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

    First draft prepared by Dr C. Dameron and colleagues at the
    National Research Centre for Environmental Toxicology, Australia,
    and by Mr P.D. Howe, Institute of Terrestrial Ecology, Monks Wood,
    United Kingdom

    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

              World Health Organization
              Geneva, 1998

         The International Programme on Chemical Safety (IPCS),
    established in 1980, is a joint venture of the United Nations
    Environment Programme (UNEP), the International Labour Organisation
    (ILO), and the World Health Organization (WHO).  The overall
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    exposure to chemicals, through international peer review processes,
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    provide technical assistance in strengthening national capacities
    for the sound management of chemicals.

         The Inter-Organization Programme for the Sound Management of
    Chemicals (IOMC) was established in 1995 by UNEP, ILO, the Food and
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    relation to human health and the environment.

    WHO Library Cataloguing in Publication Data


         (Environmental health criteria ; 200)

         1.Copper - adverse effects.        2.Copper - toxicity
         3.Environmental exposure           4.Occupational exposure
         I.International Programme on Chemical Safety II.Series

         ISBN 92 4 157200 0                 (NLM Classification: QV 65)
         ISSN 0250-863X

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    names of proprietary products are distinguished by initial capital




         1.1. Identity, physical and chemical properties
         1.2. Analytical methods
         1.3. Sources of human and environmental exposure
         1.4. Environmental transport, distribution and transformation
         1.5. Environmental levels and human exposure
         1.6. Kinetics and metabolism in laboratory animals and humans
         1.7. Effects on laboratory animals and  in vitro test systems
         1.8. Effects on humans
         1.9. Effects on other organisms in the laboratory and field
         1.10. Conclusions
              1.10.1. Human health
              1.10.2. Environmental effects


         2.1. Identity
         2.2. Physical and chemical properties
         2.3. Analytical methods
              2.3.1. Sampling and sample preparation
                 Separation and concentration
                 Sample preparation
                 "Clean" techniques for measurement
                                  of ultratrace copper levels
              2.3.2. Detection and measurement
                 Gravimetric and colorimetric methods
                 Atomic absorption, emission and mass
                                  spectrometry methods
                 Specialized methodologies
         2.4. Speciation
              2.4.1. Speciation in water and sediments
                 Detection and quantification
              2.4.2. Speciation in biological matrices


         3.1. Natural sources
         3.2. Anthropogenic sources
              3.2.1. Production levels and processes
         3.3. Copper use


         4.1. Transport and distribution between media
              4.1.1. Air
              4.1.2. Water and sediment

              4.1.3. Soil
              4.1.4. Sewage sludge inputs to land
              4.1.5. Biodegradation and abiotic degradation
         4.2. Bioaccumulation
              4.2.1. Microorganisms
              4.2.2. Aquatic plants
              4.2.3. Aquatic invertebrates
              4.2.4. Fish
              4.2.5. Terrestrial plants
              4.2.6. Terrestrial invertebrates
              4.2.7. Terrestrial mammals


         5.1. Environmental levels
              5.1.1. Air
              5.1.2. Water and sediment
              5.1.3. Soil
              5.1.4. Biota
         5.2. General population exposure
              5.2.1. Air
              5.2.2. Food and beverages
              5.2.3. Drinking-water
                 Organoleptic characteristics
                 Copper concentrations in
              5.2.4. Miscellaneous exposures
         5.3. Occupational exposures
         5.4. Total human intake of copper from all environmental


         6.1. Essentiality
         6.2. Homoeostasis
              6.2.1. Cellular basis of homoeostasis
              6.2.2. Absorption in animals and humans
              6.2.3. Transport, distribution and storage
              6.2.4. Excretion
         6.3. Methods of studying homoeostasis
              6.3.1. Analytical methods
              6.3.2. Intake
              6.3.3. Diet
              6.3.4. Balance studies
         6.4. Biochemical basis of copper toxicity
         6.5. Interactions with other dietary components
              6.5.1. Protein and amino acids
              6.5.2. Phytate and fibre
              6.5.3. Ascorbic acid
              6.5.4. Zinc
              6.5.5. Iron
              6.5.6. Carbohydrates

              6.5.7. Infant diets
              6.5.8. Other interactions (molybdenum, manganese,


         7.1. Single exposure
              7.1.1. Oral
              7.1.2. Dermal
              7.1.3. Inhalation
         7.2. Short-term exposure
              7.2.1. Oral
              7.2.2. Inhalation
                 Copper(II) sulfate
                 Copper chloride
         7.3. Repeated exposure: subchronic toxicity
              7.3.1. Oral
                 Copper(II) sulfate
                 Copper chloride
         7.4. Long-term exposure chronic toxicity or carcinogenicity
         7.5. Reproductive and developmental toxicity
         7.6. Mutagenicity and related end-points
              7.6.1. Copper sulfate
                  In vitro
                  In vivo
              7.6.2. Other copper compounds
                  In vitro
         7.7. Other studies
              7.7.1. Neurotoxicity
                 Copper sulfate
                 Copper chloride
              7.7.2. Immunotoxicity
                 Copper(II) sulfate
         7.8. Biochemical mechanisms of toxicity


         8.1. General population: copper deficiency and toxicity
         8.2. Copper deficiency
              8.2.1. Clinical manifestations of copper deficiency
              8.2.2. Biological indicators of copper deficiency:  
                        balance studies
         8.3. Toxicity of copper in humans
              8.3.1. Single exposure
              8.3.2. Repeated oral exposures
                 Gastrointestinal and hepatic effects
                 Reproduction and development
              8.3.3. Dermal exposure
         8.4. Disorders of copper homoeostasis: populations at risk
              8.4.1. Menkes disease
              8.4.2. Wilson disease

              8.4.3. Hereditary aceruloplasminaemia
              8.4.4. Indian childhood cirrhosis
              8.4.5. Idiopathic copper toxicosis, or non-Indian   
                        childhood cirrhosis
              8.4.6. Chronic liver diseases
              8.4.7. Copper in infancy
              8.4.8. Malabsorption syndromes
              8.4.9. Parenteral nutrition
              8.4.10. Haemodialysis patients
              8.4.11. Cardiovascular diseases
         8.5. Occupational exposure


         9.1. Bioavailability
              9.1.1. Bioavailability in water
                 Predicting effects of copper on fish
                                  gill function
              9.1.2. Bioavailability of metals in sediments
         9.2. Essentiality
              9.2.1. Animals
              9.2.2. Plants
                 Aquatic plants
                 Terrestrial plants
         9.3. Toxic effects: laboratory experiments
              9.3.1. Microorganisms
              9.3.2. Aquatic organisms
                 Model ecosystems and community
              9.3.3. Terrestrial organisms
         9.4. Field observations
              9.4.1. Microorganisms
              9.4.2. Aquatic organisms
              9.4.3. Terrestrial organisms
                 Copper fungicides and fertilizers


         10.1. Concepts and principles to assess risk of adverse effects
              of essential elements such as copper
              10.1.1. Human health risks
              10.1.2. Homoeostatic model

         10.2. Evaluation of risks to human health
              10.2.1. Exposure of general population
              10.2.2. Occupational exposures
         10.3. Essentiality versus toxicity in humans
              10.3.1. Risk of copper deficiency
              10.3.2. Risk from excess copper intake
                General population
                Occupational risks
         10.4. Evaluation of effects on the environment
              10.4.1. Concept of environmental risk assessment
              10.4.2. Components of risk assessment process
                        for copper
         10.5. Environmental risk assessment for copper
              10.5.1. Aquatic biota
                Overview of exposure data
                Overview of toxicity data
              10.5.2. Terrestrial biota
                Overview of exposure data
                Plant foliar levels
                Assessment of toxicity of copper in


         11.1. Human health
         11.2. Environmental protection


         12.1. Health protection
         12.2. Environmental protection






         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.

                                 *     *     *

         A detailed data profile and a legal file can be obtained from the
    International Register of Potentially Toxic Chemicals, Case postale
    356, 1219 Châtelaine, Geneva, Switzerland (telephone no. + 41
    22 - 9799111, fax no. + 41 22 - 7973460, E-mail irptc@unep.ch).

                                 *     *     *

         This publication was made possible by grant number
    5 U01 ES02617-15 from the National Institute of Environmental Health
    Sciences, National Institutes of Health, USA, and by financial support
    from the European Commission.

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    FIGURE 1



    Professor D. Culver, retired from Department of Medicine, University
    of Califomia, Califorma, USA

    Professor H. Dieter, Institute for Water, Soil and Air Hygiene,
    Federal Enviromnent Agency, Berlin, Germany

    Dr R. Erickson, US Environniental Protection Agency, Duluth,
    Minnesota, USA

    Dr G.S. Fell, Department of Pathological Biochemistry, University
    of Glasgow, Glasgow Royal Infirmary, Glasgow, Scotland

    Dr J. Fitzgerald, Environmental Health Branch, Public and
    Envircumental Health Service, South Australian Health Commission,
    Rundle Mall, Adelaide, South Australia, Australia

    Dr T.M. Florence, Centre for Environmental Health Sciences, Oyster
    Bay, New South Wales, Australia

    Professor J.L. Gollan, Brigham and Women's Hospital, Harvard Medical
    School, Gastroenterology Division, Boston, Massachusetts, USA

    Dr R.A. Goyer, University of Western Ontario, Chapel Hill, North
    Carolina, USA ( Chairman)

    Professor T.C. Hutchinson, Trent University, Environmental and
    Resource Studies Program, Peterborough, Ontario, Canada

    Ms M.E. Meek, Health Protection Branch, Environmental Health
    Directorate, Health Canada, Ottawa, Ontario, Canada

    Professor MR. Moore, National Research Centre for Environmental
    Toxicology, The University of Queensland, Coopers Plains,
    Queensland, Australia ( Co-Vice-Chairman)

    Professer A. Oskarsson, Department of Food Hygiene, Faculty of
    Veterinary Medicine, Swedish University of Agricultural Sciences,
    Uppsala, Sweden

    Dr S. Sethi, Department of Pathology, Lady Hardinge Medical College
    and S.M.T. Sucheta Kripalani Hospital, New Delhi, India

    Dr K.H. Summer, National Research Centre for Environment and
    Health, Institute of Toxicology, Neuherberg, Germany

    Dr J.H.M. Terninink, Department of Toxicology, Wageningen Agricultural
    University, Wageningen, The Netherlands ( Co-Vice-Chairman)

    Dr R. Uauy, University of Chile, Santiago, Chile

    Dr J.M. Weeks, Institute of Terrestrial Ecology, Monks Wood,
    Abbots Ripton, Huntingdon, Cambridgeshire, United Kingdom


    Dr W.J. Adams, Kennecott Utah Copper, Magna, Utah, USA (Representing

    Dr K. Bentley, Department of Health and Family Services, Environmental
    Health Policy, Canberra, Australia

    Dr K.J. Buckett, Environmental Health Service, Health Department
    of Western Australia, Perth, Western Australia, Australia

    Professor J.C. Castilla, Ecology Department, Faculty of Biological
    Sciences, Pontificia Universidad Catolica de Chile, Santiago, Chile
    (Representing the Chilean Govemment)

    Dr C. Fortin, Commercial Chemicals Evaluation Branch, Environment
    Canada, Ottawa, Ontario, Canada

    Dr R. Gaunt, RTZ Ltd, London, United Kingdom (Representing the
    European Centre for Ecotoxicology and Toxicology of Chemicals)

    Mr M. Thierry Gerschel, Trefîmetaux, Courbevoie, France (Eurometaux)

    Dr P. Imray, Environmental Health Branch, Queensland Health,
    Brisbane, Queensland, Australia

    Mr C.M. Lee, International Copper Association, New York, USA

    Dr E.V. Ohanian, Health and Ecological Criteria Division, Office of
    Water, US Environinental Protection Agency, Washington, DC, USA

    Dr J.-P. Robin, Noranda Metallurgy lue., Occupational Health & Safety,
    McGill College, Montreal, Quebec, Canada (Representing ICME)


    Dr G.C. Becking, International Programme on Chemical Safety
    Inter-regional Research Unit, World Health Organization, Research
    Triangle Park, North Carolina, USA ( Secretary)

    Mr P. Callan, Departrnent of Health and Family Services, Environmental
    Health Policy, Canberra, Australia) ( Co-rapporteur)

    Dr C. Dameron, National Research Centre for Environmental Toxicology,
    The University of Queensland, Coopers Plains, Queensland, Australia

    Mr P.D. Howe, Institute of Terrestrial Ecology, Monks Wood, Abbots
    Ripton, Huntingdon, Cambridgeshire, United Kingdom ( Co-rapporteur)

    Dr L. Tomaska, Australian and New Zealand Food Authority, Canberra,
    Australia ( Co-rapporteur)


          A WHO Task Group on Enviromnental Health Criteria for Copper met
    in Brisbane, Australia, from 24 to 28 June 1996. The meeting was
    sponsored by a consortium of Australian Commonwealth and State
    Govemments through a national steering committee chaired by Dr K.
    Bentley, Director, Health and Envirorimentai Policy, Deparünent of
    Health and Family Services, Canberra. ne meeting was co-hosted and
    organized by the Department of Health and Family Services,
    Commonwealth of Australia, the Queensland Depariments of Health,
    Environment and Heritage, and the National Research Centre for
    Environmental Toxicology. Participants were welcorned by Dr G.R.
    Neville, Principal Medical Adviser, Queensland Health on behalf of the
    host organizations. In opening the meeting, Dr G.C. Becking, on behalf
    of Dr M. Mercier, Director of the IPCS and the three cooperating
    organizations (UNEP/ILO/WHO), thanked the Australian Commonwealth and
    State Govemments for their longstanding generous support in providing
    funding for this Task Group as well as several previous IPCS Task
    Groups and consultations over the last four years. lie thanked the
    Staff of Queensland Health and the National Research Centre for
    Environmental Toxicology for their excellent work in organizing the
    Task Group for Copper. The Task Group reviewed and revised the draft
    criteria monograph, and made an evaluation of the risks to human
    heaith and the enviromnent from exposure to copper.

          The first draft of this monograph was prepared by Dr C, Dameron
    and colleagues at the National Research Centre for Environmental
    Toxicology, Australia, and by Mr P.D. Howe, Institute of Terrestrial
    Ecology, Monks Wood, United Kingdom. The Task Group draft,
    incorperating the comments received fiom the IPCS Contact Points for
    Enviromnental Health Criteria monographs, was prepared by Mr P.D. Howe
    and the Secretariat.

