finalization of the monograph, are gratefully acknowledged.

ACRONYMS AND ABBREVIATIONS

1. SUMMARY

1.1 Identity, physical and chemical properties, and analytical methods

Nitro-polycyclic aromatic hydrocarbons (nitroPAHs) are derivatives of polycyclic aromatic hydrocarbons (PAHs), which contain two or more fused aromatic rings made of carbon and hydrogen atoms. NitroPAHs occur in the environment as a mixture together with parent PAHs and hundreds of other organic compounds. NitroPAHs are usually present in much smaller quantities than PAHs.

NitroPAHs in the environment occur either in the vapour phase or adsorbed to particulate matter. NitroPAHs are insoluble or sparingly soluble in water but mostly soluble in organic solvents.

The sampling of nitroPAHs is similar to that of PAHs. Ambient air is sampled by collecting particulate matter on special filters by means of high-volume samplers. Vapour-phase nitroPAHs are commonly collected on solid sorbents such as polyurethane foam.

Solvent extraction is followed by cleanup using liquid chromatography with silica gel or alumina, high-performance liquid chromatography (HPLC) or solid-phase extraction. The nitroPAH fraction must be separated from the PAH fraction and oxygenated PAH fraction by HPLC on silica. Methods used for the separation and detection of nitroPAHs include gas chromatography with a variety of detectors, HPLC with fluorescence, chemiluminescence or electrochemical detector, and mass spectrometric techniques. Analysis is dependent on the standards available.

Another approach to analysis of complex mixtures is bioassay-directed chemical analysis, where mutagenically active fractions are bioassayed and characterized until the major class or specific compounds potentially responsible for the mutagenicity are identified. The use of bacterial tester strains selectively sensitive to nitroarenes has led to the identification of nitroPAHs as potent mutagens in complex mixtures from diverse sources. Synthetic standards are required for this type of analysis.

The nitroketone 3-nitrobenzanthrone and nitrolactones, such as 2- and 4-nitrodibenzopyranone, are nitro-oxy compounds, which have been detected together with nitroPAHs and are analysed by similar methods.

1.2 Sources of human and environmental exposure

NitroPAHs originate primarily as direct or indirect products of incomplete combustion. Only a few nitroPAHs are produced industrially; commercially produced nitronaphthalenes and 5-nitroacenaphthene, for example, are used primarily as chemical intermediates.

NitroPAHs originate from PAHs (generally adsorbed on particu-late matter and themselves products of incomplete combustion) by at least two distinct processes: (1) through nitration during combustion processes (e.g., in vehicle exhaust, particularly diesel, but also gasoline and aircraft emissions; industrial emissions; domestic residential heating/cooking; wood burning) and (2) through atmospheric formation from PAHs by either gas-phase reactions — daytime hydroxyl radical addition to the PAH followed by reaction with nitrogen dioxide and loss of a water molecule and nighttime nitrate radical addition to the PAH followed by reaction with nitrogen dioxide and loss of nitric acid — or heterogeneous gas–particle interaction of parent PAHs adsorbed onto particles with nitrating agents.

The distribution of nitroPAH isomers in samples of ambient air has been found to be significantly different from that in direct emissions from combustion. 2-Nitrofluoranthene and 2-nitropyrene are ubiquitous components of particulate matter, although they are not directly emitted from most combustion sources. The nitroPAH profile, or the relative quantities of certain "marker" PAHs, is a pointer to the source of formation of the nitroPAH. The most abundant nitro isomers of pyrene, fluorene and fluoranthene observed in diesel exhaust are 1-nitropyrene, 2-nitrofluorene and 3-nitrofluoranthene, whereas the isomers formed from the hydroxyl radical reactions of these PAHs are 2-nitropyrene, 3-nitrofluorene and 2-nitrofluoranthene.

The majority of ambient nitroPAHs are now thought to be formed in the atmosphere from the gas-phase reactions of PAHs with four rings or less.

Many mono- and some di- and trinitroPAH isomers have been identified and quantified in various samples of diesel exhaust, 1-nitropyrene usually being the most abundant. 1-Nitropyrene is the "marker" nitroPAH for diesel exhaust, and its presence in ambient air samples is a sign of pollution by diesel vehicle traffic. Diesel fuel, engine types and catalytic traps are continually being modified, so the various studies of nitroPAHs in diesel exhaust cannot be directly compared. In general, the mass emission of particles, emissions of particle-bound PAHs and nitroPAHs, and mutagenic activity levels generally decreased with the use of either particulate traps or catalytic converters.

The concentration of 1-nitropyrene was much less in gasoline exhaust particles than in diesel exhaust particles, but the concentrations of 1,3-, 1,6- and 1,8-dinitropyrenes were found to be almost the same in gasoline and diesel exhaust particles.

There is evidence of the presence of nitroPAHs in jet aeroplane exhaust.

NitroPAHs have been detected in the emissions of kerosene heaters, fuel gas and liquefied petroleum gas (LPG) burners, which are used in many countries for heating and cooking at home.

3-Nitrobenzanthrone has been detected in diesel exhaust particulate and in urban air samples. 2-Nitrodibenzopyranone and 4-nitrodibenzopyranone as well as nitropyrene lactones have been observed in ambient particulate matter.

1.3 Environmental transport, distribution and transformation

1.3.1 Environmental transport and distribution

NitroPAHs can be transported in the vapour phase or adsorbed onto particulate matter. Those with liquid-phase vapour pressures greater than 10^–4 Pa at ambient air temperature (i.e., two- to four-ring PAHs and two-ring nitroPAHs) will exist at least partially in the gas phase.

Owing to their low aqueous solubility or insolubility, nitroPAHs are not expected to be transported in water. Data available give high values for sorption coefficients (log K_oc), suggesting that nitroPAHs, similar to PAHs, adsorb onto soil and sediments. Leaching into groundwater is thought to be negligible. Some nitroPAHs may be slowly biodegradable under certain conditions.

The values for the n-octanol/water partition coefficient (log K_ow) range from 2.5 for 1-nitronaphthalene to 6.3 for 3-nitroperylene, suggesting a potential for bioaccumulation. There were no data available on biomagnification.

1.3.2 Biotransformation

Many anaerobic and aerobic bacteria reduce nitroPAHs to mutagenic aminoPAHs. Nitroreduction by intestinal microflora plays a major role in the metabolism of nitroPAHs in mammals. Although a wide variety of bacteria, fungi and algae have been shown to degrade the parent PAHs containing two to five rings, nitro-substituted PAHs are only slowly degraded by indigenous microorganisms and may persist in soils and sediments. The recalcitrance of high molecular weight nitroPAHs is due in part to the strong adsorption to soil organic matter, low solubility, large molecular size and the polar character of the nitro group.

Time course studies in microcosms showed that 1-nitropyrene was degraded slowly under aerobic and anaerobic conditions in estuarine sediments.

Sphingomonas paucimobilis strain EPA 505 (a soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy) biodegraded 1-nitropyrene to 48.6% after 6 h.

The filamentous fungus Cunninghamella elegans has been shown to oxidatively metabolize, via a cytochrome P450 monooxygenase, a number of nitroPAHs (1-nitropyrene, 2-nitrofluorene, 2- and 3-nitrofluoranthene, 6-nitrochrysene, 1-nitrobenzo[e]pyrene and 6-nitrobenzo[a]pyrene) to products that are less mutagenic than the nitroPAHs themselves.