          Dr G.C. Becking (IPCS Central Unit, Interregional Research Unit)
    and Ms K. Lyle (Sheffield, England) were responsible for the overall
    scientific content and technical editing, respectively, of this

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


    AAS       atomic absorption spectroscopy

    ALAD      aminolaevulinic acid dehydratase

    ALAT      alanine aminotransferase

    AROI      acceptable range of oral intake

    ASAT      aspartate arninotransferase

    ASV       anodic stripping voltammetry

    AVS       acid volatile suffides

    CEC       cation exchange capacity

    CNS       central nervous system

    CSV       cathodic stripping voltarrimetry

    CTMAX     critical thermal maxima

    DT-OCEE   deficiency toxicity optimum concentration for essential

    EDTA      ethylene diamine tetraacetic acid

    EPA       Enviromnental Protection Agency (USA)

    ER        endoplasmic reticulum

    FI-AAS    flow-injection atornic absorption spectroscopy

    GF-AAS    graphite fumace atomic absorption spectroscopy

    GLC       gas liquid chromatography

    GLC-MS    gas liquid chromatography-mass spectrorrietry

    HDL       high density lipoprotein

    HPLC      high performance liquid chromatography

    IC        ion chrornatography

    ICC       Indian childhood cirrhosis

    ICP-AES   inductively coupled plasma-atornic emission spectroscopy

    ICP-ES    inductively coupled plasrna-emission Spectroscopy

    ICP-MS    inductively coupled plasma-mass spectrometry

    ICT       idiopathic copper toxicosis

    LBW       low birth weight

    LDL       low density lipoprotein

    LEC       Long-Evans Cinnamon (rat)

    LOEC      lowest-observed-effect concentration

    MATC      maximum acceptable toxicant concentration

    MRE       metal responsive element

    NMR       nuelcar magnetic resonance

    NOAEL     no-observed-adverse-effect level

    NOEC      no-observed-effect concentration

    NOEL      no-observed-effect level

    NTA       nitrilotriacetic acid

    OCEE      optimal concentration of essential elements

    PIXE      proton-induced X-ray fluorescence - PTDI
              provisional tolerable daily intake

    RER       rough endoplasmic reticulum

    SAAM      standard algal assay medium

    SER       smooth endopiasmic reticulurn

    SOD       superoxide dismutase

    TIMS      thermal ionization mass spectrometry

    UV        ultraviolet

    XRF       X-ray fluorescence


    1.1  Identity, physical and chemical properties

         Copper is a reddish-brown, ductile and malleable metal.  It
    belongs to group IB of the Periodic Table.  In compounds found in the
    environment it usually has a valence of 2 but can exist in the
    metallic, +1 and +3 valence states.  Copper is found naturally in a
    wide variety of mineral salts and organic compounds, and in the
    metallic form.  The metal is sparingly soluble in water, salt or
    mildly acidic solutions, but can be dissolved in nitric and sulfuric
    acids as well as basic solutions of ammonium hydroxide or carbonate.

         Copper possesses high electrical and thermal conductivity and
    resists corrosion.

    1.2  Analytical methods

         The wide range of copper species, inorganic and organic, has led
    to the development of an array of sampling techniques, preparation and
    analytical methods to quantify the element in environmental and
    biological samples.  Contamination of the samples with copper from
    air, dusts, vessels or reagents during sampling and preparation is a
    major source of analytical errors, and "clean" techniques are

         Colorimetric and gravimetric methods for the measurement of
    copper are simple to use and are inexpensive; however, their
    usefulness is limited to situations where extreme sensitivity is not
    essential.  For measurement of low concentrations of copper in various
    matrices, atomic absorption spectrophotometric (AAS) methods are the
    most widely used.  A dramatic increase in sensitivity is obtained by
    the utilization of graphite furnace atomic absorption
    spectrophotometry (GF-AAS) rather than flame AAS.  Depending upon
    sample pretreatment, separation and concentration procedures,
    detection limits of about 1 µg/litre in water by GF-AAS and 20
    µg/litre by AAS have been reported and levels of 0.05-0.2 µg/g of
    tissue have been detected by GF-AAS.  Greater sensitivities can be
    achieved through the use of emission techniques such as high
    temperature inductively coupled argon plasma techniques followed by
    atomic emission spectroscopy (ICP-AES) or a mass spectrometer
    (ICP-MS).  Other more sensitive and specialized methodologies are
    available such as X-ray fluorescence, ion-selective electrodes and
    potentiometric methods, and anodic stripping and cathodic stripping

    1.3  Sources of human and environmental exposure

         Natural sources of copper exposure include windblown dust,
    volcanoes, decaying vegetation, forest fires and sea spray.
    Anthropogenic emissions include smelters, iron foundries, power
    stations and combustion sources such as municipal incinerators.  The
    major release of copper to land is from tailings and overburdens from

    copper mines and sewage sludge.  Agricultural use of copper products
    accounts for 2% of copper released to soil.

         Copper ores are mined, smelted and refined to produce many
    industrial and commercial products.  Copper is widely used in cooking
    utensils and water distribution systems, as well as fertilizers,
    bactericides, fungicides, algicides and antifouling paints.  It is
    also used in animal feed additives and growth promoters, as well as
    for disease control in livestock and poultry.  Copper is used in
    industry as an activator in froth flotation of sulfide ores,
    production of wood preservatives, electroplating, azo-dye manufacture,
    as a mordant for textile dyes, in petroleum refining and the
    manufacture of copper compounds.

    1.4  Environmental transport, distribution and transformation

         Copper is released to the atmosphere in association with
    particulate matter.  It is removed by gravitational settling, dry
    deposition, washout by rain and rainout.  Removal rate and distance
    travelled from the source depend on source characteristics, particle
    size and wind velocity.

         Copper is released to water as a result of natural weathering of
    soil and discharges from industries and sewage treatment plants.
    Copper compounds may also be intentionally applied to water to kill
    algae.  Several processes influence the fate of copper in the aqueous
    environment.  These include complex formation, sorption to hydrous
    metal oxides, clays and organic materials, and bioaccumulation.
    Information on the physicochemical forms of copper (speciation) is
    more informative than total copper concentrations.  Much of the copper
    discharged to water is in particulate form and tends to settle out,
    precipitate out or be adsorbed by organic matter, hydrous iron,
    manganese oxides and clay in the sediment or water column.  In the
    aquatic environment the concentration of copper and its
    bioavailability depend on factors such as water hardness and
    alkalinity, ionic strength, pH and redox potential, complexing
    ligands, suspended particulate matter and carbon, and the interaction
    between sediments and water.

         The largest release of copper is to land; the major sources of
    release are mining operations, agriculture, solid waste and sludge
    from treatment works.  Most copper deposited in soil is strongly
    adsorbed and remains in the upper few centimetres of soil.  Copper
    adsorbs to organic matter, carbonate minerals, clay minerals, hydrous
    iron and manganese oxides. The greatest amount of leaching occurs from
    sandy acidic soils. In the terrestrial environment a number of
    important factors influence the fate of copper in soil.  These include
    the nature of the soil itself, pH, presence of oxides, redox
    potential, charged surfaces, organic matter and cation exchange.

         Bioaccumulation of copper from the environment occurs if the
    copper is biologically available.  Accumulation factors vary greatly
    between different organisms, but tend to be higher at lower exposure
    concentrations. Accumulation may lead to exceptionally high body
    burdens in certain animals (such as bivalves) and terrestrial plants
    (such as those growing on contaminated soils).  However, many
    organisms are capable of regulating their body copper concentration.

    1.5  Environmental levels and human exposure

         The concentration of copper in air depends on the proximity of
    the site to major sources such as smelters, power plants and
    incinerators.  Copper is widely distributed in water because it is a
    naturally occurring element.  However, care must be taken when
    interpreting copper concentrations in the aquatic environment.  In
    aquatic systems the environmental levels of copper are usually
    measured as either total or dissolved concentrations, with the latter
    being more representative of the bioavailability of the metal.

         Average background concentrations of copper in air in rural areas
    range from 5 to 50 ng/m3.  Copper levels in seawater of 0.15 µg/litre
    and in fresh water of 1-20 µg/litre are found in uncontaminated areas.
    Sediment is an important sink and reservoir for copper.  Background
    levels of copper in natural freshwater sediments range from 16 to 5000
    mg/kg (dry weight).  Copper levels in marine sediments range from 2 to
    740 mg/kg (dry weight).  In anoxic sediments copper is bound strongly
    by sulfide and therefore not bioavailable.  Median copper
    concentrations in uncontaminated soil were reported to be 30 mg/kg
    (range 2-250 mg/kg).  Copper is accumulated by plants, invertebrates
    and fish.  Higher concentrations of copper have been reported in
    organisms from copper-contaminated sites than in those from
    non-contaminated sites.

         For healthy, non-occupationally-exposed humans the major route of
    exposure to copper is oral.  The mean daily dietary intake of copper
    in adults ranges between 0.9 and 2.2 mg.  A majority of studies have
    found intakes to be at the lower end of that range.  The variation
    reflects different dietary habits as well as different agricultural
    and food processing practices used worldwide.  In some cases,
    drinking-water may make a substantial additional contribution to the
    total daily intake of copper, particularly in households where
    corrosive waters have stood in copper pipes.  In homes without copper
    piping or with noncorrosive water, copper intake from drinking-water
    seldom exceeds 0.1 mg/day, although intakes greater than a few mg per
    day can result from corrosive water distributed through copper pipes.
    In general, total daily oral intakes of copper (food plus
    drinking-water) are between 1 and 2 mg/day, although they may
    occasionally exceed 5 mg/day.  All other intakes of copper (inhalation
    and dermal) are insignificant in comparison to the oral route.
    Inhalation adds 0.3-2.0 µm/day from dusts and smoke.  Women using
    copper IUDs are exposed to only 80 µg or less of copper per day from
    this source.

    1.6  Kinetics and metabolism in laboratory animals and humans

         The homoeostasis of copper involves the dual essentiality and
    toxicity of the element.  Its essentiality arises from its specific
    incorporation into a large number of proteins for catalytic and
    structural purposes.  The cellular pathways of uptake, incorporation
    into protein and export of copper are conserved in mammals and
    modulated by the metal itself.

         Copper is mainly absorbed through the gastrointestinal tract.
    From 20 to 60% of the dietary copper is absorbed, with the rest being
    excreted through the faeces.  Once the metal passes through the
    basolateral membrane it is transported to the liver bound to serum
    albumin.  The liver is the critical organ for copper homoeostasis.
    The copper is partitioned for excretion through the bile or
    incorporation into intra- and extracellular proteins.  The primary
    route of excretion is through the bile.  The transport of copper to
    the peripheral tissues is accomplished through the plasma attached to
    serum albumin, ceruloplasmin or low-molecular-weight complexes.

         The methods used to study copper homoeostasis in mammals include
    dietary analyses and balance studies. Isotope and standardized
    biochemical analyses of these processes are essential to understand
    copper deficiency and excess.

         The biochemical toxicity of copper, when it exceeds homoeostatic
    control, is derived from its effects on the structure and function of
    biomolecules such as DNA, membranes and proteins directly or through
    oxygen-radical mechanisms.

    1.7  Effects on laboratory animals and in vitro test systems

         The toxicity of a single oral dose of copper varies widely
    between species (LD50 range 15-1664 mg Cu/kg body weight).  The more
    soluble salts (copper(II) sulfate, copper(II) chloride) are generally
    more toxic than the less soluble salts (copper(II) hydroxide,
    copper(II) oxide). Death is preceded by gastric haemorrhage,
    tachycardia, hypotension, haemolytic crisis, convulsions and
    paralysis.  LD50 values for dermal exposure were reported at > 1124
    and > 2058 mg Cu/kg body weight in rats and rabbits respectively.
    The inhalation LC50 (exposure duration unspecified) was > 1303 mg
    Cu/kg body weight in rabbits, and respiratory function was impaired in
    guinea-pigs exposed to 1.3 mg Cu/m3 for 1 h.

         Rats given up to 305 mg Cu/kg per day orally in the diet as
    copper(II) sulfate for 15 days showed alterations in blood
    biochemistry and haematology (particularly anaemia) and adverse
    effects on the liver, kidney and lungs. Effects were qualitatively
    similar with other copper compounds and in other species.  The
    no-observed-effect level (NOEL) in this study was 23 mg Cu/kg body
    weight per day.  However, sheep were particularly sensitive and
    repeated doses of 1.5-7.5 mg Cu/kg body weight per day as copper(II)
    sulfate or copper(II) acetate resulted in progressive liver damage,
    haemolytic crisis and ultimately death.

         Long-term exposure in rats and mice showed no overt signs of
    toxicity other than a dose-related reduction in growth after ingestion
    of 138 mg Cu/kg body weight per day (rats) and 1000 mg Cu/kg body
    weight per day (mice).  The no-observed-adverse-effect level (NOAEL)
    was 17 mg Cu/kg body weight per day in rats, and 44 and 126 mg Cu/kg
    body weight per day in male and female mice, respectively. The effects
    included inflammation of the liver and degeneration of kidney tubule

         Studies of reproductive and developmental toxicity were limited.
    Some testicular degeneration and reduced neonatal body and organ
    weights were seen in rats at dose levels in excess of 30 mg Cu/kg body
    weight per day over extended time periods, and fetotoxic effects and
    malformations were seen at high dose levels (> 80 mg Cu/kg body
    weight per day).

         Copper(II) sulfate was not mutagenic in bacterial assays.
    However, a dose-related increase in unscheduled DNA synthesis was seen
    in rat hepatocytes.  In the mouse micronucleus assay, one study showed
    a significant increase in chromosome breaks at the highest intravenous
    dose (1.7 mg Cu/kg body weight) but no effect was seen in another
    study at intravenous doses up to 5.1 mg Cu/kg body weight.

         Studies of neurotoxicity have not shown effects on behaviour but
    neurochemical changes have been reported after oral administration of
    20-40 mg Cu/kg body weight per day.  A limited number of
    immunotoxicity studies showed humoral and cell-mediated immune
    function impairment in mice after oral intakes from drinking-water of
    about 10 mg Cu/kg body weight per day.

    1.8  Effects on humans

         Copper is an essential element and adverse health effects are
    related to deficiency as well as excess.  Copper deficiency is
    associated with anaemia, neutropenia and bone abnormalities but
    clinically evident deficiency is relatively infrequent in humans.
    Balance data may be used to anticipate clinical effects, whereas serum
    copper and ceruloplasmin levels are useful measures of moderate to
    severe deficiency but less sensitive measures of marginal deficiency.