A plant cell culture derived from alligator weed (Alternanthera philoxeroides) detoxified 1-nitropyrene and 1,3-, 1,6- and 1,8-dinitropyrene, all direct-acting mutagens, when incubated with them, as shown by mutagenicity response in the Salmonella typhimurium TA98 assay.

1.3.3 Abiotic degradation

The photolysis of nitroPAHs has been studied under varied conditions of irradiation. The rate of photolysis depends not only on the conditions of irradiation but also on whether the nitroPAH is in the gaseous phase (e.g., 1-and 2-nitronaphthalene), in solution (type of solvent) or bound to solids/particles. In the latter case, the type and age of the particle seem to influence the photochemistry of the respective nitroPAH. The rate of photodecomposition, identification of photolytic products and resulting loss or gain of metabolic activity as determined by the S. typhimurium assay have been the main end-points studied.

Calculated atmospheric lifetimes of nitroPAHs due to photolysis and gas-phase reactions with hydroxyl and nitrate radicals and with ozone under atmospheric conditions show that the dominant loss process for nitroPAHs (e.g., 1- and 2-nitronaphthalene) is photolysis. Particle oxidation of nitroPAHs by ozone may be the main loss process at night.

1.4 Environmental levels and human exposure

NitroPAHs that have been detected in ambient air include 1- and 2-nitronaphthalene and methylnitronaphthalenes (predominantly in the vapour phase), 2-nitrofluorene, 9-nitroanthracene, 9-nitrophenanthrene, 2-, 3- and 8-nitrofluoranthene, 1- and 2-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene and 6-nitrochrysene.

At remote and forest sites, nitroPAHs were either not detected or detected in the low picogram per cubic metre range (e.g., 17 pg/m³ for 2-nitrofluoranthene; 4 pg/m³ for 1-nitropyrene). The concentration of nitroPAHs in the atmosphere of urban regions depends on the season, the type of heating used and the number and regulation of traffic vehicles. Reported levels in air do not usually exceed 1 ng/m³, although maxima of up to 13 ng/m³ have been reported.

Various studies have been performed monitoring certain isomeric nitroPAHs. Investigators have concentrated on the nitroPAHs that seem to be of quantitative/environmental (e.g., nitroPAHs of relative molecular mass 247: 1-nitropyrene, 2-nitropyrene, 2-nitrofluoranthene) or carcinogenic (e.g., 1-nitropyrene, 2-nitrofluorene, dinitropyrenes) importance.

Studies of daytime/nighttime concentrations of specific isomeric nitroPAHs in certain regions (in particular California, USA) and parallel environmental chamber studies have led to an understanding of the atmospheric formation of certain nitroPAHs (2-nitrofluoranthene and 2-nitropyrene). Concurrent studies of certain nitroPAHs (1-nitropyrene, dinitropyrenes) and traffic volume have confirmed that traffic emission is a source of nitroPAHs.

Most seasonal studies show higher winter/spring concentrations of marker nitroPAHs, which parallels the use of domestic heating, although this is not always the case.

1.4.1 Indoor air

As nitroPAHs have been detected in the emissions of kerosene heaters, fuel gas and LPG burners used for heating and cooking at home, as well as in the fumes of cooking oils, there is therefore a potential indoor exposure to nitroPAHs in poorly ventilated conditions.

Concentrations of polyaromatic compounds, including nitroPAHs, were measured in a study of indoor and outdoor air levels associated with 33 homes located in two US cities: Columbus, Ohio, and Azusa, California. The overall levels were much higher in homes occupied by smokers, but the use of natural gas heating and cooking appliances also appeared to increase the nitroPAH levels slightly.

1-Nitropyrene (4.2–25 600 ng/litre) was detected in 36 of 55 samples of wastewater from oil–water separating tanks of gasoline stations and in used crankcase oil.

1- and 2-nitronaphthalene and 1,3- and 1,5-dinitronaphthalene were detected in river water in Japan at concentrations of 1.3, 11.7, 1.7 and 3.2 ng/litre, respectively. In another water sample, 1-nitropyrene was identified.

There are only limited data on the presence of nitroPAHs in samples of soil, sewage sludge, sediment and incinerator ash (e.g., for 1-nitropyrene, 0.03–0.8 µg/kg dry weight in soil, 0.68 µg/kg in sewage sludge, 25.2 µg/kg in sediment and <0.01–0.89 mg/kg in incinerator ash).

1.4.2 Food and beverages

With the exception of spices, smoked and grilled foods and peanuts, the concentrations of nitroPAHs in foods are below 5 µg/kg.

In a study in the United Kingdom, foodstuffs were monitored for the presence of 9-nitroanthracene and 1-nitropyrene. Twenty-five out of 28 foods contained no detectable levels of these nitroPAHs. 9-Nitroanthracene was tentatively identified in peated malt, at 0.9 µg/kg, and 1-nitropyrene in two samples of tea leaves, at 1.7 and 0.17 µg/kg.

Another survey of nitroPAH levels in various foods in Austria showed mostly detectable levels of 2-nitrofluorene, 1-nitropyrene and 2-nitronaphthalene. The highest concentrations were found in spices, smoked foods and teas, in particular Mate tea, which is roasted. NitroPAHs were also detected in vegetables and fruits, probably due to atmospheric pollution.

1-Nitropyrene was detected in grilled corn, mackerel and (in considerable amounts) pork and yakitori (grilled chicken) grilled with sauce (up to 43 ng/g).

1.4.3 Other products

In 1980, studies showed that extracts of selected xerographic toners and paper photocopies were mutagenic. The fraction of the carbon black B responsible for 80% of the mutagenicity contained 1-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene, 1,3,6-trinitropyrene and 1,3,6,8-tetranitropyrene. As a result of this finding, the manufacturers modified the production of carbon black B, substantially reducing the levels of nitropyrenes.

1.4.4 Occupational exposure

Occupational exposure to nitroPAHs has been demonstrated in workplaces associated with the use of diesel engines. For example, concentrations of 1-nitropyrene in air were measured in various workplaces associated with the use of diesel engines. The highest levels (42 ng/m³) reported were determined in the breathing zones of the underground workers (drivers of diesel-powered excavators) at an oil shale mine in Estonia.

1.5 Kinetics and metabolism in laboratory animals and humans

1-Nitropyrene and 2-nitrofluorene administered by various routes are rapidly absorbed, and the resulting metabolites are conjugated and excreted. Radiolabelled 1-nitropyrene was found to be widely distributed in the body of rats and mice following administration by all routes. Other nitroPAHs have not been as well studied.

The metabolism of nitroPAHs is complex. It seems that there are at least five metabolic activation pathways through which mutations can be induced by nitroPAHs in bacterial and mammalian systems and/or through which DNA binding occurs. These are 1) nitroreduction; 2) nitroreduction followed by esterification (in particular acetylation); 3) ring oxidation; 4) ring oxidation and nitroreduction; and 5) ring oxidation and nitroreduction followed by esterification. In bacteria, nitroreduction seems to be the major metabolic pathway, whereas the fungus Cunninghamella elegans is an example of a species in which nitroPAHs are metabolized by ring oxidation.

Nitroreduction of nitroPAHs in vivo probably occurs mainly by bacteria in the intestinal tract. In oxidative metabolism, the first step is transformation to phase I primary metabolites such as epoxides, phenols and dihydrodiols, and then to secondary metabolites, such as diol epoxides, tetrahydrotetrols and phenol epoxides. In mammalian systems, the phase I metabolites are then conjugated with glutathione, sulfate or glucuronic acid to form phase II metabolites, which are more polar and water-soluble than the parent hydrocarbons. On reaching the intestine, the conjugated metabolites can be deconjugated by the intestinal microflora and absorbed, entering enterohepatic circulation. Nitroreduction and N-acetylation can occur, resulting in the excretion in urine and faeces of metabolites such as acetylaminopyrenols after 1-nitropyrene administration.

Different cytochrome P450 enzymes may be involved in the metabolism of a specific nitroPAH, and these may differ in the related isomers, resulting in possibly different kinetics and pathways. Cytochrome P450 enzymes responsible for the metabolism of nitroPAHs may vary between species and in different target organs and in different cell types within target organs.

All nitroPAHs do not follow the same activation pathways. Some are mutagenic when reduced to an arylhydroxylamine (e.g., 1-nitropyrene is metabolized mainly by hydroxylation of the aromatic moiety, followed by nitroreduction and N-acetylation); others (e.g., 1,8- and 1,6-dinitropyrene) are reduced to the arylhydroxylamine and then require further O-esterification (in particular O-acetylation) to an acyloxy ester for mutagenicity. Some may be mutagenic only after activation by oxidation to reactive epoxides or dihydrodiol epoxides (as possibly in 6-nitrobenzo[a]pyrene, similar to benzo[a]pyrene, or BaP). The main DNA adducts detected with nitroPAHs in vivo and in vitro are C8-substituted deoxyguanosine adducts; however, N²-substituted deoxyguanosine and C8-substituted deoxyadenosine derivatives have also been detected and may predominate in nitroPAHs with greater hydrocarbon character (e.g., 3-nitrobenzo[a]pyrene and 6-nitrochrysene). DNA adducts of dinitropyrenes are formed only via nitroreduction, presumably owing to the high electron deficiency in the aromatic rings caused by the presence of two nitro groups. The DNA adducts resulting from the nitroreduction of nitroPAHs are better characterized than those arising from oxidative metabolism, although the latter may be of more importance in mammalian metabolism.

1.6 Effects on laboratory mammals and in vitro test systems

Only six nitroPAHs have been tested for acute toxicity. In rats, an LD₅₀ of 86 mg/kg of body weight (kg bw) after intraperitoneal (i.p.) application was reported for 1-nitronaphthalene; in mice, an oral LD₅₀ of 1300 mg/kg bw was reported for 2-nitronaphthalene. In further studies on both substances, systemic effects on the target organs lung and liver were observed after single high doses; however, 2-nitronaphthalene seemed to be less toxic than 1-nitronaphthalene. 5-Nitroacenaphthene at an i.p. dose of 1700 mg/kg bw was lethal to all treated rats. For 2-nitrofluorene, an oral LD₅₀ of 1600 mg/kg bw in mice was reported, whereas gavaging with up to 5000 mg 1-nitropyrene/kg bw resulted in no observable toxic effects. Local inflammation and ulceration were seen in rats after subcutaneous (s.c.) injection of 8 mg 3-nitrofluoranthene/kg bw.

Data on systemic or local non-neoplastic effects caused by short-term or long-term treatment with nitroPAHs are limited, as the end-point of most studies has been carcinogenicity. In most cases, non-neoplastic toxic effects were observed at doses at which carcinogenic responses are also manifested. Systemic non-neoplastic toxic effects, such as reduced body weight or increased mortality, appeared presumably independently of carcinogenic effects in feeding studies with 5-nitroacenaphthene at a dose level of 500 mg/kg bw per day (rat) or 40 mg/kg bw per day (mice) and with 2-nitrofluorene at a dose of 25 mg/kg bw per day (rat). Medium-term exposure via inhalation to 1-nitropyrene resulted in metaplasia of the upper respiratory tract at concentrations of >0.5 mg/m³.

No data are available on skin and eye irritation, sensitization or reproductive toxicity.

Data on genotoxicity in vitro are available on 95 nitroPAHs; for 74 nitroPAHs, however, only one or two end-points, mainly in bacterial test systems, were investigated. A sufficient database, including eukaryotic test systems, has been found only with 21 nitroPAHs. Most of these substances (67 out of 95) showed positive results, but the results were derived from a small database. Clearly positive results were obtained for 19 nitroPAHs, and questionable results for 8 nitroPAHs. With none of the nitroPAHs were clearly negative results obtained.

For 86 nitroPAHs, data on the S. typhimurium microsome test are available. In contrast to the parent PAHs, most nitroPAHs were clearly more effective in the Salmonella microsome test without metabolic activation. There are five nitroPAHs that showed exceptionally high mutagenic potency (>100 000 revertants/nmol) in this test system: 3,7- and 3,9-dinitrofluoranthene, 1,6- and 1,8-dinitropyrene, and 3,6-dinitrobenzo[a]pyrene.

Bacterial nitroreductase and acetyltransferase are involved in the metabolic activation of nitroPAHs, but not all nitroPAHs follow the same metabolic activation pathways. Furthermore, there is no uniform mutagenic effect of the different nitroPAHs, as they produce both frameshift and base pair substitutions in the S. typhimurium microsome test. There is evidence that nitroPAHs with nitro groups perpendicular to the aromatic ring are not as mutagenic as isomers having parallel nitro orientation.

Data on the in vivo genotoxicity of nitroPAHs are available for 15 nitroPAHs. All nitroPAHs that gave positive results in vivo were also positive in vitro. Four nitroPAHs that were positive in in vitro genotoxicity tests revealed inconsistent or inconclusive genotoxicity (2-nitronaphthalene, 5-nitroacenaphthene and 3-nitrofluoranthene) or negative genotoxicity (2,7-dinitrofluorene; limited validity) results in vivo.

3-Nitrobenzanthrone, like 1,6- and 1,8-dinitropyrene, is highly mutagenic in bacteria through nitroreduction and O-esterification. 3-Nitrobenzanthrone is also an effective gene mutagen and causes micronuclei formation in human cells in vitro and in mice in vivo.

2-Nitrodibenzopyranone was reported to be highly mutagenic in the S. typhimurium microsome test in strain TA98 (–S9), being more mutagenic than 2-nitrofluorene and 1-nitropyrene. 1- and 3-nitropyrene lactones have been found to be highly mutagenic in the S. typhimurium microsome test.

Studies on the in vitro genotoxicity of 2-nitrodibenzopyranone in forward mutation assays using two human B-lymphoblastoid cell lines are conflicting. Nitropyrene lactones were found to induce mutations at the tk and hprt loci in both cell lines. Further, they induced kinetochore-positive and -negative micronuclei in the CREST modified micronucleus assay, which detects chromosomal loss and breakage events.

Data on carcinogenic effects are available for 28 nitroPAHs. Although inhalation is the main exposure route in humans, no long-term inhalation study on any nitroPAH is available. Most studies examined the carcinogenic effects of nitroPAHs by oral administration, topical application, pulmonary implantation or intratracheal administration.

Owing to the limitations in experimental design, none of the negative studies confirmed the absence of carcinogenic effects in animals. However, results showed carcinogenic effects in experimental animals for 5-nitroacenaphthene, 2-nitrofluorene, 3-nitrofluoranthene, 3,7- and 3,9-dinitrofluoranthene, 1- and 4-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene and 6-nitrochrysene. Some carcinogenic effects in experimental animals were observed for 2-nitropyrene, 7-nitrobenz[a]anthracene, 2- and 6-nitrobenzo[a]pyrene, 3,6-dinitrobenzo[a]pyrene, 7-nitrodibenz[a,h]anthracene and 3-nitroperylene. For the remaining 10 nitroPAHs tested, not enough data were available with which to evaluate their carcinogenicity in experimental animals.