         Except for occasional acute incidents of copper poisoning, few
    effects are noted in normal populations.  Effects of single exposure
    following suicidal or accidental oral exposure have been reported as
    metallic taste, epigastric pain, headache, nausea, dizziness, vomiting
    and diarrhoea, tachycardia, respiratory difficulty, haemolytic
    anaemia, haematuria, massive gastrointestinal bleeding, liver and
    kidney failure, and death.  Gastrointestinal effects have also
    resulted from single and repeated ingestion of drinking-water
    containing high copper concentrations, and liver failure has been
    reported following chronic ingestion of copper.  Dermal exposure has
    not been associated with systemic toxicity but copper may induce
    allergic responses in sensitive individuals.  Metal fume fever from
    inhalation of high concentrations in the air in the occupational

    setting has been reported and, although other respiratory effects have
    been attributed to exposure to mixtures containing copper (e.g.
    Bordeaux mix, mining and smelting), the role of copper has not been
    demonstrated. Workers apparently exposed to high air levels resulting
    in an estimated intake of 200 mg Cu/day developed signs suggesting
    copper toxicity (e.g. elevated serum copper levels, hepatomegaly).
    Available data on reproductive toxicity and carcinogenicity are
    inadequate for risk assessment.

         A number of groups are described where apparent disorders in
    copper homoeostasis result in greater sensitivity to copper deficit or
    excess than the general population.  Some disorders have a
    well-defined genetic basis.  These include Menkes disease, a generally
    fatal manifestation of copper deficiency; Wilson disease
    (hepatolenticular degeneration), a condition leading to progressive
    accumulation of copper; and hereditary aceruloplasminaemia, with
    clinical symptoms of iron overload.  Indian childhood cirrhosis (ICC)
    and idiopathic copper toxicosis (ICT) are conditions related to excess
    copper which may be associated with genetically based copper
    sensitivity, although this has not been demonstrated unequivocally.
    These are fatal liver conditions in early childhood where copper
    accumulates in the liver. Incidences of the diseases were related to
    high copper intake, at least in some cases.

         Other groups potentially sensitive to copper excess are
    haemodialysis patients and subjects with chronic liver disease.
    Groups at risk of copper deficiency include infants (particularly low
    birth weight/preterm babies, children recovering from malnutrition,
    and babies fed exclusively with cow's milk), people with malabsorption
    syndromes (e.g. coeliac disease, sprue, cystic fibrosis), and patients
    on total parenteral nutrition.  Copper deficiency has been implicated
    in the pathogenesis of cardiovascular disease.

    1.9  Effects on other organisms in the laboratory and field

         The adverse effects of copper must be balanced against its
    essentiality.  Copper is an essential element for all biota, and care
    must be taken to ensure the copper nutritional needs of organisms are
    met.  At least 12 major proteins require copper as an integral part of
    their structure. It is essential for the utilization of iron in the
    formation of haemoglobin, and most crustaceans and molluscs possess
    the copper-containing haemocyanin as their main oxygen-carrying blood
    protein.  In plants copper is a component of several enzymes involved
    in carbohydrate, nitrogen and cell wall metabolism.

         A critical factor in assessing the hazard of copper is its
    bioavailability.  Adsorption of copper to particles and complexation
    by organic matter can greatly limit the degree to which copper will be
    accumulated and elicit effects.  Other cations and pH can also
    significantly affect bioavailability.

         Copper has been shown to exert adverse reproductive, biochemical,
    physiological and behavioural effects on a variety of aquatic
    organisms.  Copper concentrations as low as 1-2 µg/litre have been
    shown to have adverse effects on aquatic organisms;  however, large
    variations due to species sensitivity and bioavailability must be
    considered in the interpretation and application of this information.

         In natural phytoplankton communities chlorophyll  a and nitrogen
    fixation were significantly reduced at copper concentrations of
    > 20 µg/litre and carbon fixation was significantly reduced at
    > 10 µg/litre.  EC50s (72 h) for algae, based on growth
    inhibition, range from 47 to 120 µg Cu/litre.

          For freshwater invertebrates, 48-h L(E)C50s range from 5 µg
    Cu/litre for a daphnid species to 5300 µg Cu/litre for an ostracod.
    For marine invertebrates 96-h LC50s range from 29 µg Cu/litre for the
    bay scallop to 9400 µg Cu/litre for the fiddler crab.  The acute
    toxicity of copper to freshwater and marine fish is highly variable.
    For freshwater fish 96-h LC50s range from 3 µg Cu/litre (Arctic
    grayling) to 7340 µg Cu/litre (bluegill).  For marine fish 96-h LC50s
    range from 60 µg Cu/litre for chinook salmon to 1400 µg Cu/litre for
    grey mullet.

         Although plants require copper as a trace element, at high soil
    levels copper can be extremely toxic. Generally visible symptoms of
    metal toxicity are small chlorotic leaves and early leaf fall.  Growth
    is stunted and initiation of roots and development of root laterals
    are poor. Reduced root development may result in a lowered water and
    nutrient uptake which leads to disturbances in the metabolism and
    growth retardation. At the cellular level, copper inhibits a large
    number of enzymes and interferes with several aspects of plant
    biochemistry (including photosynthesis, pigment synthesis and membrane
    integrity) and physiology (including interference with fatty acids,
    protein metabolism and inhibition of respiration and nitrogen fixation

         Toxic effects have been observed in laboratory studies of
    earthworms exposed to copper in soil; cocoon production is the most
    sensitive parameter measured, with significant adverse effects at
    50-60 mg Cu/kg.

         Adverse field effects on soil microorganisms have been correlated
    with enhanced copper concentrations in areas where copper-containing
    fertilizers have been applied and in areas near to copper-zinc
    smelters. In citrus-growing areas, to which copper-containing
    fungicides have been applied, leaf chlorosis has been found to be
    significantly correlated with soil copper levels.

         Tolerance to copper has been demonstrated in the environment for
    phytoplankton, aquatic and terrestrial invertebrates, fish and
    terrestrial plants.  Tolerance mechanisms which have been proposed in
    plants include binding of metal to cell wall material, presence of

    metal-tolerant enzymes, complex formation with organic acids with
    subsequent removal to the vacuole, and binding to specialized
    thiol-rich proteins or phytochelatins.

    1.10  Conclusions

    1.10.1  Human health

         The lower limit of the acceptable range of oral intake (AROI) is
    20 µg Cu/kg body weight per day.  This figure is arrived at from the
    adult basal requirement with an allowance for variations in copper
    absorption, retention and storage (WHO, 1996).  In infancy, this
    figure is 50 µg Cu/kg body weight per day.

         The upper limit of the AROI in adults is uncertain but it is most
    likely in the range of several but not many mg per day in adults
    (several meaning more than 2-3 mg/day).  This evaluation is based
    solely on studies of gastrointestinal effects of copper-contaminated
    drinking-water.  A more specific value for the upper AROI could not be
    confirmed for any segment of the general population.  We have limited
    information on the level of ingestion of copper from food that would
    provoke adverse health effects.

         The available data on toxicity in animals were considered
    unhelpful in establishing the upper limit of the AROI, owing to
    uncertainty about an appropriate model for humans.  Moreover,
    traditional methodology for safety assessment, based on application of
    uncertainty factors to data in animals, does not adequately address
    the special attributes of essential elements such as copper.

         From available data on human exposures worldwide, but
    particularly in Europe and the Americas, there is greater risk of
    health effects from deficiency of copper intake than from excess
    copper intake.

    1.10.2  Environmental effects

         Protection of aquatic life in waters with high bioavailability
    will require limiting total dissolved copper to some concentration
    less than 10 µg/litre; however, the appropriate concentration limit
    will depend on the biota and exposure conditions at sites of concern
    and should be set based on further evaluation of all relevant data.

         At many sites, physicochemical factors limiting bioavailability
    will warrant higher copper limits.  Regulatory criteria should take
    into account the speciation of copper if dischargers can demonstrate
    that the bioavailability of copper in the receiving water can be
    measured reliably.

         When sampling and analysing environmental media for copper, it is
    essential that "clean" techniques be employed.

         Because copper is an essential element, procedures to prevent
    toxic levels of copper should not incorporate safety factors that
    result in recommended concentrations being below natural levels.


    2.1  Identity

         Copper, the 29th element and the first in group IB of the
    Periodic Table, displays four oxidation states: metallic copper Cu0,
    cuprous ion Cu+, cupric Cu2+ and trivalent copper ion Cu3+.
    Copper also forms organometallic compounds.  The natural isotopic
    abundance is 69.17% 63Cu and 30.83% 65Cu, giving the element an
    average relative atomic mass of 63.546 (Lide & Frederikse, 1993b).
    The limited range of stable isotopes and their common distribution has
    inhibited isotopic distribution studies.  Useful radioactive copper
    isotopes are 64Cu (12.701 h half-life) and 67Cu (61.92 h half-life);
    they decay with the production of ß-particles and gamma-rays (Lide &
    Frederikse, 1993b) and are produced in synchrotrons for physical and
    biological studies.

         Copper is found in a wide variety of mineral salts and organic
    compounds, and can also be found naturally in the elemental or
    metallic form.  The metal is a dull lustrous reddish-brown in colour,
    malleable, a good thermal conductor and an excellent electrical
    conductor.  The metallic form is very stable to dry air at low
    temperatures but undergoes a slow reaction in moist air to produce a
    hydroxycarbonate or hydroxysulfate that forms a greenish-grey
    amorphous film over the surface which protects the underlying metal
    from further attack.  The metal is sparingly soluble in water, in salt
    solutions and in mildly acidic solutions, but can be dissolved in
    nitric acid and sulfuric acid as well as in basic solutions of
    ammonium hydroxide, ammonium carbonate and cyanide in the presence of
    oxygen (Cotton & Wilkinson, 1989).

         The electronic configuration of the metallic (Cu0) form is
    1s22s22p63s23p63d104p1.  The common solution oxidation states
    are the cuprous (Cu(I) 3d10) or the cupric (Cu(II) 3d9) forms.  The
    chemistry of the element, especially in biological systems, is
    profoundly affected by the electronic/oxidation state.  The facile
    exchange between oxidation states endows the element with redox
    properties which may be of an essential or deleterious nature in
    biological systems.

         The most important oxidation state in natural, aqueous
    environments is copper(II).  Any copper(I) present is quickly oxidized
    by any oxidizing reagent present, or in a disproportionation reaction,
    unless it is stabilized by complex formation.  The copper(II) ion
    binds preferentially via oxygen to inorganic ligands such as H2O, OH-,
    CO32-, SO42-, etc. and to organic ligands via phenolic and
    carboxylic groups (Cotton & Wilkinson, 1989).  Thus, almost all of the
    copper in natural samples is complexed with organic compounds
    (Neubecker & Allen, 1983; Nor, 1987; Allen & Hansen, 1996).

         Many cupric compounds and complexes are soluble in water and have
    a characteristic aqua-blue-green colour.  The trivalent form of copper
    is found in only a few compounds and is a strong oxidizing agent
    (Cotton & Wilkinson, 1989).  In environmental and mineral environments

    the divalent oxidation state readily adsorbs to a variety of hydrated
    metal oxides including those of iron, aluminium and manganese (Grant
    et al., 1990).

         Identification, quantification and speciation of copper is
    described in sections 2.3 and 2.4 and the influences on the speciation
    in water and soil are described in section 2.4.1.

    2.2  Physical and chemical properties

         The physical and chemical properties of copper and some of its
    salts are summarized in Table 1.

    2.3  Analytical methods

         The wide range of copper species, inorganic and organic, has lead
    to the development of an array of sampling techniques and preparative
    and analytical methods to quantify the element in environmental and
    biological samples.  The following sections offer a brief overview of
    these methodologies.

    2.3.1  Sampling and sample preparation

         Sampling and the subsequent work-up is highly dependent on the
    type of sample being analysed and the level of detail needed to
    evaluate it.  Most of the techniques described below suffer at some
    level from the effects of the surrounding milieu or matrix.
    Qualitative analysis to determine the presence of copper in a sample,
    for instance, may or may not require consideration of the matrix,
    whereas quantitation of metals usually does.  Quantitation of the
    various forms of copper requires a detailed evaluation of the matrix
    and the techniques being used.  Sampling

         Owing to the abundance of copper in the environment, the
    collection of samples for copper analysis requires precautions to
    avoid accidental contamination.  Most plastics and glassware are
    relatively free of copper contamination but care should be taken to
    avoid heavily pigmented plastics that could contain copper or other
    metals that might compromise the analysis.  Interference by
    contaminating metals is more likely to be a problem in colorimetric
    analyses.  Vessels to be used in the collection of samples for copper
    analysis should be cleaned of dust and debris and washed with a dilute
    metal-free mineral acid such as 0.1 mol/litre hydrochloric or nitric
    acid, rinsed copiously with clean distilled water and dried in a
    dust-free area.  Copper is frequently and naturally found in
    industrial and household dusts (Kim & Fergusson, 1993) so care should
    be taken that the samples are not contaminated.  Removal of copper
    from washing and rinsing water, and even distilled water, can be
    compromised by the use of copper plumbing and brass fixtures.  Removal
    of metals and other ions can be accomplished through the use of
    ion-exchange resins.

        Table 1.  Physical and chemical properties of copper and some of its saltsa

                              Copper        Copper(II)           Cuprous(I)       Copper(II)         Copper(II)         Oxine-copperb
                                            sulfate              oxide            hydroxide          chloride

    CAS registry number       7440-50-8     7758-98-7            1317-39-1        20427-59-2         7447-39-4          10280-28-6

    Molecular formula         Cu            CuSO4                Cu2O             Cu(OH)2            CuCl2              C18H12CuN2O2

    Relative molecular mass   63.55         159.6                141.3            97.56              134.45             351.9

    Boiling point (°C)        2567          decomposes to                         decomposes at      decomposes at
                                            CuO at 650 °C                         140 °C             993 °C

    Melting point (°C)        1083.4        slightly decomposes  1235             decomposes         620                decomposes
                                            at > 200°C                                                                  at 270°C

    Vapour pressure (kPa)     1.33 at
                              1870 °C

    Water solubility          insoluble     143 g/litre          practically      2.9 mg/litre       706 g/litre        insoluble
                                            at 0°C               insoluble        at 25 °C

    a Lide & Frederikse (1993)
    b Copper 8-hydroxyquinolinate.  Separation and concentration

         It is not generally necessary that the metal itself be isolated
    before analysis, but frequently the metal or at least the inorganic
    portion of the sample must be concentrated.  The requirement for
    concentration of the sample depends on the sensitivity of analytical
    method to be employed.

         Particulates (dust, smoke, spray) are sampled from air on filters
    before analysis.  Aqueous samples may need to be dried or concentrated
    using an ion-exchange procedure (Vermeiren et al., 1990; Chakrabarti
    et al., 1994).