Besides local effects at the site of injection, nitroPAHs induced mainly systemic tumours in mammary tissue, lung, liver and the haematopoietic system. 6-Nitrochrysene appears to be the most carcinogenic of the nitroPAHs considered here. With systemic effects after s.c. or i.p. injection, 1-nitropyrene was more carcinogenic than the dinitropyrenes. The carcinogenicity of 1-nitropyrene and dinitropyrenes varies, depending on the route of administration.

Nitrated benzo[a]pyrenes are generally less potent carcinogens than the parent compound BaP. However, the mono- or dinitrated pyrenes are more carcinogenic than pyrene. Similar results were presented for 3-nitroperylene compared with perylene and for 6-nitrochrysene compared with chrysene; with local effects after dermal exposure, however, 6-nitrochrysene was less active than chrysene.

Data were available on carcinogenic effects of some metabolites of 2-nitrofluorene, 1-nitropyrene and 6-nitrochrysene. Comparing 2-nitrofluorene with its metabolites in rats, the highest carcinogenic potency was shown by 2-acetylaminofluorene. 1-Nitropyrene was significantly more carcinogenic after oral application in rats than either 1-nitrosopyrene or 1-aminopyrene. In contrast, 1-nitrosopyrene induced a higher incidence of liver tumours in mice after i.p. application than 1-nitropyrene; no effects were observed with ring hydroxylated metabolites. 6-Nitrosochrysene and 6-aminochrysene were inactive, in contrast to the ring hydroxylated metabolites, which showed carcinogenic activity in the liver similar to that of the parent compound 6-nitrochrysene; this indicates that the metabolic activation of 6-nitrochrysene occurs by ring oxidation and/or a combination of ring oxidation and nitroreduction.

1.7 Effects on humans

There are no reports on the effects of individual nitroPAHs on humans. As would be expected, since nitroPAHs occur in complex mixtures in the atmosphere and exhaust, the exact contribution of nitroPAHs to the adverse health consequences of exposure to polluted atmospheres and to exhaust cannot be elucidated.

At present, investigations on the effects of nitroPAHs on human health are being carried out using biomarkers of exposure. Several reports have described the development of methods for and provided data on the evaluation of 1-nitropyrene as a biomarker for occupational exposure to diesel exhaust. Urinary metabolites of PAHs and nitroPAHs were determined in the urine of diesel mechanics using the enzyme-linked immunosorbent assay (ELISA). In another study, metabolites of 1-nitropyrene (namely, N-acetyl-1-aminopyren-6-ol and N-acetyl-1-aminopyren-8-ol) were measured in the urine of workers in a shipping department. Several studies have focused on measuring the haemoglobin and plasma adducts of metabolites of 1-nitropyrene and other nitroPAHs and may provide appropriate biomarkers in future molecular epidemiological investigations.

1.8 Effects on other organisms in the laboratory and field

Data on the acute toxicity of nitroPAHs to aquatic organisms are available only for 1-nitronaphthalene. An LC₅₀ (96 h) of 9.0 mg/litre was reported for the fathead minnow (Pimephales promelas). Furthermore, this nitroPAH inhibited the growth of the ciliate Tetrahymena pyriformis, with an EC₅₀ (60 h) of 17.3 mg/litre.

Some studies have been concerned with the effect of nitroPAHs on the metabolism of some aquatic species — for example, the subcellular and tissue distribution of two- and one-electron NAD(P)H-dependent nitroreductase activity in marine invertebrates from three phyla: mussel (Mytilus edulis), crab (Carcinus maenas) and starfish (Asteria rubens). NADPH-dependent two-electron nitroreductase activity, occurring only under anaerobic conditions, was detected in the microsomal and cytosolic fractions of the major digestive tissues of mussel (digestive gland) and crab, but not in the gills of either species. 1-Aminopyrene was the only metabolite identified. No activity was detectable in the pyloric caeca or stomach region of the starfish. NAD(P)H-dependent one-electron nitroreduction was present in all subcellular fractions of the major digestive tissues of the three species.

In the presence of calf thymus DNA, adducts derived from 1-nitropyrene were detected in vitro using hepatic S9 fractions prepared from fish. The ability of 1-nitropyrene to form DNA adducts was also established in vivo using brown trout (Salmo trutta) and turbot (Scophthalmus maximus). These DNA adducts were comparable to those obtained in Wistar rats treated with 1-nitropyrene.

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS

2.1 Identity

Nitro-polycyclic aromatic hydrocarbons (nitroPAHs) are derivatives of polycyclic aromatic hydrocarbons (PAHs), which contain two or more fused aromatic rings made of carbon and hydrogen atoms, formed as a result of incomplete combustion (see IPCS, 1998). NitroPAHs occur in the environment as a mixture together with parent PAHs and hundreds of other organic compounds (see chapter 3). NitroPAHs are usually present in smaller quantities (by 2 orders of magnitude) than PAHs.

Interest was focused on nitroPAHs in the early 1980s as correlations were found between the presence of nitroPAHs in diesel exhaust and environmental extracts and mutagenic activity. A large number of groups of nitro, oxy and mixed nitro-oxy compounds eluted together in the mutagenic fractions. Analytical methods were developed to separate and identify these compounds and to specify their isometric composition, as the biological action of these compounds also depends on their stereospecificity (see chapters 6 and 7). As it would be impossible to evaluate all these compounds in one document, a decision was made to include mono- and dinitroPAHs (2–5 rings) but, in general, not methylated or hydroxylated nitroPAHs. Some nitro-oxyPAHs are also included: nitroketones (3-nitrobenzanthrone) and selected nitrolactones (e.g., the nitrophenanthrene lactones: 2- and 4-nitrodibenzopyranone [2- and 4-nitro-6H-dibenzo[b,d]pyran-6-one] and nitropyrene lactones), which have recently been shown to be present in the extracts of the polar fractions of diesel exhaust and airborne particulates.

The nomenclature, molecular formula, relative molecular mass and Chemical Abstracts Service (CAS) number of selected nitroPAHs and nitro-oxyPAHs are given in Table 1. The structural formulas of some selected nitroPAHs and nitro-oxyPAHs are shown in Figure 1.

Fig. 1. Structural formulas of some nitroPAHs and some nitro-oxyPAHs.

Table 1. Nomenclature, molecular formulas, relative molecular mass and CAS numbers of selected nitroPAHs and their oxygen-containing derivatives

2.2 Physical and chemical properties

At ambient temperatures, nitroPAHs are yellowish to orange solids that tend to sublime (White, 1985). NitroPAHs in the environment occur in the vapour phase or are absorbed and/or adsorbed to particulate matter, depending upon their vapour pressure and the ambient conditions. Two- to four-ring nitroPAHs are present partially in the vapour phase under certain conditions; for example, 1-nitronaphthalene (in certain climates) occurs mainly in the vapour phase (Arey et al., 1987), whereas 2-nitrofluorene occurs equally in both vapour and particulate phases, and 1-nitropyrene occurs in the particulate phase (Schuetzle & Frazier, 1986).

NitroPAHs are insoluble or sparingly soluble in water but are mostly soluble in organic solvents such as acetone, benzene, dimethyl sulfoxide (DMSO) and methylene chloride.

Table 2 gives details of some environmentally relevant physical and chemical properties of nitroPAHs together with those of the parent PAHs (see IPCS, 1998). Nitrodibenzopyranones have a lower vapour pressure than nitroPAHs and therefore are to be found predominantly in the particulate phase.