          Total copper (in water) includes all forms of copper
    irrespective of form, whether dissolved or bound.   Suspended copper 
    refers to copper attached to suspended particles in water large enough
    to be filtered by a 0.45 µm membrane filter.   Dissolved copper  is
    defined operationally as all forms of copper which pass through a 0.45
    µm membrane filter (ATSDR, 1990).  Separation of dissolved and
    suspended forms of copper requires filtering.  Special measures must
    be taken to avoid sample contamination when filtering.  First, the
    membrane filter and filter holder must be acid cleaned.  The filter
    must be discarded and the filter holder should be acid rinsed between
    samples and subsequently rinsed with metal-free water.  Second, glass
    fibre filters must not be used.  Third, the filter holder and membrane
    filter must be conditioned with the sample, i.e. an initial portion of
    the sample filtered and discarded.  Lastly, if positive pressure
    filtration is used, the gas must be passed through a 0.2 µm in-line
    filter.  Sample preparation

         Direct analysis of metals with little modification or preparation
    of the sample is desirable but frequently not achievable.   Direct
    analysis of copper is appropriate when relatively concentrated samples
    are analysed (0.1-2 mg/litre or higher), provided they are very low in
    interfering inorganics and especially organic materials.  More dilute
    samples can be concentrated as described above. Concentrated samples
    can be diluted with appropriate diluents, usually distilled water or
    dilute copper-free mineral acid solutions.  Care should be taken to
    keep the pH near or below neutral to avoid the formation of insoluble
    copper hydroxides.

         Sample preparation for the most widely utilized analytical
    techniques, or where the removal of the organic matrix is required, is
    generally achievable by means of a preceding open vessel oxidative
    degradation step involving nitric acid or acid mixtures such as aqua
    regia or sulfuric acid/hydrogen peroxide. (Perchloric acid is less
    frequently used because of its explosive nature.)  A procedure using a
    mixture of nitric, perchloric and hydrofluoric acids was reported to
    give good recoveries of metals including cadmium, chromium, copper,
    manganese, nickel, lead and zinc in estuarine sediments (Bello et al.,
    1994).  Recently, oxidative UV photolysis (Kolb et al., 1992) and

    microwave-assisted acid digestion in a closed vessel have become more
    popular in sample preparation for various sample matrices prior to
    elemental analyses.  Microwave-assisted digestion has been employed as
    a sample preparation procedure prior to the measurement of copper
    level in human bone (Baranowska et al., 1995), in duck eggs (Jeng &
    Yang, 1995), in sediments by anodic stripping voltametry (Olsen et
    al., 1994), in marine biological tissues such as mollusc, fish and
    crustacean by AAS (Baldwin et al., 1994), in steels and copper alloys
    by ICP-AES (Borszeki et al., 1994), and in plant materials (Matejovic
    & Durackova, 1994).  The microwave digestion procedure is fast
    becoming the method of choice because sample preparation is rapid and
    the values of blanks are significantly lower than in the traditional
    wet and dry mineralization methods (Matejovic & Durackova, 1994).  A
    fast and quantitative on-line microwave digestion/extraction of copper
    from different solid matrices, such as vegetables, powdery dietary
    products and sewage sludge, was developed using a flow
    injection-atomic absorption system (FI-AAS) (Delaguardia et al.,
    1993).  A similar FI-AAS method for the determination of copper in
    whole blood was also reported by Burguera et al. (1993).  "Clean" techniques for measurement of ultratrace copper levels

         Information provided by Shiller & Boyle (1987), Windom et al.
    (1991) and Hurley et al. (1996) has raised questions concerning the
    quality of data collected and reported for trace metals analysis over
    the past several decades.  The concern is that insufficient care in
    sampling, sample preparation and analysis have resulted in samples
    being contaminated and the values reported in the sub-mg/litre range
    have questionable accuracy.  It has been shown that many published
    literature values for surface waters are biased on the high side owing
    to contamination and/or matrix interferences.  Matrix interferences
    commonly encountered in copper analyses are chemical, spectral,
    ionization and high dissolved solids.  Copper determination by ICP
    emission spectroscopy (ICP-ES) can suffer from interference by iron,
    thallium and vanadium (US EPA, 1986).  Copper determination by ICP-MS
    emission spectroscopy is susceptible to interference from chlorides,
    although procedures have been developed to overcome this interference
    in blood serum samples, for example (Lyon & Fell, 1990).  Both ICP-ES
    and ICP-MS are excellent techniques for measuring copper if care is
    taken to eliminate interferences.  "Clean" techniques (Prothro, 1993;
    US EPA, 1995) address the problem associated with making accurate and
    precise trace determinations of metals particularly when attempting to
    lower detection limits and report microgram/litre and
    sub-microgram/litre concentrations.  "Clean" techniques require
    special attention to be paid in seven areas:

    1.   use of "clean" techniques during collecting, handling, storing,
         preparing and analysing samples to avoid contamination
    2.   use of analytical methods that have sufficiently low detection
    3.   avoidance of interference in the quantification step
    4.   use of blanks to assess contamination
    5.   use of matrix spikes and certified reference materials (CRMs) to
         assess interference and contamination

    6.   use of replicates to assess precision
    7.   use of certified standards.

         To achieve accurate and precise measurement of any particular
    sample, it is recommended that both the detection limit and the blank
    value should be less than one-tenth the sample concentration.  This is
    a stringent requirement, but one that is especially important in
    measuring metals at concentrations near the method detection limit and
    at environmentally relevant concentrations.  The methods employed to
    attain these goals seek to increase sensitivity, decrease
    contamination and decrease interference.  The specific recommendations
    used to achieve these goals and address the seven items above are
    provided in Prothro (1993).

    2.3.2  Detection and measurement  Gravimetric and colorimetric methods

         Gravimetric and colorimetric methods were the earliest procedures
    used for the measurement of copper.  Gravimetric methods are
    non-specific and may precipitate other cations including zinc,
    cadmium, cobalt and nickel.  Useful spectrophotometric reagents for
    copper include cuprizone (biscyclohexanoneoxalydihydrazone) (Peterson
    & Bollier, 1955), bathrocuproinedisulfonic acid
    (2,9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulfonic acid) (Zak,
    1958), bathocuproine (dimethyl-4,7-diphenyl-1,10-phenanthroline)
    (Wharton & Rader, 1970) and more recently 1-(2-pyridylazo)-2-naphthol
    (Malvankar & Shinde, 1991), BPKQH (benzyl 2-pyridyl ketone
    2-quinolylhydrazone (Garcia-Sanchez et al., 1990) and
    2,2'-bichinchioninic acid (Brenner & Harris, 1995).  The bathocuproine
    method can achieve a limit of detection of 2 µg Cu/litre in water

         Although colorimetric methods can suffer from lack of
    specificity, they are nevertheless useful, especially in laboratories
    where more sophisticated instrumentation is not available.  Beyond a
    spectrophotometer and an analytical balance, no specialized equipment
    is required.  In addition, the methods are, in general, simple,
    inexpensive, easily taught and rapidly carried out.  Because of these
    advantages they should be considered in situations where extreme
    sensitivity is not essential.  Atomic absorption, emission and mass spectrometry methods

         Atomic absorption spectrophotometric (AAS) methods are the most
    widely used for the determination of copper in various matrices.  A
    dramatic increase in sensitivity over that obtained by flame AAS is
    obtained with GF-AAS.  Increasingly more common is the use of emission
    methods in which the sample is introduced into a high temperature
    inductively coupled argon plasma (ICP) where the element is rapidly
    vaporized and ionized.  The element is detected and quantified by
    atomic emission spectroscopy (ICP-AES).

         A further increase in sensitivity is obtained through the
    coupling of the ICP to a mass spectrometer (ICP-MS).  The attraction
    of the ICP methods is the ability to do multielemental analysis
    (Vollkopf & Barnes, 1995) which is the obvious advantage over other
    spectroscopic techniques.  The ICP-MS technique has the additional
    advantage that isotopic information can be obtained, which is
    especially useful if stable isotopes of copper are used for
    bioavailability and other studies (Lyon et al., 1988, 1995, 1996).  An
    isotope dilution ICP-MS method (Beary et al., 1994) reported precision
    of less than 0.15% for copper and cadmium in zinc ore and for copper
    and molybdenum in domestic sludge; others (Lu et al., 1993) reported a
    more conservative precision of less than 1% and a detection limit of
    58 ng/litre for copper in a number of biological and environmental
    reference materials.  The International Standards Organization have
    published procedures using AAS for the analysis of copper in water
    between 0.05 and 200 µg/litre (ISO, 1986).  Detection limits are
    summarized in Table 2.  Specialized methodologies

         Many X-ray fluorescence (XRF) methods, which are nondestructive
    techniques, have been published for the determination of trace
    elements including copper.  XRF has for a long time been used as a
    rapid and convenient method for trace element determination although
    its sensitivity is somewhat lower than anodic stripping voltametry
    (ASV) (Viksna et al., 1995).  The technique can be used for a variety
    of sample types, such as human serum (Viksna et al., 1995),
    electrolyte purification solutions (Davidson et al., 1994), human
    kidney tumours (Hamilton et al., 1972) and contaminated soils (Wilson
    et al., 1995).  Field instruments are available for scans of
    contaminated sites to estimate the metal in the surface layer of the
    soil.  A proton-induced X-ray fluorescence technique (PIXE) was also
    reported for the measurement of trace elements in amniotic fluid
    (Napolitano et al., 1994).

         Ion-selective electrode and potentiometric methods have been used
    for copper speciation in soil (Town & Powell, 1993), and in seawater
    (Román & Rivera, 1992; Soares et al., 1994).  Voltammetric methods
    have comparable sensitivity to conventional AAS, but also offer
    speciation capability (Scarano et al., 1990; Chakrabarti et al., 1994;
    Cheng et al., 1994).  Voltammetric/potentiometric analyses offer
    sensitivity in the parts per billion (µg/kg) range for copper and some
    other metals.  Potentiometric analysis relies on the elements
    electrochemical properties.  An attraction of potentiometric methods
    is their ability to help in the speciation of copper and limited
    multielement detection.  ASV has been used to analyse copper in foods
    (Holak, 1983).  Cathodic stripping voltametry (CSV) is an extremely
    sensitive method for copper in both seawater and fresh water, with a
    limit of detection of 0.005 µg/litre (Donat et al., 1994).

         Some analytical methods for the detection of copper in different
    media are summarized in Table 2.

        Table 2. Analytical methods for the detection of copper
    Medium          Sample                   Methoda       Detection         Reference
                    preparation                            limit
    Air             filter collection on     ICP-AES       1 µg              ATSDR
                    0.8 µm membrane;                                         (1990)
                    acid digestion

                    filter collection on     AAS           0.05 µg           ATSDR
                    0.8 µm membrane;                                         (1990)
                    acid digestion

    Fresh           acidify with 1:1         AAS           20 µg/litre       US EPA
     water          HNO3 to a pH < 2                                         (1986)

                    sample solutions         GF-AAS        1 µg/litre        US EPA
                    should contain 0.5%                                      (1986)

                    filter and acidity       ICP           2-10 µg/litre     US EPA
                    sample                                                   (1986)

                    filter and acidity       ICP-AES       6 µg/litre        ATSDR
                    sample                                                   (1990)

                    acid digestion with      ICP-MS        0.01 µg/litre     US EPA
                    HNO3, reflux and                                         (1994)
                    dilute with type 1

    Sediment        acid digestion           AAS           1.0 µg/g          US EPA
                    acid digestion           GF-AAS        0.05-0.20 µg/g    (1986)
                    acid digestion           ICP           0.20-0.50 µg/g    US EPA
                    acid digestion           ICP-MS        0.025-0.005 µg/g  (1986)

    Tissue          acid digestion           AAS           0.5-1.0 µg/g      US EPA
                    acid digestion           GF-AAS        0.05-0.20 µg/g    (1986)
                    acid digestion           GF-AAS        0.25 µg/g         Lowe et
                                                           wet weight        al. (1985)
                    acid digestion           ICP           0.04-0.1 µg/g     US EPA
                    acid digestion           ICP-MS        0.025-0.05 µg/g   (1986)
                    acid digestion           ICP-AES       0.2 µg/g tissue   NIOSH
                                                           1 µg/100 ml blood (1987)

    Food            closed system            ASV           0.32 µg/g         Holak
                    digestion                                                (1983)

    a               See list of abbreviations on p. xxii.

    2.4  Speciation

         Developing an objective assessment of the hazard that copper
    poses to humans and the environment depends on an intimate
    understanding of its bioavailability. Bioavailability, defined as the
    extent to which the metal is taken up by an organism upon exposure,
    depends on the species of the metal or metallo complex and/or how
    easily it can be transformed to a more or less bioavailable species.

    2.4.1  Speciation in water and sediments

         In natural waters, only very small percentages of copper are
    present as the "free" aquo ion (Cu2+); rather, most copper is
    adsorbed to suspended particles or complexed with various ligands
    (Florence & Batley, 1980).  Inorganic ligands of greatest importance
    are hydroxide, carbonate and, in saline waters, chloride (Bodek et
    al., 1988).  Binding of copper to fulvic and humic acids and to other
    organic compounds can be very strong, so that a large proportion of
    dissolved copper is often organically complexed (Neubecker et al.,
    1983; Coale & Bruland, 1988; Allen & Hansen, 1996).  In air, copper is
    present in particulate form.  In sediments and soils, most copper is
    also on or in particles, either as a constituent of mineral phases or
    adsorbed to oxide surfaces or organic matter; formation of copper
    sulfide can be particularly important in anoxic sediments (DiToro et
    al., 1990).  Copper speciation in interstitial water can be affected
    by high concentrations of inorganic and organic ligands.

         Speciation, the identification and quantitation of a metal in its
    various oxidation states, inorganic forms and organometallic
    complexes, is afforded through a wide variety of techniques (ICME,
    1995).  Detection and quantification

    a)   Electrochemical methods

         Electrochemical techniques, especially ASV, have been widely used
    to measure the "electrochemically labile" fraction of copper in water
    samples, with the assumption that the electrochemically labile
    fraction is an approximation of the bioavailable fraction of copper
    (Neubecker & Allen, 1983; Bruland et al., 1985; Buckley & van den
    Berg, 1986; Morrison & Florence, 1989; Florence et al., 1992; Donat et
    al., 1994).  It has been shown that if the ASV measurement is carried
    out in a manner such that the copper complexing agents in the water
    sample affect only the efficiency of electrochemical deposition, but
    not the stripping process, then ASV-labile copper correlates very well
    with bioavailable copper as measured by algal assay (Florence et al.,
    1992).  Simple ASV analysis of a water sample at the natural pH where
    complexing agents affect both the deposition and stripping processes
    tends to underestimate the bioavailable fraction of copper (Zhang &
    Florence, 1987; Morrison & Florence, 1989).