Table 2. Physical and chemical properties of nitroPAHs and their parent PAHs^a

2.3 Conversion factors

Atmospheric concentrations of nitroPAHs are usually expressed as micrograms, nanograms or picograms per cubic metre. At 25 °C and 101.3 kPa, the conversion factors for a compound of given molecular mass are obtained as follows:

ppb = µg/m³ × 24.45/relative molecular mass

µg/m³ = ppb × relative molecular mass/24.45

where ppb is parts per billion, and one billion is 10⁹.

For example, for 1-nitropyrene, 1 ppb = 10.1 µg/m³, and 1 µg/m³ = 0.099 ppb.

2.4 Analytical methods

A direct analysis of nitroPAHs from environmental sources is not possible, as all environmental matrices are very complex. The samples often contain thousands of combustion products, including parent PAHs and other closely related derivatives (in particular oxygenated PAHs such as aldehydes, ketones and carboxylic acids), which tend to co-elute with nitroPAHs under a variety of liquid and gas chromatographic conditions and are present at concentrations 1 or 2 orders of magnitude higher than those of the nitro-substituted compounds (Schuetzle, 1983; Vincenti et al., 1996; see Table 3). An extensive sample cleanup and prefractionation of the sample are tedious but necessary prerequisites for trace analysis of nitroPAHs.

Isomer-specific identification is necessary, as the biological activity depends on the position of the nitro substituent. The source of the nitroPAH (e.g., from combustion or from atmospheric reactions; see chapter 3) may determine the isomeric specificity of the nitroPAH.

Although 1- and 2-nitronaphthalene are expected to be found predominantly in the gas phase, other semivolatile nitroPAHs will be distributed between the particulate and gas phases, depending upon the ambient temperature. Thus, many ambient measurements will underestimate the total nitroPAHs present unless both gas- and particulate-associated species have been measured.

Analysis is hindered by a lack of adequate instrumental sensitivity or selectivity and limited availability of native and isotope-labelled standards (Chiu & Miles, 1996).

Bioassay-directed fractionation (usually using the Ames test or modifications of this with specific Salmonella typhimurium strains) and subsequent chemical characterization have been used for the identification of nitroPAHs in a number of complex mixtures (see section 2.4.5).

As most nitroPAHs are mutagenic, special precautions should be taken, even at ultratrace concentrations.

Table 3. Relative concentrations of various PAH compounds and PAH derivatives in the non-polar and moderately polar fractions of a diesel particle extract, showing only the nitroPAH fraction in detail^a

^a Adapted from Schuetzle (1983).

It should be noted that many nitroPAHs, in particular 9-nitro-anthracene, are unstable in the presence of light; therefore, reduced light conditions should be used (see chapter 4).

Details of methods used for the analysis of nitroPAHs in different matrices are given in Table 4. Reviews on the analysis of nitroPAHs are given by White (1985), Vincenti et al. (1996), CONCAWE (1998) and Hayakawa (2000).

Table 4. Analysis of nitroPAHs^a

Sample type	Extraction		Cleanup	Analysis	Detector	Detection limitb	References
Air
Ambient air	Soxhlet (DCM)		NP-LC	GC	MS in SIM		Arey et al. (1987)
Reaction chamber study	Soxhlet (DCM)		NP-LC	GC RP-LC	MS in SIM UV		Atkinson et al. (1987a)
Ambient air	Ultrasonication (DCM)		Extensive, including NP-LC	GC	FPD; NPD; MS EI		Fernández & Bayona (1992)
Air particulate	Ultrasonication (DCM)		Silica gel	HPLC	Electrochemical		Galceran & Moyano (1993)
Air particulate	Ultrasonication (benzene/ethanol)		NP-LC	HPLC (with on-line reduction)	CL	0.3–5 fmol	Murahashi & Hayakawa (1997)
Air particulate	Ultrasonication (DCM)		Silica/cyclohexane	HPLC switching technique GC ELISA	FL HRMS	20 fmol (1-nitropyrene)	Zühlke et al. (1998)
Diesel exhaust
Diesel	Soxhlet		NP-LC	GC	NPD (FL)		Schuetzle & Perez (1983)
Diesel particulate	Soxhlet (DCM)		NP-LC	GC GC	NPD MS	0.5 ppm 5 ppm	Paputa-Peck et al. (1983)
Diesel or air particulates	Soxhlet (DCM)		Extensive; also NP-LC	GC GC	FID TEA		Niles & Tan (1989)
Diesel or air particulates	Ultrasonication (acetone)		SPE and on-line reduction	GC	MS EI	1 ng/g	Scheepers et al. (1994a)
Air and diesel particulates	Soxhlet (DCM)		NP-LC	LC On-line reduction	Electrochemical FL	60 pg 10 pg	MacCrehan et al. (1988)
Diesel or air particulates and gaseous	DCM		SPE	HRGC	HRMS	low ng/g (diesel particulate); pg/m3 range	Chiu & Miles (1996)
Vehicle or air particulates	Ultrasonication (benzene/ethanol)		Precolumn reduction	HPLC (column switching)	CL with on-line reduction		Hayakawa et al. (1999a)
Water
River water			DCM; florisil column	GC	MS		Takahashi et al. (1995)
River water	Concentration by blue chitin column; methanol			GC	MS		Nagai et al. (1999)
Wastewater	Fractionation into diethyl ether-soluble neutral, acidic and basic fractions			HPLC	UV and FL		Manabe et al. (1984)
Crankcase oil	Extraction with methanol, concentration and dissolution in water; then as above			HPLC	UV and FL		Manabe et al. (1984)
Soil, sewage sludge, sediment
Urban dust, residual of incinerator, soil	Soxhlet (acetonitrile)		SPE	GC	NPD		Librando et al. (1993)
Sewage sludge	Dimethyl formamide		LSE or SPE	GC	FID; GC-MS		Bodzek & Janoszka (1995)
River sediment	Ether; sodium hydroxide or benzene methanol; chloroform		Silica gel column; reduced/acylated	GC	ECD		Sato et al. (1985)
Soil	Methanol		Silica gel column; hexane/benzene/ chloroform; HPLC ODS columns	Analytical ODS column for HPLC	Reduction to amino derivatives; fluorescence	Dinitropyrenes 0.7–4 pg	Watanabe et al. (1999)
Soil	Methanol/acetone		SPE; HPLC	GC	MS EI or NICI	30 ng/kg dry weight	Niederer (1998)
Biological samples
Rat tissue		Homogenate treated with acetonitrile; evaporated under nitrogen; residue dissolved in methanol; extraction with Blue Rayon		HPLC; on-line reduction	Fluorescence		van Bekkum et al. (1999)
Human lung specimens		Ultrasonication (DCM)		Bioassay-directed chemical analysis, HPLC, GC	MS		Tokiwa et al. (1993a, 1998a)
Food and beverages
Various food samples		Homogenization; acetonitrile; hexane	Silica gel column; DCM	GC	TEA	12 pg	Dennis et al. (1984)
Various food samples				GC GC	MS NPD	5 pg	Schlemitz & Pfannhauser (1996a)
Grilled and smoked food samples				HPLC; on-line reduction	Fluorescence		Schlemitz & Pfannhauser (1996b)
Fish, meat products and cheese				HPLC; on-line reduction	Fluorescence	50 ng/kg	Dafflon et al. (2000)

2.4.1 Sampling

Methods of collecting air particulates include a) Mega sampler with 50% cut-off point of 20 µm and typically 6-h sampling periods (used in Claremont, California, USA); b) an electrostatic precipitator with an impact stage designed for a 15-µm cut-off (used in Aurskog, Norway); and c) specially designed filter baghouses collected over a period of a year (National Institute of Standards and Technology [NIST] Standard Reference Materials [SRMs]) (Ramdahl et al., 1986). A 10-µm size-selective inlet (for particulate matter less than 10 µm in diameter, or PM₁₀) sampler is also reported (Nishioka & Lewtas, 1992) .