         Electrochemical titrations using ASV can provide information on
    the "complexing capacity" of a water sample, as well as quantitative
    data on the conditional formation constants of copper with the ligands
    present in the sample.  Complexing capacity is defined as the total
    concentration of ligands, both organic and inorganic, in a water
    sample that will bind copper in nonlabile complexes (Donat et al.,

    b)   Equilibration methods

         Together with electrochemical methods, equilibration techniques
    are among the most popular and successful methods used for speciation
    studies.  The equilibration methods mostly use ion-exchange resins or
    weak inorganic exchangers and complexing ligand.  The equilibrium
    constant of both the resin and the complex has to be satisfied
    simultaneously.  The distribution ratio for a fixed resin
    concentration is measured in the presence of a competing ligand with
    known metal equilibria, which determines the partition coefficient for
    the resin.  Stability constants and ligand concentrations of unknown
    solutions can then be measured (Neubecker & Allen, 1983).

         The total concentration of most biologically important trace
    metals including copper in seawater is in the range 10-10-10-8
    mol/litre and hence the concentration of any individual metal organic
    complex must be considerably lower. Characterization and
    identification of individual compounds at these concentrations in
    seawater by chemical techniques is very difficult, if not impossible.
    The methodology usually involves first extracting and concentrating
    the compounds from sample matrices on to a resin, followed by
    fractionation according to different chemical and physical properties.
    Since the compounds may not be volatile, the most useful technique is
    high performance liquid chromatography (HPLC); alternatively, the
    compounds can be made volatile by some derivatization steps then
    determined by gas liquid chromatography (GLC), or gas liquid
    chromatography-mass spectrophotometry (GLC-MS).  Thompson & Houk
    (1986) reported an HPLC-ICP-MS method of multielemental analysis and
    speciation with a limit of detection of 4 ng of copper.  Recently, the
    sensitivity for copper was increased by using an ion
    chromatography-ICP-MS (IC-ICP-MS) technique (McLaren et al., 1993).
    The aluminium hydroxide-cation exchange mini-column technique (Zhang &
    Florence, 1987) provides a rapid and simple method for determining
    bioavailable copper in both seawater and fresh water samples.

    2.4.2  Speciation in biological matrices

         The speciation of copper in tissue and blood samples has been
    studied (Florence & Batley, 1980; Brouwer et al., 1989; Florence et
    al., 1992).  In particular, techniques have been developed for the
    separation and determination of caeruloplasmin in blood plasma (Lyon &
    Fell, 1990) and for metallothioneins in tissue samples (Florence et
    al., 1992).


    3.1  Natural sources

         Metal oxides, silicates and other materials are the building
    blocks of rocks forming the earth's crust and it is the weathering of
    these rocks that creates soils and sediment.  Copper oxide, copper
    sulfide and other ores are among these components.  Copper, along with
    other metals, is distributed through the environment by precipitation
    and resulting riverine flows which transport the particles.  Depending
    on the flow dynamics, these particles settle out and form sedimentary
    deposits.  Volcanic activity injects dust and particles into the
    atmosphere; they then settle out on soil and water surfaces.  Wind is
    a significant factor in moving metal-laden soil particles around the
    land surface of the earth, which they can also reach from atmospheric
    sources by both wet (rain washout) and dry deposition.  An important
    source of copper in aquatic sediments is from dead organisms which
    settle out and contribute both copper and organic material.  This can
    be a significant source in the oceans, for example.

         Copper has a natural abundance of approximately 60 mg/kg in the
    earth's crust and 2.5 × 10-4 mg/litre in the sea (Lide & Frederikse,
    1993).  It occurs naturally in many minerals such as cuprite (Cu2O),
    malachite (Cu2CO3.Cu(OH)2), azurite (2CuCO3.Cu(OH)2),
    chalcopyrite (CuFeS2), chalcocite (Cu2S), and bornite (Cu5FeS4).
    Copper is also found naturally in its metal form (Tuddenham & Dougall,
    1978).  The copper content of ore deposits ranges from 0.5 to 5% by
    weight, whereas igneous rock contains 0.010% (Duby, 1980) and
    crystalline rock 0.0055% by weight.  The most important sources of
    copper are chalcocite, chalcopyrite and malachite (Weant, 1985).

         Figures from Cannon et al. (1978) indicate a range of 4-200 mg
    Cu/kg and a range of mean concentrations of 2-90 mg Cu/kg in igneous
    and sedimentary rocks.  Nriagu (1989) estimated mean worldwide
    emissions of copper from natural sources as follows: windblown dusts,
    0.9-15 × 103 tonnes; forest fires, 0.1-7.5 × 103 tonnes; volcanic
    particles, 0.9-18 × 103 tonnes; biogenic processes, 0.1-6.4 × 103
    tonnes; sea salt spray, 0.2-6.9 × 103 tonnes.

         Average background concentrations of copper in air in rural areas
    range from 5 to 50 ng/m3.  Copper levels in seawater of 0.15 µg/litre
    and in freshwater of 1.0-20 µg/litre are found in uncontaminated areas
    (Nriagu, 1979b).  Background levels of copper in uncontaminated
    sediments range from 800 to 5000 mg/kg (dry weight) (Forstner &
    Wittmann, 1979).  Copper levels in marine sediments range from 2 to
    740 mg/kg (dry weight). Median copper concentrations in uncontaminated
    soil were reported to average 30 mg Cu/kg with a range of 2-250 mg/kg
    (Bowen, 1985).  Detailed information on concentrations in the
    environment is presented in section 5.1.  Copper is found as a natural
    component of foods eaten by humans and animals.

    3.2  Anthropogenic sources

         Anthropogenic sources of copper include emissions from mines,
    smelters and foundries producing or utilizing copper, zinc, silver,
    gold and lead.  Environmental copper can also arise from the burning
    of coal for power generation and from municipal waste incinerators.  A
    major release of copper to land comes from mine tailings and
    overburden from mining operations.  Other anthropogenic sources of
    copper include its use as an antifouling agent in paints, agriculture
    (fertilizers, algicides, feed supplements) and animal and human
    excreta (animal manure and human sewage sludge).  Copper is also
    intentionally released into some water bodies to control the growth of
    algae (Slooff et al., 1989; ATSDR, 1990).

         Although it was estimated that 66% of copper emissions to the
    environment in 1983 were from anthropogenic sources (Nriagu, 1989),
    there is evidence that industrial emissions are decreasing owing to
    stringent controls developed in facilities manufacturing and using
    copper (Dann, 1994).

    3.2.1  Production levels and processes

         The mining and refining of copper takes place on all six
    continents.  Mines in Chile, USA and Canada account for over 50% of
    the annual worldwide production of 11 × 106 tonnes of refined copper
    metal (ICSG, 1996).  Other major areas for copper mining include
    Russia, Australia, Zambia, Indonesia, Peru, China and Poland.  It is
    estimated that about 40% of the copper used worldwide (approximately
    15 × 106 tonnes) comes from recycled metal (ATSDR, 1990).  Release of
    airborne copper from smelters is currently one of the major sources of
    copper to the environment.

         The majority of copper metal is produced by smelting of the
    copper sulfide ore followed by electrolytic refining (ATSDR, 1990).
    Some 106 tonnes were produced in Chile and North America using
    solvent extraction technology.  The process involves extraction of
    copper from acidic leach solutions using organic reagents followed by
    electrolytic extraction.  The principal sources of copper for this
    process are conventional mining of oxide ores in open pits, leaching
    of mine dump low-grade ore, and mill tailings and mine water run-off.
    Extraction of mine tailings and dumps in this way reduces the
    environmental impact of mine wastes by reducing the copper
    concentrations in these sources.

    3.3  Copper use

         The world uses approximately 15 × 106 tonnes of copper a year.
    Of this about one-third is derived from recycled metal, and the rest
    is supplied from the mining of ore bodies and refining of the
    extracted copper.

         The unique combination of properties of copper, including
    durability, ductility, malleability and electrical and thermal
    conductivity, determine its uses in a vast range of applications. A
    summary of these uses in the USA, Western Europe and Japan is given in
    Table 3, compiled from Marco (1989).

         Worldwide, the largest use of copper is in electrical wire and
    cable and other electronic applications, which can account for as much
    as 65% (9.75 × 106 tonnes) of total annual copper consumption.
    Rolled copper is also extensively used in architectural applications
    for roofing, rainwater goods and cladding, while rolled copper and
    brass are also used for vehicle radiators.  Overall, the major
    industrialized countries consume over 1.5 × 106 tonnes of rolled
    product per year.  Approxi mately 15% (2.25 × 106 tonnes) of copper
    is used annually in building and construction, including plumbing,
    architectural applications such as roofing, guttering and flashing,
    and in fixtures and fittings.  The remaining 20% (3 × 106 tonnes)
    goes to transport equipment, air-conditioning and refrigeration as
    well as general and light engineering uses such as machine parts, and
    process equipment, coinage, ordnance and consumer goods, such as
    domestic appliances as well as production of bronze and brass alloys.

         Extruded brass is a raw material for the forging and machining
    sectors, and is turned into a wide range of components such as taps,
    valves and water fittings, and instrument and machine parts.  Over 1.7
    × 106 tonnes of extruded copper alloy products are consumed by the
    major industrialized countries annually.

         Tubes in copper and copper alloys are widely and increasingly
    used for domestic plumbing and heating systems, air conditioning,
    refrigeration and industrial applications. Over 1.5 × 106 tonnes of
    tubes are consumed annually by the major industrialized countries.

         A small percentage of copper production goes into the manufacture
    of copper compounds, particularly copper sulfate which is used
    primarily for industrial and agricultural purposes.  In industry,
    copper sulfate is used as an activator in the froth flotation of
    sulfide ores, production of chromated copper arsenate wood
    preservatives, electroplating, azo-dye manufacture, as a mordant for
    textile dyes, in petroleum refining and in the manufacture of other
    inorganic and organometallic compounds (ATSDR, 1990).  Other copper
    compounds find uses as pigments, paints, dyes, glasses, catalysts and
    fungicides.  Copper is finding increasing use as the active ingredient
    in antifouling paints.  In this context it is also used in paints for
    operating theatres and other hospital facilities to reduce inadvertent
    contamination of surfaces and transmission of disease-causing

        Table 3.  Copper consumption in 1988a (in thousands of tonnes)

    Use                        Building and       Electrical/        Industrial
                               construction       electronics

    Copper wire                0                  4293               0
    Copper rod                 5                  164                34
    Copper sheet and strip     240                140                225
    Copper tube                551                0                  424
    Alloy wire                 7                  9                  65
    Alloy rod                  338                114                462
    Alloy sheet and strip      66                 123                443
    Alloy tube                 14                 8                  110
    Castings                   142                58                 292

    Totals                     1363               4909               2055

    a    Based on figures from the USA, western Europe and Japan (about 75%
         of world consumption of 11 090 000 tonnes) (Marco, 1989)

         In agriculture, copper compounds, especially copper sulfate, are
    used as fungicides, pesticides, algicides, nutritional supplements in
    animal feeds, and fertilizers.  Copper fungicides are used to treat
    foliage, seeds, wood, fabric and leather as a protectant against
    blights, downy mildews and rusts (ATSDR, 1990).  One of the principle
    mixtures used to treat foliage for mildew and fungal infections is the
    Bordeaux mixture used to spray vines which typically contains 0.05-2%
    copper neutralized with soda lime (Pimentel & Marques, 1969).  Copper
    sulfate is used throughout the world to kill and inhibit the growth of
    algae in municipal reservoirs, irrigation equipment and piping,
    swimming pools and industrial cooling systems.  It is also used in
    animal feed additives and growth promoters, as well as for disease
    control in livestock and poultry (Grant et al., 1990).

         Copper enjoys limited use in human and veterinary medicine,
    having been largely replaced by other compounds and treatments.
    Copper is, however, a major constituent of many of the metallic
    amalgams (e.g. mercury amalgams) used in dentistry.  It is also used
    to prepare intrauterine devices (IUDs).


    4.1  Transport and distribution between media

         The information reviewed in this section describes the environ
    mental fate of copper.  The factors affecting the distribution of
    copper in air, water, sediment and soil are first described.  This is
    followed by a review of the factors influencing the bioaccumulation of
    copper.  This review is not intended to be exhaustive but rather to
    present selected representative papers.

    4.1.1  Air

         Copper is released to the atmosphere in the form of particulate
    matter or adsorbed to particulate matter.  It is removed by
    gravitational settling (bulk deposition), dry deposition (inertial
    impaction characterized by a deposition velocity), washout by rain
    (attachment to droplets within clouds), and rainout (scrubbing action
    below clouds) (Schroeder et al., 1987).  Removal rate and distance
    travelled from the source depend on source characteristics, particle
    size and wind velocity.  Gravitational settling governs the removal of
    large particles (> 5 µm), whereas smaller particles are removed by
    other forms of dry and wet deposition.  The relative importance of wet
    as compared to dry deposition generally increases with decreasing
    particle size (ATSDR, 1990).

         Chakrabarti et al. (1993) analysed samples of rainwater (pH 5.3)
    and snow (pH 4.7) in Canada; the total copper concentrations were 30.3
    µg/litre in the rainwater and 24.6 µg/litre in the snow.  In the
    rainwater sample 98.3% of the copper was in the soluble phase (< 0.45
    µm) and 1.7% in the particulate phase (> 0.45 µm) whereas in the snow
    sample 80.5% was found in the particulate phase and 4.8% in the
    soluble phase.  Another snow sample (pH 3.9) was analysed and revealed
    a copper concentration of 5.7 µg/litre with 4.7 µg/litre in the
    soluble phase and 1.08 µg/litre in the particulate phase.  Kinetic
    results suggested that the copper in the snow sample was probably
    bound to different sites having different bonding energies in
    polyfunctional complexing agents.  Four different copper species
    having different dissociation rate constants were observed
    (3.1 × 10-2, 1.6 × 10-3, 6.2 × 10-5 and 8.8 × 10-6/s).  Cheng et al.
    (1994) found that the distribution of copper species in rainwater
    collected in Ottawa, Canada, was very similar to that in the
    previously reported snow sample.  The rainwater sample contained 7.10
    µg Cu/litre of which 2.03 µg/litre was in the particulate phase and
    5.07 µg/litre in the soluble phase (< 0.45 µm).  The scavenging ratio
    of the copper concentration in precipitation (mg/litre) to air
    concentrations (µg/m3) for large particles displays a seasonal
    variation reflecting the more effective scavenging of snow compared
    with rain (Chan et al., 1986).

         There is large temporal and spatial variability in copper
    deposition.  Schroeder et al. (1987)  reviewed deposition rates and
    washout ratios for copper.  Copper deposition rates in urban areas

    were estimated to be 0.119 and 0.164 kg Cu/ha per year for dry and wet
    deposition, respectively.  Bulk deposition was reported to range from
    0.002 to 3.01 kg Cu/ha per year.  In rural areas bulk deposition was
    reported to range from 0.018 to 0.5 kg Cu/ha per year and wet
    deposition was 0.033 kg Cu/ha per year.  The washout ratio is
    114 000-612 000 (µg Cu/m3 rain)/(µg Cu/m3 air) [(140-751 µg Cu/kg
    rain)/(µg Cu/kg air)].

         Ottley & Harrison (1993) calculated the dry deposition flux of
    copper to the North Sea to be 350 tonnes Cu/year.  Migon et al. (1991)
    studied the input of copper through rainfall and dry deposition to the
    Ligurian Sea (Mediterranean) over a period of two years.  The total
    flux was calculated to be 1.85 kg Cu/km2 per year.  A mean yearly
    atmospheric input for copper was calculated at 98 tonnes.  Fergusson &
    Stewart (1992) estimated deposition flux for copper in the insoluble
    component of bulk deposition derived from Christchurch city, New
    Zealand.  Copper fluxes followed approximately exponential decay
    curves away from the city.  Deposition rates varied from 0.83 µg
    Cu/m2 per day (a remote site) to 21 µg Cu/m2 per day (an inner city
    site).  In the city and nearby rural areas soil is not a major source
    of atmospheric copper, whereas at remote sites atmospheric copper is
    mostly soil-derived.

         The atmospheric wet deposition of copper at Chesapeake Bay, USA,
    was examined during 1990 and 1991.  The monthly integrated atmospheric
    fluxes exhibited a high degree of spatial and temporal variability.
    The arithmetically averaged annual wet flux was 260 µg Cu/m2
    (Scudlark et al., 1994), and this was derived predominantly from
    anthropogenic sources.  Wu et al. (1994) calculated the dry deposition
    flux for Chesapeake Bay to be 290-810 µm Cu/m2 per year.  Dry
    deposition fluxes for Lake Michigan were estimated at 690 and 800 µm
    Cu/m2 per year.

         Migon (1993) compared riverine and atmospheric inputs of copper
    with the Ligurian Sea (Mediterranean).  Atmospheric inputs were found
    to be higher, with a ratio of 16.3 to 32.6.

         Chan et al. (1986) reported that in southern Ontario, Canada
    during 1982, the mean concentration of copper in precipitation was
    1.57 µg Cu/litre of which 1.36 mg Cu/m2 was from wet deposition.  The
    mean concentrations of copper in precipitation were 1.36 and 1.58 µg
    Cu/litre for central and northern Ontario, respectively.  In both
    areas the annual wet deposition averaged 1.13 mg Cu/m2.

         Remoudaki et al. (1991) calculated the seasonal copper
    atmospheric deposition to the western Mediterranean.  Atmospheric
    deposition of copper during the wet season ranged from 0.0004 to
    0.0005 µg Cu/cm2 per day and during the dry season 0.0007 to 0.0014
    µg Cu/cm2 per day.

         Gorzelska (1989) analysed snowpack samples from 18 sites in the
    vicinity of Inuvik, Canada during 1985 and 1986.  Copper
    concentrations ranged from 0.1 µg Cu/kg 20 km north of the town to

    0.54 µg Cu/kg near a power plant.  In all the samples the trace metals
    were enriched with respect to crustal material.  Mass balance
    calculations have shown that most of the copper emitted by the local
    sources is transported outside the immediate vicinity of the town.

    4.1.2  Water and sediment

         Several processes influence the fate of copper in aquatic
    systems.  These include complexation to inorganic and organic ligands,
    sorption to metal oxides, clays, and particulate organic material,
    bioaccumulation and exchange between sediment and water (Stiff, 1971;
    Callahan et al., 1979).

         Much of the copper discharged to water is in particulate form and
    tends to settle out, precipitate out or be adsorbed by organic matter,
    hydrous iron, manganese oxides and clay in the sediment or water
    column. Equilibrium is normally reached within 24 h.  Copper
    discharged into a river leading into Chesapeake Bay contained 53 µg
    Cu/litre, of which 36 µg/litre was in the form of settleable solids
    (Helz et al., 1975). The concentration of copper 2-3 km downstream
    from the outfall had fallen to 7 µg/litre.  Copper in particulate form
    includes precipitates, insoluble organic complexes and copper adsorbed
    to clay and other mineral solids (Stiff, 1971).

         Owing to unacceptable past practices, Macquarie Harbour on the
    west coast of Tasmania, Australia contains dissolved copper levels as
    high as 560 µg/litre as a result of riverine transport in dissolved
    and particulate forms from the Mount Lyell copper mine (Carbon, 1996).
    Some 97 × 106 tonnes of mine tailings and 1.4 × 106 tonnes of slag
    were deposited into the Queen and King river system over a 78-year
    period before closure of the mine.

         The copper(I) ion is unstable in aqueous solution, tending to
    disproportionate to copper(II) and copper metal unless a stabilizing
    ligand is present (Callahan et al., 1979).  The only cuprous compounds
    stable in water are insoluble ones such as the sulfide, cyanide and
    fluoride.  In its copper(II) state, copper forms coordination
    compounds or complexes with both inorganic and organic ligands.
    Ammonia and chloride ions are examples of species that form stable
    ligands with copper.  Copper also forms stable complexes with organic
    ligands such as humic acids.  In seawater, organic matter is generally
    the most important complexing agent.  Samples collected from the
    surface waters (< 200 m) of the northeast Pacific revealed that over
    99.7% of the total dissolved copper was associated with organically
    complexed forms.  At depths of 1000 m approximately 50-70% of the
    copper was in the organically complexed form.  Copper complexation
    gave rise to very low cupric ion activities in surface waters, around
    1 pg Cu2+/litre.  The authors reported that two classes of
    copper-binding ligands were identified: an extremely strong ligand at
    low concentrations dominated in surface waters and a weaker class of
    ligand at higher concentrations was found throughout the water column
    (Coale & Bruland, 1988).

         Tan et al. (1988) collected freshwater river samples from the
    Linggi river basin, Malaysia.  Samples were separated into colloidal
    fractions and soluble fractions.  Soluble fractions were classified
    according to the lability of the copper forms in the water.
    Categories range from very labile (e.g. free metal ion) to nonlabile
    (e.g. colloidally bound metal).  In this study 18-70% of the dissolved
    copper was moderately labile and 13-30% was slowly labile.

         Copper in the fresh and estuarine waters of the Cochin estuary,
    India, was found to be extensively associated with organic colloidal
    matter.  The relationship between exchangeable and total particulate
    copper did not show a significant correlation during the study,
    emphasizing the role of lattice-incorporated copper as distinct from
    particulate scavenged/adsorbed exchangeable copper (Shibu et al.,

         A detailed study of the Tamar estuary, United Kingdom, revealed a
    decrease in the alpha-coefficient for complexation of Cu2+ by natural
    organic ligands (log alpha CuL) from 10.8 to 8.3 with increasing
    salinity, demonstrating that major cations compete with copper for the
    complexing sites.  The free Cu2+ concentrations were very low (16.2
    < pCu(II) < 18.2) throughout the estuary even though the total
    dissolved copper concentrations were high (up to 300 nmol/litre),
    probably because of complexation to dissolved organic complex (Van den
    Berg et al., 1990).

         Giesy et al. (1986) isolated dissolved organic carbon from nine
    surface waters in the southeastern USA and found that the binding of
    copper by humate occurs with different strengths at a number of sites,
    the binding strength at the sites varying by two orders of magnitude,
    dependent on the ratio of copper to total organic ligand.

         Organic compounds form complexes with 94-98% of dissolved copper
    in the surface waters of the North Sea.  In all samples strong
    copper-chelating compounds were found at concentrations of 4-10 µg
    Cu/litre (60-150 nmol/litre).  The major inorganic complexes in the
    seawater samples were CuCO30 (60%), CuOH+ (16%) and Cu(OH)20
    (16%) (Van den Berg, 1984).

         Mackey & Higgins (1988) found that the strong copper-complexing
    capacity of seawater can vary by more than three orders of magnitude.
    Copper-complexing capacity was related to the phytoplankton biomass.
    High values were associated with high phytoplankton mass, whereas when
    the biomass was low the copper-complexing capacity was also low.  The
    authors found that in nutrient-limiting, oligotrophic waters of low
    average productivity the copper-complexing capacity was variable.

         Midorikawa et al. (1992) identified three classes of natural
    organic ligands in coastal seawater classified by differences in their
    complexing abilities for copper.

         Gardner & Ravenscroft (1991) studied the behaviour of copper
    complexation in rivers and estuaries of northeast England.  They found
    that copper speciation in rivers and estuaries is dominated by organic
    complexation.  The authors found a mixture of ligands of different
    affinities for copper in natural waters.  The complexation of copper
    discharged to rivers and estuaries occurred very rapidly. Complexation
    capacities were consistently in the range 10-25 µg Cu/litre (150-400
    nmol/litre).  The copper-complexing capacity of Linggi river water
    (Malaysia) was in the range 26-74 µg Cu/litre (410-1160 nmol/litre)
    (Tan et al., 1988).

         Sharma & Millero (1988) measured the oxidation of copper(I) in
    air-saturated solutions of seawater as a function of pH (5.3-8.6),
    temperature (5-45 °C) and salinity (5-44%).  The rate of reaction
    increased with pH and temperature, and decreased with salinity (ionic
    strength).  The results indicate that the rates are controlled by the
    concentration of Mg2+, Ca2+, Cl- and HCO3- through complex
    formation and ligand exchange.

         Bradley & Cox (1988) found that 80% of the measurable copper in
    standard river sediment SRM 1645 was in the organic fraction.  In
    Yamuna river sediments, India, copper is mainly associated with the
    organic matter owing to its high complexing tendency for organic
    matter.  A high percentage of copper is also found in the residual
    fraction, and much lower concentrations are associated with the
    carbonate and iron-manganese oxide phases (Gadh et al., 1993).

         Calmano et al. (1993) studied the mobilization of copper from
    contaminated sediments.  The dominant mobilizing factor was pH with
    mobilization increasing with increasing acidity.  At pH values
    of < 4.5 there was a strong influence of pH on mobilization.  At
    identical pH values the mobilized portions of copper from the oxic
    sediment are tenfold higher than those from anoxic sediment.

         Samanidou & Fytianos (1990) estimated a mobilization of 10-15% of
    copper due to NTA and EDTA in two rivers in northern Greece, with no
    consideration of the biodegradation of metal complexes.  Samanidou et
    al. (1991) estimated that humic substances (~2-3 mg/litre) were able
    to cause the long-term release of 70-80% of copper in the same rivers.
    In experimental studies copper was remobilized by synthetic complexing
    agents more readily than other metals tested (cadmium, lead, manganese
    and chromium).

    4.1.3  Soil

         In the terrestrial environment, a number of important factors
    influence the fate of copper in the soil.  These include the nature of
    the soil itself, its pH, the type and distribution of organic matter,
    the soil redox potential, the presence of oxides, the base status of
    the soil and its cation exchange capacity (CEC), the rate of litter
    decomposition and the proportions of clay to silt to sand particles.
    The residence time of copper in the soil is also a function of overall
    climate and of the vegetation present at a site.

         Most copper deposited on soil from the atmosphere, from
    agricultural applications and from sewage sludge amendments is
    strongly adsorbed to the upper few centimetres of the soil. It is
    especially bound to the organic matter, as well as being adsorbed by
    carbonate minerals and hydrous iron and manganese oxides.  Copper
    binds more strongly than most other metals and is less influenced by
    pH as a result.  The greatest amount of leaching of copper occurs from
    sandy soils, compared with clays and peats, whereas acidic conditions
    favour copper leaching to the groundwater from the soil.

         Lehmann & Harter (1984) studied the kinetics of copper desorption
    from the A horizon of Paxton soil (surface soil), USA, following
    addition of copper at rates ranging from 100 to 500 mg/kg.  When 500
    mg Cu/kg is added to this soil, about 94% is adsorbed within 15 min.
    The copper appears to be preferentially adsorbed to high energy sites.
    It appears that this soil is capable of retaining about 100 mg Cu/kg
    on high-energy bonding sites.  If the copper is present in excess of
    the high energy sites, the surplus fills low-energy sites.  This more
    loosely bonded fraction continues to react for several hours.  After 1
    day this latter process reaches equilibrium, although the soil
    continues to adsorb copper very slowly from solution for up to 4 days.

         Assaad & Nielsen (1984) studied the adsorption of copper in three
    Danish soil types (two orthic luvisols and a eutric fluvisol).  The
    Langmuir adsorption equation was found to be the best to describe
    copper adsorption in these soils. Copper adsorption increased with
    increasing soil pH (pH 4.91-8.48) and decreased with increasing
    temperature (5-25 °C).

         Petruzzelli et al. (1988) found that fly ash (10%) and humic acid
    (1%) increased the adsorption of copper (up to 100 µg/ml) in histosol.
    The addition of sewage sludge to a sandy loam soil increased the
    sorption of copper solutions of differing concentrations (0.1-1.5 µmol
    Cu/cm3).  The authors suggested that new adsorbing sites become
    available on the solid phase of the soil following "low metal" sludge
    addition (Petruzzelli et al., 1994).

         King (1988) incubated 13 soil types (10 mineral and 3 organic)
    collected from the southeastern USA with 70 mg Cu/kg for 6 days.  The
    amount of copper adsorbed ranged from 36% to 100%.  Removal of copper
    from solution was much higher in surface soils than in subsurface
    sandy soils.  Nonexchangeable copper was relatively high (up to 100%)
    in all but some of the acid subsoils.  In the B and C horizons 96% of
    the variation in sorbed copper was explained by pH, whereas copper in
    the A horizon (surface soil) was unaffected by pH.  The soil/water
    partition coefficient for copper was > 64 for mineral soils and 403
    for organic soils.

         Elliott et al. (1986) studied pH-dependent adsorption of copper,
    cadmium, zinc and lead on to four soils with differing chemical
    properties.  Copper and lead were more strongly retained under acidic
    conditions (pH 5.0) than cadmium and zinc.  Adsorption increased with

    pH (pH 3-5).  The removal of organic matter from the soils
    substantially reduced the adsorption of copper.