For investigations into the formation and photochemistry of nitroPAHs, three different collection media for ambient air sampling have been used: Tenax-GC solid adsorbent, polyurethane foam (PUF) and filters for high-volume sampling (Arey et al., 1991).

Three methods for the sampling of the semivolatile phase of diesel exhaust were compared: cryogenic sampling, adsorbent sampler with XAD-2 and PUF. The PUF technique gave the highest recovery of PAHs and mutagenic activity. The three sampling techniques for the semivolatile phase resulted in extracts with different chemical composition, different mutagenic potency and different mutagenicity profiles (Westerholm et al., 1991).

In general, vapour-phase constituents are collected on solid sorbents such as XAD and PUF, and particles are collected on Teflon-impregnated glass fibre filters (see Table 4).

For sampling of diesel exhaust particulates, a dilution tunnel method is mainly used (Hayakawa, 2000). The exhaust is diluted by the filtered air in the tunnel to simulate the real road conditions, and an aliquot is sampled on the filter. Gaseous substances are trapped in PUF. The sampling and analytical methods are reviewed by Levsen (1988).

Blue cotton bearing covalently linked copper phthalocyanine trisulfonates as a ligand adsorbs polyaromatic compounds and preconcentrates several nitroPAHs in water (Hayatsu, 1992).

2.4.2 Extraction

Extraction of filter and PUF samples can be carried out by dichloromethane (DCM) in a Soxhlet apparatus for 16 h, the Soxhlet body being loosely covered in aluminium foil to exclude light (Chiu & Miles, 1996). DCM was found to be the most efficient solvent for extraction of mutagenic compounds from diesel particles (Montreuil et al., 1992).

Toluene as solvent has also been reported (Spitzer, 1993; Vincenti et al., 1996). Supercritical fluid extraction of nitroPAHs from diesel exhaust particulate matter using carbon dioxide–chlorodifluoromethane (HCFC-22) or carbon dioxide–toluene has been demonstrated (Paschke et al., 1992).

Soil sample extraction has been described using the Soxhlet device using 1:1 (v/v) toluene:methanol (Vincenti et al., 1996) or 5% ethanol in toluene (Spitzer, 1993).

2.4.3 Cleanup

Various procedures for fractionation of particulate extracts have been described:

• open-column liquid chromatography with either silica gel or alumina (Yu et al., 1984; Lindskog et al., 1987; Lewtas, 1988; Kamiura et al., 1991; Galceran & Moyano, 1993; Spitzer, 1993);
• normal-phase high-performance liquid chromatography (HPLC), or NP-LC (Oehme et al., 1982; Nielsen, 1983; Paputa-Peck et al., 1983; Stray et al., 1984; Tomkins et al., 1984; Pitts et al., 1985a; Arey et al., 1986, 1987, 1988a; Ramdahl et al., 1986; Ciccioli et al., 1988; Niles & Tan, 1989; Veigl et al., 1994); or
• solid-phase microextraction (Xu et al., 1982; Jin & Rappaport, 1983; LaCourse & Jensen, 1986).

From open-column liquid chromatography, four main fractions are obtained by eluting with different solvents (in parentheses): aliphatics (hexane), aromatics (hexane/benzene), moderately polar (DCM) and highly polar (methanol) (Lewtas, 1988). The nitroPAHs are to be found in the moderately polar fraction, but together with a number of oxy derivatives (e.g., aldehydes, ketones, quinones), which may interfere with them. Therefore, a more effective fractionation is achieved by NP-LC, but here the nitroPAHs are not collected in a single fraction but are distributed in several fractions (Vincenti et al., 1996). Another approach to separate nitroPAHs from hydroxyPAHs uses a sequential cleanup with silica and alumina (Moyano & Galceran, 1997). Nitrated and hydroxylated PAHs extracted from air particulates could be fractionated from other micropollutants by semipreparative packed-column supercritical fluid chromatography on silica gel (Medvedovici et al., 1998).

Figure 2 shows a schematic diagram of an analytical method used for the cleanup and separation of particulate organic matter (Ciccioli et al., 1996).

2.4.4 Analytical separation and detection

The most frequently used techniques for the detection of nitroPAHs are (Vincenti et al., 1996):

• gas chromatography (GC) combined with a thermionic or nitrogen–phosphorus selective detector (Nielsen, 1983; Paputa-Peck et al., 1983; Liberti et al., 1984; Nielsen et al., 1984; Matsushita & Iida, 1986; Warzecha, 1996), chemiluminescence-based thermal energy analyser (Tomkins et al., 1984; Yu et al., 1984; Robbat et al., 1986; Niles & Tan, 1989) or electron capture detector (ECD) (Oehme et al., 1982; LaCourse & Jensen, 1986; Spitzer, 1993);
• HPLC with fluorescence (Tejada et al., 1986; MacCrehan et al., 1988; Kamiura et al., 1991; Hayakawa et al., 1993; Veigl et al., 1994), chemiluminescence (Sigvardson & Birks, 1984; Hayakawa et al., 1992, 1995a) or electrochemical (Jin & Rappaport, 1983; MacCrehan et al., 1988; Galceran & Moyano, 1993) detector; for determination of 1,3-, 1,6- and 1,8-dinitropyrenes, HPLC using chemiluminescence after precolumn reduction has been developed, giving detection limits 2 orders lower than those with fluorescence (Hayakawa et al., 1992, 1995a; Maeda et al., 1994; Murahashi & Hayakawa, 1997);
• GC/mass spectrometry (GC-MS) in electron impact (EI) (Paputa-Peck et al., 1983; Liberti et al., 1984; Yu et al., 1984; Pitts et al., 1985a; Arey et al., 1986, 1987; Ramdahl et al., 1986; Niles & Tan, 1989; Paschke et al., 1992), positive ion chemical ionization (Schuetzle et al., 1982) or negative ion chemical ionization (NICI) electron capture (EC) (Oehme et al., 1982; Ramdahl & Urdal, 1982; Henderson et al., 1983; Liberti et al., 1984; Nielsen et al., 1984; Stray et al., 1984);
• EC-MS, which also provides adequate sensitivity and selectivity for the analysis of nitroPAHs in most matrices; however, there is still difficulty due to interference from oxyPAHs (Lewtas & Nishioka, 1990); and
• tandem mass spectrometry (MS/MS) (Schuetzle et al., 1982; Henderson et al., 1983). Combining this technique with GC-EC gives four "separation" stages: a chromatographic separation, a selective ionization method and two mass spectrometric analyses (Schilhabel & Levsen, 1989). The method has now been refined, and the high selectivity of the instrumentation techniques means that the preliminary sampling is no longer required on air particle samples (Vincenti et al., 1996).