         Sanders & McGrath (1988) studied the extent of copper complex
    formation by soluble organic matter extracted from an organic soil, a
    clay and two sandy loams.  Copper was extensively complexed in these
    solutions.  The percentage of copper existing as Cu2+ fell as the pH
    increased, and also fell as the total copper concentration decreased.
    Weight for weight, organic matter from the sandy loams was most
    effective at forming complexes with copper within the experimental pH
    range (pH 4-7) followed by the organic soil and then the clay.

         Allard et al. (1991) studied the distribution of copper within an
    illitic clay formation beneath an old (approx. 150 years) deposit of
    sulfidic mine tailings.  The adsorption in the lower pH range had
    little impact on the mobility of copper: at pH levels in excess of 5,
    copper is immobilized.  The results suggest that transport of copper
    originating from the tailings is diffusion controlled.

         Tyler & McBride (1982) studied the relative mobility of copper
    added to several mineral and organic soils and the simultaneous
    desorption and leaching of metals determined by eluting soil columns
    with 0.01 mol/litre calcium chloride.  Copper was eluted much more
    slowly and in much smaller quantities than zinc, cadmium or nickel.

         Berggren (1992) studied the factors affecting the mobilization of
    copper in spruce, beech and birch forest soil profiles (podzols and
    cambisols) at two sites in Sweden.  At a depth of 15 cm almost all of
    the copper was found to be organically bound.  The results also
    indicate that organically-complexed copper constituted the predominant
    copper form in soil solutions at 50 cm despite the relatively low
    dissolved organic carbon (3-14 mg/litre) and the highly
    aluminium-saturated organic compounds.

         Strain et al. (1984) studied the leaching of copper by simulated
    "acid" rain (pH 2.8-4.2) applied in rainwater to soil from Swedish
    spruce forest polluted by a brass mill.  Leaching of copper increased
    considerably when water at pH < 3.4 was applied to the soil.

         Campanella et al. (1989) found that UV (mercury lamp) irradiation
    of urban sludge resulted in an increased mobility of copper eluted
    with sulfuric acid; this was attributed to degradation of organic
    matter through radical reactions which provoked the formation of
    smaller molecules acting as more soluble metal carriers.

         Wong et al. (1993) found that a copper(II)-accumulating bacterial
    strain  (Pseudomonas putida II-11) isolated from electroplating
    effluent removed a significantly high amount of copper(II) from growth
    medium and buffer.  The adsorption was pH dependent with a maximum at
    pH 8.0.

         Groudev & Groudeva (1993) studied the microflora of four
    industrial copper dump leaching operations.  It was found that copper
    solubilization depended mainly on the amount and activity of the
    mesophyllic acidophilic chemolithotrophic bacteria which occurred in
    the ore dumps.

    4.1.4  Sewage sludge inputs to land

         Land treatment is increasingly being utilized as a method of
    waste disposal for sewage effluent and sludge.  The intent is to
    combine the benefits of fertilizer effects and organic additions to
    soils, with safe land disposal of the large quantities of domestic
    sewage being generated (Brown et al., 1983; Juste & Mench, 1992; Henry
    & Harrison, 1992).  Sewage effluent and sludges vary greatly in their
    content of metals and especially when domestic sewage is not separated
    from industrial sources the metal levels can be high (e.g. for
    chromium, copper, zinc, nickel, cadmium) and can pose potential
    hazards as a result of metal accumulation if applied to land at high
    rates over the long term.  There are a number of sources of copper in
    sewage effluent and sludge including human excreta, from the corrosion
    of copper pipes in domestic water supplies and from direct additions
    from industrial processes.  In view of the recent interest in the
    sustainability of agricultural land focus has been on the potential of
    land treatment to cause elevated and toxic levels in the soils.
    Present national and regional guidelines are aimed at protecting such
    amended land into the future (Table 4).

         Copper concentrations in sewage sludge vary greatly. For example,
    Hedberg et al. (1996) quote copper concentrations from 0 to 16 000
    mg/kg per day sludge for Finland, with a median value of 214 mg Cu/kg.
    In nine different sewage districts in Norway the levels in sludge
    varied from 100 to 500 mg Cu/kg d.s.  For this Norwegian data set,
    there was a relationship between the copper content in the sewage
    sludge and the pH of the drinking-water.  The average copper content
    in the sludge was 140 mg Cu/kg d.s. for those drinking-water plants
    with pH adjustments (pH increased to 8-8.5) while the average  copper
    content in the sewage sludge which had received water without pH
    adjustments was 320 mg Cu/kg d.s.  Attempts to reduce the corrosivity
    of piped water supplies can lead to changes in the copper (and iron)
    in sewage sludge.

         Copper, like other metals applied to land by sludge or effluent
    amendments, is rather strongly adsorbed in the upper surfaces,
    especially by organic matter, for prolonged periods.  It is already
    organically bound and, upon release by respiratory breakdown, is then
    re-absorbed.  Juste & Mench (1992) examined the long-term effects of
    sewage sludge applications (10 years or more in duration) on metal
    distribution in the soil profile as well as crop responses and metal
    uptake from field trials in the EC and the USA.  In almost all cases,
    sludge-borne metals appeared to remain in the zone of sludge
    incorporation to soils (0-15 cm).  Mass balances on metal recoveries
    from soil additions ranged from 30% to 90%.  Lateral soil movement was
    the main explanation of the progressive disappearance of metal from

        Table 4.  Directives for maximum allowed metal concentrations in sewage sludge
    used as a soil improvement agent in agriculture (From: Hedberg et al., 1996)

    Country/             Maximum allowed metal concentration (mg/kg dry weight)

                         Copper          Zinc           Lead          Cadmium
    EUa                  1000-1750       2500-4000      750-1200      20-40
    Denmark              1000            4000           120           0.8
    Germany              800             2500           900           10
    Finland              600             1500           100           1.5
    France               1000            3000           800           20
    Netherlands          75              300            100           1.25
    Norwaya              1000-1500       1500-3000      100-300       4-10
    Sweden               600             800            100           2
    USA (EPA)            1500-4300       2800           300-840       89

    a  The higher level is valid for application on greenlands

    experimental plots.  Copper was a good deal less bioavailable to crops
    from sludge amendments than cadmium, nickel and zinc, but somewhat
    more mobile and bioavailable than lead.

         In forest soils the retentivity of copper in the profile may be
    even greater from sludge amendments than in agriculture systems.  For
    example, Zabowski & Zasoski (1987) equilibrated three soil horizons
    (A, B2 and C) of an acidic forest soil with copper solutions in the
    presence and absence of municipal sewage sludge leachate.  Copper
    binding to the soils in each of the three horizons was greater than
    that of cadmium or zinc.  Sludge leachate reduced copper adsorption in
    all three horizons.

         In the great majority of sludge metal studies done to date,
    although copper is a constituent of the sludge, it is very rarely the
    element which imposes the limits for addition of sludges or sewage
    effluent to land.

    4.1.5  Biodegradation and abiotic degradation

         Copper is transformed in the environment to forms that are either
    more or less bioavailable, depending upon the physical and chemical
    conditions present in the environment of interest.  For information on
    the speciation of copper, see section 2.4.

    4.2  Bioaccumulation

         Bioaccumulation is defined as the net uptake of copper by
    microorganisms, plants or animals from their surrounding environment
    (water, sediment, soil and diet).  The species of copper present in
    environmental media and its associated bioavailability, together with
    differences in plant and animal uptake and excretion rates, determine
    the extent of bioaccumulation.  For aquatic organisms bioconcentration
    refers specifically to water.

    4.2.1  Microorganisms

         Sahoo et al. (1992) found that a bacterial  (Bacillus circulans)
    biomass of 1.48-1.52 g/litre (dry weight) removed 80% of copper in a
    495 mg Cu/litre solution.  A reduction of the pH was detrimental to
    the accumulating capacity of the bacteria.

         Bengtsson et al. (1983) grew the hyphomycete (fungus)
     Verticillium bulbillosum in agar containing 15, 45 or 150 mg
    Cu/litre for one week.  Mean copper concentrations in the mycelium
    were, respectively, 1296, 2608 and 3245 mg/kg for the three exposure

    4.2.2  Aquatic plants

         Bioaccumulation factors have been calculated for over 20 species
    of marine macroalgae showing maximum values up to 27 000, depending on
    the exposure concentration (Bryan & Hummerstone, 1973;  Phillips,
    1977; Malea et al., 1994; Correa et al., 1996).

         Hall et al. (1979) found that a nontolerant strain of the brown
    alga  Ectocarpus siliculosus exposed to various copper concentrations
    (up to 250 µg/litre) displayed higher accumulation values than did a
    tolerant strain.  At 72 h incubation, the tolerant strain accumulated
    mean copper values of 20 mg/kg (wet weight) with no added copper and
    234 µg/kg at 250 µg Cu/litre in the medium (Hall, 1981).  The same
    strain incubated for 14 days displayed accumulation values of 13 mg/kg
    with no added copper and 1075 mg/kg at 250 µg Cu/litre in the medium.
    Reed & Moffat (1983) exposed the green alga  Enteromorpha compressa 
    to copper concentrations of up to 610 µg/litre (9.6 µmol/litre) for 6
    days.  Copper accumulation was linearly dependent on the exposure
    concentration and the pattern was similar in both the tolerant and
    non-tolerant strains.  Mean maximum concentrations in the algae were
    22.2 mg Cu/kg (0.35 µmol/g) (fresh weight) for the nontolerant strain
    and 25.4 mg Cu/kg (0.4 µmol/g) for the tolerant strain.  Equilibrium
    was not reached within the experimental time period.

         Mersch et al. (1993) maintained the aquatic moss
     Rhynchostegium riparoides in water containing copper levels ranging
    from 4.5 to 50 µg/litre for 27 days.  Accumulation was rapid and
    reached a plateau after 18 days.  At the end of the 14-day depuration
    phase the moss had lost 50% of the accumulated copper.  Claveri et al.
    (1994) studied the uptake of copper (5-342 µg/litre) by

     R. riparoides  for periods of up to 168 h.  The accumulation of
    copper occurred predominantly during the initial 96 h and had reached
    equilibrium within 168 h.  Copper concentrations in the mosses ranged
    from 30 to 2500 mg/kg (dry weight). During the 10 day depuration
    period there was a rapid decrease in copper levels during the first 72
    h after which copper concentrations in the mosses approached
    equilibrium values ranging from 32 to 700 mg/kg (dry weight).

         Sinha & Chandra (1990) studied the accumulation of copper
    (0.05-5.0 mg/litre) by the aquatic plant  Bacopa monnieri for 168
    days.  Accumulation was directly related to the exposure
    concentration.  Copper concentrations in shoots ranged from 20 to 721
    mg/kg (dry weight) and in roots from 195 to 3821 mg/kg.

         The uptake of copper by duckweed  (Lemna minor) and water velvet
     (Azolla pinnata) was investigated by Jain et al. (1989).  Plants
    were grown in copper solutions of 1, 2, 4 or 8 mg/litre under static
    renewal conditions for 14 days.  Copper concentrations in the plants
    ranged from 979 to 6714 mg/kg (dry weight) for duckweed, and from 1159
    to 7725 mg/kg for water velvet.  Uptake rate was highest at the lower
    exposure concentrations; concentration factors ranged from 51 to 60
    for duckweed, and from 58 to 66 for water velvet.  Dirilgen & Inel
    (1994) grew duckweed  (Lemna minor) in Jacob nutrient medium at
    copper concentrations ranging from 0.23 to 2.03 mg/litre for 7 days.
    Bioconcentration factors, based on copper content of plants on a dry
    weight basis, were 1447, 444 and 314 at copper concentrations of 0.23,
    1.03 and 2.03 mg/litre, respectively.

         Kay et al. (1984) exposed water hyacinths
     (Eichhornia crassipes) to copper (0.5-5.0 mg/litre) for 6 weeks.  At
    the highest copper concentration levels in leaves, stems roots and
    dead tissue were 321, 710, 8160 and 5151 mg/kg (dry weight)
    respectively; bioconcentration factors ranged from 64 to 1632.  Nor &
    Cheng (1986) grew water hyacinths in 2 mg/litre copper solutions.
    Fulvic acid (10-50 mg/litre) did not affect the uptake of copper by
     Eichornia; however, humic acid (20 and 50 mg/litre) strongly
    inhibited copper uptake.  In the absence of ligands  Eichornia 
    accumulated 204 and 2451 mg/kg (dry weight) from copper solutions of 1
    and 10 mg/litre, respectively.

    4.2.3  Aquatic invertebrates

         Hansen et al. (1995) exposed the marine demosponge
     Halichondria panicea to dissolved copper concentrations ranging from
    0.45 (control) to 1000 µg/litre for 14 days.  The sponge accumulated
    copper in direct proportion to the concentration of the dissolved
    metal in the surrounding medium.  Final body copper concentrations
    were 236 and 818 mg/kg (dry weight) at exposure concentrations of 300
    and 1000 µg dissolved Cu/litre, respectively.  There was no
    significant loss of copper during an 8 day depuration period.  The
    authors proposed this species as a suitable biomonitoring organism.

         Elliott et al. (1985) found that the marine mussel
     Mytilus edulis exposed either continually, or in a 2 day cycle, to
    copper (10 µg/litre) exhibited a linear accumulation over a 40 day
    period.  Mussels exposed under cycled conditions showed a lower rate
    of accumulation.  Copper accumulation was not in direct proportion to
    the time exposed to the elevated concentration.  The presence of
    cadmium reduced the accumulation factor by 50%.

         Holwerda (1991) exposed freshwater clams  (Anodonta cygnea) to
    copper (47 µg/litre) for 6.5 weeks.  An accumulation factor of 55 was
    calculated for the exposure period.  Crecelius et al. (1982) exposed
    clams  (Macoma inquinata) and shrimps  (Pandalus danae) to copper
    concentrations ranging from 5 to 30 µg/litre for one month.  Body
    burdens ranged from 25 to 97 mg Cu/kg (dry weight) for clams and from
    146 to 322 mg Cu/kg for shrimps.  Ageing of the solutions prior to
    exposure reduced the bioavailability of copper.  In a static system
    with added sediment more than 50% of the added Cu2+ became bound to
    the organic fraction of the sediment and was unavailable to
    suspension-feeding clams  (Protothaca staminea); however,
    deposit-feeding clams  (Macoma inquinata) placed in the sediment
    doubled their copper body burden within 2 months.

         Biological half-lives for depuration of copper from "green"
    oysters  (Crassostrea gigas) and mussels  (Mytilus smarangdium) from
    a copper-contaminated area, and "normal" oysters were 11.6, 6.4 and
    25.1 days, respectively (Han et al., 1993).