A thin-layer chromatography method using plates coated with a silica gel layer has been developed for analysing nitroPAHs. DCM and DCM with n-hexane and methanol were used for the mobile phases. The chromatograms developed using a mixture of n-hexane–DCM (1:1 v/v) were observed under ultraviolet (UV) light before and after being sprayed with a reducing agent (sodium borohydride dissolved in methanol and copper chloride solution). Light at lambda = 254 nm induced green fluorescence for 1-nitropyrene and 1,3-dinitropyrene only and a violet colour for the remaining compounds. Carbon disulfide quenched fluorescence (observed at lambda_exc = 365 nm) for 1-nitronaphthalene, 9-nitroanthracene, 1-nitropyrene and 1,3-dinitropyrene only (Tyrpien, 1993; Tyrpien et al., 1997 ). Janoszka et al. (1997) used acetonitrile/water as the mobile phase. The method is suggested as a simple and quick method for identifying the above nitroPAHs in airborne particulate matter after separation in moderately polar fractions by column chromatography on silica gel. Isomeric nitroPAHs cannot be separated.

More recent developments in this field include a) selective detection of several nitroPAHs by using time-of-flight MS (Bentz et al., 1995; Dotter et al., 1996; Bezabeh et al., 1997); b) a method using supercritical fluid extraction and on-line multidimensional chromatographic methods (NP-LC coupled to a high-resolution GC) and ion trap detector MS (Lewis et al., 1995a,b; Feilberg et al., 2001); and c) particle beam liquid chromatography–MS with NICI mode (Bonfanti et al., 1996). High-resolution NICI is reported to be at least 20 times more sensitive than the low-resolution NICI or EI for determination of nitroPAHs in air samples (Chiu & Miles, 1996). A method has been developed involving the derivatization of nitroPAHs to their corresponding fluorinated derivatives, followed by GC-ECD analysis. The sensitivity of the method is an order of magnitude higher than that of direct GC-ECD analysis of nitroPAHs themselves. This method is suitable for routine monitoring of nitroPAHs in air samples (Jinhui & Lee, 2001).

2.4.4.1 Difficulties in analysis

Owing to small differences in GC retention times for 2- and 3-nitrofluoranthene, the 2-nitrofluoranthene present in some samples was incorrectly reported as 3-nitrofluoranthene in earlier reports. After reanalysis, the original reports were corrected (Ramdahl et al., 1986; Nishioka et al., 1988). Use of more selective stationary phases enabled better separation of isomeric pairs (Ciccioli et al., 1988). For the best separation of all of the nitrofluoranthene and nitropyrene isomers, the use of a DB13 (or SP30-type) column is recommended (A. Cecinato, personal communication, 2002).

Sampling gas-phase PAHs from environmental chambers onto Tenax adsorbent under conditions not typical of ambient atmospheres (e.g., 19 mg nitrogen dioxide/m³, 44 mg nitrogen pentoxide/m³) can lead to artificial formation of nitro derivatives via reactions with the Tenax adsorbent (Zielinska et al., 1986a).

2.4.4.2 Complex mixtures

The chemical analysis of trace levels of organic mutagens in ambient air is more complex than comparable analyses of emissions from specific sources. Source-emitted pollutants account for only part of the whole ambient air sample. Aging of air samples introduces unknown, highly variable chemical and meteorological factors. Further, the concentrations of pollutants are much lower in ambient air than at the sources (Greenberg et al., 1993).

2.4.4.3 Analysis of nitro-oxyPAHs

The procedures followed for investigating nitro-oxyPAHs in emissions and ambient air are, in general, the same as those adopted for nitroPAHs.

2.4.5 Use of bioassay (mutagenicity) fractionation and chemical analysis

Bioassay-directed fractionation closely coupled to chemical characterization has been developed as a method of determining nitroPAHs in complex mixtures (see Figure 3) (Schuetzle & Lewtas, 1986; Lewtas, 1988; Lewtas & Nishioka, 1990; Legzdins et al., 1995; Enya et al., 1997). In this approach, the complex mixture is fractionated, and each fraction is bioassayed; the mutagenic activity for each (HPLC) fraction is plotted in a manner analogous to a conventional chromatogram, and the plot is referred to as a mutagram (mutagenicity profile = mutagram).

Fig. 3. Bioassay-directed chemical analysis scheme for the determination of air particulate matter. The numbers under each fraction represent the percent distribution of mass and mutagenicity in Salmonella tester strains (in parentheses); adapted from Schuetzle & Lewtas (1986) and Lewtas et al. (1990a).

Mutagenically active fractions are further fractionated, bioassayed and characterized until the major class of compounds or specific com-pounds potentially responsible for the mutagenicity are identified. Use of bacterial tester strains selectively sensitive to nitroarenes has led to the identification of nitroPAHs as potent mutagens in complex mixtures from diverse sources.

Non-polar fractions of an extract of diesel particulates (SRM 1650) accounted for less than 2–3% of the mutagenicity in the total extract. The distribution of mutagenicity in the moderately polar and polar fractions was dependent on the source sample (Schuetzle & Lewtas, 1986).

Studies using this bioassay-directed fractionation and chemical characterization (see also section 7.5.5) include, for example, studies of diesel exhaust extracts (Schuetzle et al., 1981; Nishioka et al., 1982; Claxton et al., 1992; Legzdins et al., 1994; Enya et al., 1997; Hayakawa et al., 1997), xerographic toners (Rosenkranz et al., 1980), cigarette smoke (Kier et al., 1974), ambient atmospheric particles (Nishioka et al., 1988; Lewtas et al., 1990a; Arey et al., 1992; Casellas et al., 1995; Hayakawa et al., 1995b) and the metabolites of 1-nitropyrene (Lewtas et al., 1990b).

For example, Casellas et al. (1995) made a detailed chemical analysis of mutagenic fractions using bioassay-directed chemical analysis in urban airborne particulate matter in Barcelona, Spain. The first fractionation of the solvent (DCM) extractable organic matter was achieved by semipreparative gel permeation chromatography (GPC). The second fractionation was achieved with NP-LC. The collected fractions were tested for mutagenicity using the S. typhimurium microsome assay with strains TA98, TA98NR– and TA98AT– (for more details, see chapter 7). The chemical characterization of mutagenic fractions was carried out by an extensive application of capillary GC-MS in the EI and NICI modes. Those fractions exhibiting the highest levels of mutagenicity were subjected to a third level of fractionation by reversed-phase HPLC (RP-LC) and analysed by GC-MS. Two sampling sites in Barcelona were monitored during 1990. Samples (24 h) of air particulate matter over periods of 1 week per season were processed.

The direct mutagenicity in the fractions NP-LC 3 (and 4) isolated from the GPC-2 fraction of airborne particulate matter collected in Barcelona (autumn 1990) seemed to be accounted for by nitrated arenes, 9-nitroanthracene, 2-nitrofluoranthene and 2-nitropyrene. 6-Nitrobenzo[a]pyrene in polar fraction 5 needed application of metabolic activation (+S9) for mutagenicity. In order to evaluate the contribution of nitro derivatives to the total mutagenicity, the NP-LC fractions were tested against TA98NR– and TA98AT–. Generally, a remarkable decrease in mutagenic activity was observed in all fractions; these decreases were more apparent in fractions NP-LC3 to NP-LC7, thus suggesting a significant contribution of nitroarenes to the mutagenicity of these fractions of medium and high polarity. Subfraction NP-LC2 was fractionated further. The expected composition was PAHs, but two nitro derivatives, 2-nitrofluoranthene and 1-nitropyrene, were identified in subfraction RP-LC3, which may be responsible for the high direct-acting mutagenic activity observed in this fraction and in the NP-LC2 fraction (Casellas et al., 1995).

NitroPAHs originate primarily as direct or indirect products of incomplete combustion. Only a few nitroPAHs are produced industrially (e.g., nitronaphthalenes and 5-nitroacenaphthene).

3.1.1 Production levels and processes

Treatment of naphthalene with mixed sulfuric/nitric acids at 60 °C yields 95% 1-nitronaphthalene and 5% 2-nitronaphthalene, together with traces of dinitronaphthalene and dinaphthol. Higher temperatures (80–100 °C) result in a mixture of 1,5- and 1,8-dinitronaphthalene in about a 2:3 ratio (Booth, 1991). The dinitro isomers can then be further separated for specific uses.

Nitronaphthalenes are produced in Germany and Japan. The production capacity of dinitronaphthalenes in Japan was 1200 tonnes per year (no year given; Booth, 1991).

3.1.2 Uses of commercially produced nitroPAHs

1-Nitronaphthalene is used almost exclusively for catalytic reduction to 1-naphthylamine. Further uses, such as use as a deblooming agent for petroleum and oils and as a component in the formulation of explosives, are of historical interest only (Booth, 1991).

1,5-Dinitronaphthalene is an intermediate in the production of naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) and 1,5-naphthalenediamine, which is mainly converted to naphthalene 1,5-diisocyanate. It is further used as a sensitizing agent for ammonium nitrate explosives (Booth, 1991).

1,8-Dinitronaphthalene is catalytically hydrogenated to 1,8-naphthalenediamine for use mainly as a colorant intermediate for naphthperinones (Booth, 1991).

5-Nitroacenaphthene is reported to be an intermediate in the synthesis of naphthalimide dyes that are used as fluorescent whitening agents and photochemical agents (Yahagi et al., 1975; IARC, 1978).

NitroPAHs in the environment originate from direct emissions from combustion sources and nitration of PAHs, primarily in the atmosphere.

The nitroPAHs emitted from combustion sources are nitroPAHs that would be formed through electrophilic nitration (e.g., 1-nitropyrene and 2-nitrofluorene; see section 3.2.1). NitroPAHs have been observed in vehicle exhaust (particularly diesel), industrial emissions and emissions from domestic residential heating/cooking and wood burning.

NitroPAHs are formed in the atmosphere from PAHs by the following reactions:

• daytime gas-phase reaction: through hydroxyl radical addition to the PAH followed by reaction with nitrogen dioxide and loss of a water molecule (formation of 2-nitrofluoranthene and 2-nitropyrene);
• nighttime gas-phase reaction: nitrate radical addition to the PAH followed by reaction with nitrogen dioxide and loss of nitric acid (formation of high yields of 2-nitrofluoranthene, nitronaphthalenes and methylnitronaphthalenes); and
• heterogeneous gas–particle interaction of parent PAHs adsorbed onto particles with nitrating agents (would be expected to produce electrophilic nitration products). This has been shown to occur in the laboratory under conditions, for example, of very high concentrations of nitrogen dioxide. Concerns that artefactual formation of nitroPAHs during ambient sampling could occur via heterogeneous reactions with nitrogen oxides have been shown to be unfounded (Arey et al., 1988a; Dimashki et al., 2000). Recent ambient measurements suggest that some 9-nitroanthracene may be formed from heterogeneous reactions during transport (Feilberg et al., 2001).

These processes are described in more detail in section 3.2.2. Other less important pathways, which are briefly mentioned here, include:

• endogenous formation of nitroPAHs in the body due to reaction of PAHs ingested in food or inhaled in ambient air with nitrogen dioxide from, for example, cigarette smoke. This has been suggested in animal experiments where mutagens have been formed from intraperitoneal (i.p.) administration of PAHs during exposure to higher than ambient levels of nitrogen dioxide (1 ppm = 1.88 mg/m³) — for example, 5–10 ppm (Kanoh et al., 1990), 10 ppm (Tokiwa et al., 1981a) and 20 ppm (Miyanishi et al., 1996); and
• formation in the water phase with nitrite as a donor of the nitro group, using mercury lamps as a UV radiation source (Ohe, 1984; Suzuki et al., 1987).

The distribution of nitroPAH isomers in samples of ambient air has been found to be significantly different from that in direct emissions from combustion (compare Table 5 and chapter 5). For example, 2-nitrofluoranthene and 2-nitropyrene are ubiquitous components of particulate matter that have been detected in urban, suburban, forest and remote areas located in Europe, America, Asia and Antarctica (Ciccioli et al., 1996), although they are not directly emitted from most combustion sources (see chapter 5). The nitroPAH profile, or the relative quantities of certain "marker" PAHs, is a pointer to the source of formation of nitroPAHs — for example, markers of direct emissions from combustion, in particular diesel exhaust, are 1-nitropyrene and 2-nitrofluorene, whereas the presence of 2-nitrofluoranthene and 2-nitropyrene points to atmospheric transformation.

Table 5. NitroPAHs detected in diesel emissions^a,b

Parent PAH; nitro derivative

Concentration (ppm = µg/g, unless otherwise stated)

a) E

b) E

c) P

d) E

e) P

f) E

g) P

h) P

i) E

j) E

k) P

l) E

m) P µg/m3

n) µg/km

o) P pmol/mg

Naphthalene

1-Nitro-

X (nd)

X

X

0.7

0.88

0.013

2-Nitro-

nd

X

X

0.02

0.039

1,3-Dinitro-

X

X

1,5-Dinitro-

X (nd)

X

X

1,8-Dinitro-

X (nd)

Acenaphthene

3-Nitro-

X*

Fluorene

1-Nitro-

5.91*

0.08*

2-Nitro-

X

X

X

nd

0.11

4.1

<0.01

0.27

0.001

X

27

8.77

0.99

nd

2,5-Dinitro-

X (nd)

2,7-Dinitro-

X (nd)

0.13

Anthracene

1-Nitro-

X*

300*

4.6

X*

2-Nitro-

X

X

10.1

9-Nitro-

X

X

X

2.8

6.02

10

1.19

1.36

7

63

1.00

Phenanthrene

1-Nitro-

0.5

2-Nitro-

X

X

X

16.5*

1.8*

3-Nitro-

9.3

4-Nitro-

0.7

9-Nitro-

2.3

0.37

0.18

0.27

Fluoranthene

1-Nitro-

X (nd)

X

4.1

X*

2-Nitro-

nd

X

0.06

3-Nitro-

X (nd)

X

1

10

0.33

1.34

1.47

<1

10

0.06

7-Nitro-

X (nd)

X

1.6

8-Nitro-

X (nd)

X

2.0

0.25

Pyrene

1-Nitro-

75

X

X

590

2.84

43

18; 19.6*

20

16.6; 18.0*

16.4

19

450

0.2 (+1,3-DNP)

3.43

6.49

2-Nitro-

nd

4-Nitro-

nd

0.07

0.04

1,3-Dinitro-

0.30

X

0.6

<0.1

0.58

0.6

with 7-NP

0.12 all DNP

0.17

1,6-Dinitro-

0.40

0.6

<0.1

0.64

1.39

0.37

0.04

1,8-Dinitro-

0.53

0.4

<0.1

0.49

1.55

0.07

Benz[a]anthracene

7-Nitro-

nd

X

8*

0.45

2.8

1.99

1.62

<1

10

0.61

Chrysene

1-Nitro-

X (nd)

0.8*

2.9*

5-Nitro-

4.6

6-Nitro-

X

0.04

0.87

0.782

X

4

0.08

0.10

Benzo[a]pyrene

1-Nitro-

1- + 3-

10*

1.2*

1.4*

6-Nitro-

X

X

5.8

1.4

1.6

0.641

X

0.12

0.30

Benzo[e]pyrene

1-Nitro-

0.83

3-Nitro-

2.2

Perylene

3-Nitro-

X

X

0.64