         Rainbow & White (1989) exposed decapods  (Palaemon elegans), 
    amphipods  (Echinogammarus pirloti) and barnacles
     (Elminius modestus) to copper at concentrations ranging from 31.62
    to 3162 µg/litre for 28 days.  Whole-body copper levels (129.3 mg/kg)
    are regulated in the decapods at exposures up to and including 100
    µg/litre and at higher exposures there is net accumulation.  In
    amphipods and barnacles there was net accumulation of copper at all
    exposures with no apparent regulation of copper levels.

         Weeks & Rainbow (1991) exposed the talitrid amphipods
     Orchestia gammarellus and  O. mediterranea to copper concentrations
    ranging from 31.6 to 3162 µg/litre for 21 days.  Mean rates of copper
    accumulation (measured as net accumulation of total copper) ranged
    from 0.9 to 77.0 µg/g per day for  O. gammarellus in a dose-related
    manner; rates of accumulation in  O. mediterranea ranged from 1.19 to
    28.1 µg/g per day showing an increase with copper exposure at
    concentrations < 100 µg/litre. Weeks & Rainbow (1993) fed the
    talitrid amphipods  O. gammarellus and  O. mediterranea on discs of
    algae treated with copper (16.3-2070 mg/kg) for 21 days.
     O. gammarellus  accumulated whole-body copper concentrations
    ranging from 104 to 163 mg/kg; haemolymph concentrations ranged from
    525 to 677 mg/kg (dry weight). Rates of accumulation ranged from 0.52
    to 4.71 µg/g per day, increasing with increasing copper exposure. The
    rates of accumulation for  O. mediterranea remained fairly constant
    at all exposure concentrations (0.28-0.37 µg/g per day) except the
    highest (1.61 µg/g per day).  It was concluded that for

     O. gammarellus  accumulation of copper from food was a more important
    route than accumulation of copper from solution.   O. mediterranea
    was unable to satisfy its copper requirements from a food source but
    was able to do so from solution.

         Weeks et al. (1993) exposed shore crabs  (Carcinus maenus) to
    750 µg Cu/litre for up to 7 days at various salinities.  Copper
    accumulated in the gills and midgut gland but not in muscle.  The
    accumulation of copper in gill tissue was positively correlated with

         Ozoh (1994) exposed ragworms  (Hediste diversicolor) to a copper
    concentration of 200 µg/litre for up to 15 days.  At 12 °C, low
    salinity (7.5%) increased the availability of copper to the worms and
    more copper was accumulated, copper concentrations ranging from 83.27
    to 183.12 mg/kg (dry weight).  Increasing salinities of 15.25 and
    30.5% reduced the accumulation of copper.  At 17 and 22 °C more copper
    was accumulated than at 12 °C, with copper concentrations ranging from
    58.7 to 784 mg/kg.  The addition of sediment to the test system
    reduced the accumulation of copper by the worms (Ozoh, 1992b).

         Zia & Alikhan (1989) found that crayfish  (Cambarus bartoni) 
    accumulated copper concentrations ranging from 130 to 296 mg/kg after
    exposure to copper concentrations ranging from 125 to 500 µg/litre for
    4 weeks.  Copper was predominantly accumulated in the gills and

         Winner (1984) exposed  Daphnia magna to copper (30 µg/litre) for
    7 days; during this period daphnids accumulated whole-body copper
    residues of 70.7 mg/kg (dry weight).  The addition of 0.75 mg humic
    acid/litre had no significant effect on the accumulation of copper.

         Giesy et al. (1983) found that the presence of organic matter
    decreased the accumulation of copper by the softwater cladoceran
     Simocephalus serrulatus.  When bioconcentration factors (BCF) were
    calculated using Cu2+ the BCFs were similar for the different water
    types tested, while when based on total copper concentrations they
    varied greatly owing to varying amounts of organic matter.  The
    authors concluded that most of the copper accumulated by this species
    was Cu2+ or the labile aquatic forms and that a decrease in Cu2+ due
    to binding of copper by organic matter reduced accumulation.

         Vogt & Quinitio (1994) exposed juvenile giant tiger prawns
     (Penaeus monodon) to 1 mg Cu/litre for 10 days.  Copper deposition
    was investigated by histochemistry and electron microscopy.  Copper
    granules were accumulated in large quantities in the hepatopancreas
    tubules, the amount and size of the granules increasing along the
    tubules in relation to the cells' age.  The granules were released by
    discharge of senescent hepatopancreas cells and were added to the

         Timmermans & Walker (1989) exposed fourth instar larvae of the
    midge  Chironomus riparius to copper (50 or 100 µg/litre).  Larvae

    accumulated copper with increasing levels of exposure, but very small
    amounts were recovered in pupae or imagines. Average body burdens were
    approximately 425 and 750 ng copper, respectively, for the two

         Dodge & Theis (1979) reported that copper (85 or 325 µg/litre)
    was accumulated from solutions by midge larvae  (Chironomus tentans) 
    in which the dominant aqueous forms were free Cu2+ ion and a copper
    hydroxy complex reaching concentrations in excess of 200 mg/kg (dry
    weight).  No significant uptake was observed when copper-glycine and
    copper-NTA complexes were dominant.

    4.2.4  Fish

         Peres & Pihan (1991b) exposed carp  (Cyprinus carpio) for up to
    3 weeks to copper concentrations of 20, 40 and 120 µg/litre at water
    hardnesses of 50, 100 and 300 mg CaCO3/litre, respectively.  Accumu
    lation in gills after 3 weeks was 53, 58 and 78 mg/kg dry weight for
    the three exposure conditions, compared to 13 mg/kg initially.

         Daramola & Oladimeji (1989) exposed the freshwater fish
     Clarius anguillaris and  Oreochromis niloticus to copper for 8
    weeks.  For  C. anguillaris, whole body accumulation was 15.7, 21.8
    and 31.2 µg Cu/g dry weight for exposure concentrations of 27, 55 and
    110 µg Cu/litre, compared to 6.9 µg Cu/g in control fish. For
     O. niloticus, accumulation was 34.7, 36.1 and 81.0 at exposures of
    0.05, 0.10 and 0.20 µg Cu/litre, respectively, compared to 17.6 µg
    Cu/g in controls.

         Playle et al. (1992) studied the accumulation of copper (16
    µg/litre) on the gill of fathead minnow  (Pimephales promelas) 
    exposed for 2-3 h.  The addition of Ca2+ (2100 or 4200 µeq/litre)
    reduced gill copper accumulation during exposures at pH 4.8 but not at
    pH 6.3.  EDTA eliminated copper deposition at both pH levels when
    equimolar with copper, but reduced copper deposition by 50% when half
    equimolar at pH 4.8.  The authors concluded that copper accumulation
    on the fish gills was reduced by Ca2+ and H+ competition at the
    gill surface, and by EDTA complexation of copper in the ambient water.

         Buckley et al. (1982) exposed coho salmon
     (Oncorhynchus kisutch) to copper at concentrations of 70 and 140
    µg/litre for 15 weeks.  Copper accumulation in liver was greatly
    elevated, averaging approximately 180 and 320 µg Cu/g dry weight
    versus 60 µg Cu/g in control fish in the latter half of the
    experiment.  Gill concentrations were also significantly elevated,
    averaging 5.6 µg Cu/g and 9.5 µg Cu/g compared to 3.2 µg Cu/g in
    controls.  Copper concentrations in plasma were not significantly
    elevated by copper exposure except during the first day, while
    concentrations in kidney were only slightly elevated (6.6, 7.2 and 9.4
    µg Cu/g dry weight for controls, low and high exposures,

         Lanno et al. (1985) fed rainbow trout  (Oncorhynchus mykiss) 
    diets containing various levels of copper.  For an 8 week exposure,
    copper concentrations in liver ranged from 127 µg/g dry weight for a
    diet containing 8.5 mg/kg dry weight to 3200 µg Cu/g for a diet of
    3100 mg Cu/kg.  For a 24 week exposure, accumulation in liver ranged
    from 295 µg Cu/g for a diet of 8.5 µg Cu/g to 1640 µg Cu/g for a diet
    of 660 µg Cu/g, while concentrations in kidney ranged only from 8.5 to
    21.8 µg Cu/g.

         Mount et al. (1994) fed rainbow trout  (Oncorhynchus mykiss) on
    a brine shrimp  (Artemia sp.) diet containing 9.4, 440, 830 or 1000
    mg Cu/kg (dry weight) for up to 60 days.  After 35 days whole-body
    copper concentrations were 5.9, 36, 43.5 and 57.5 mg Cu/kg (dry
    weight) for the control and three doses, respectively, but after 60
    days copper levels had fallen to 3.6, 19.6, 22.4 and 27.7 mg Cu/kg.
    In a second experiment fish were fed diets containing copper
    concentrations ranging from 7.8 to 320 mg Cu/kg.  Whole-body copper
    concentrations ranged from 2.7 to 35.8 mg Cu/kg after 35 days, and
    from 2.3 to 8.8 mg Cu/kg after 60 days.

    4.2.5  Terrestrial plants

         Terrestrial plants respond in a number of ways to copper in the
    soils on which they grow.  Rooted species are subject to exposures
    which vary seasonally and over the plants' lifetime.  Perennial and
    especially long-lived species may experience wide changes in exposure
    over time.  Species differ both in their requirements and in their
    tolerances for copper.  Indeed, some terrestrial species are well
    known and used in mineral prospecting as copper indicators.  These
    include both mosses and higher plants.  Others are hyperaccumulators
    (Brooks, 1977; Baker & Brooks, 1989; Brooks et al., 1992).  Among the
    metal accumulators, a number of species from widely different plant
    families can accumulate from 2000 to 14 000 µg Cu/g (dry weight) in
    foliage, compared with 20-40 µg Cu/g (dry weight) in other species
    (Baker & Brooks, 1989).

         In Austria, the average copper level in soils was 17 µg/g and
    that in vegetation 12 µg/g; for Belgium it averaged 17 µg/g for soil
    and 17 µg/g for vegetation; in Finland, 4.3 µg/g for soil and 6.1 µg/g
    for vegetation; and for Germany 22 µg/g for soil and 24.5 µg/g for
    vegetation (Angelone & Bini, 1992).

         In studies of copper tolerant and sensitive strains (varieties)
    of the forage grass  (Festuca rubra) Wong et al. (1994) showed that
    copper concentrations in hydroponic solution of 50 µg/g allowed growth
    of a tolerant variety whereas even 5 µg Cu/g inhibited a sensitive
    strain.  Root copper concentrations reached 750 µg/g in the tolerant
    strain exposed to 1 µg Cu/g, whereas in the sensitive strain they were
    about 390 µg/g at the same exposure.  In contrast, in the shoots of
    these same plants exposed to 1 µg Cu/g the tolerant plants contained
    18 µg Cu/g and the sensitive plants 10 µg Cu/g. Higher root than shoot
    concentrations of copper are normal in terrestrial plants.

         In contrast to the situation for aquatic biota, copper levels in
    soils can vary over a wide range of concentrations and plant genetic
    tolerances allow an equally wide range of responses to these copper
    exposures.  Copper levels in foliage can be below the soil
    concentrations over which they grow or can be very much higher in
    accumulator species.

    4.2.6  Terrestrial invertebrates

         Moser & Wieser (1979) fed snails  (Helix pomatia) on a diet
    containing 230 or 1390 mg Cu/kg for 3 weeks.  Animals exposed during
    the summer accumulated copper concentrations ranging from 76 mg/kg
    (dry weight) (buccal mass and oesophagus) to 238 mg Cu/kg (intestine).
    Copper contents of midgut gland and foot were 44.7 and 56.0 µg/kg (dry
    weight) respectively.  In snails exposed during the winter months much
    higher concentrations were accumulated, ranging from 106 mg Cu/kg in
    the buccal mass and oesophagus to 1621 mg Cu/kg in the intestine.  In
    short-term (2-10 days) feeding experiments with lettuce containing
    1390 mg Cu/kg, about 97% of the metal ingested remained in the snail.
    Berger & Dallinger (1989) fed terrestrial snails  (Arianta arbustorum)
    on copper-enriched agar at concentrations of 209 mg Cu/kg or 723 mg
    Cu/kg (dry weight).  The highest concentrations of copper following
    exposure to the lower concentration for 21 days were in the midgut
    (492 mg Cu/kg).  The copper concentration of the faeces increased
    continuously during the experiment but the highest value recorded at
    69.5 mg Cu/kg was only one-third of the concentration in the food.
    In a 14-day copper balance study utilizing the higher dose
    (723 mg/kg) the mean rate of copper uptake was 6 µg/day.  The main
    site of copper storage seemed to be the foot/mantle tissues where 49%
    of the ingested copper was found.  The efficiency of copper
    assimilation always exceeded 95%.  Dallinger & Wieser (1984)
    maintained snails  (Helix pomatia) on a diet of lettuce enriched with
    533.8 mg Cu/kg for 32 days.  Copper contents of foot (0.579 g dry
    weight), midgut gland (0.326 g dry weight) and posterior gland (0.057
    g dry weight) were 90.1, 42.7 and 15.0 µg after 32 days; copper
    contents in foot and midgut gland had fallen to 39.6 and 23.1 µg after
    38-48 days on a "clean" diet. Copper was distributed more evenly in
    the organs of the snail than the other metals investigated (lead, zinc
    and cadmium); the midgut gland did not play such a dominant role in
    the storage of copper.

         Dallinger & Wieser (1977) exposed three species of isopods to
    copper concentrations of 340 and 5200 mg/kg (dry weight) in food
    (birch litter) for 14 days.  When feeding on natural litter with a low
    concentration (20 mg Cu/kg) all three species lost more copper through
    their faeces than they ingested.  When fed artificially enriched
    litter the efficiency of assimilation increased, so that at the
    highest concentration tested between 80% and 99% of the ingested
    copper was assimilated.  Isopods are capable of digesting even tightly
    bound copper during one passage of food through the gut.  However,
    they are unable to resorb more copper than they lose unless the food
    is enriched with soluble copper or the rate of food passage through
    the gut is slowed down.

    4.2.7  Terrestrial mammals

         Dodds-Smith et al. (1992a) maintained shrews  (Sorex araneus) on
    a diet containing copper at an intake of 2.13 mg/day for 12 weeks.
    Mean whole-body copper concentrations were 23.6 mg/kg (dry weight) in
    males and 64.8 mg/kg in females; mean total body burden was 64.7 µg Cu
    in males and 150.1 µg Cu in females.  Mean copper concentrations were
    31.0 and 23.4 mg/kg in kidneys of males and females, and 192.5 and
    820.5 mg/kg in livers of males and females, respectively (Dodds-Smith
    et al., 1992b).


    5.1  Environmental levels

         There is a very large amount of information on the levels of
    total copper in the various environmental compartments but little
    information on speciation.  Therefore, an attempt has been made to
    summarize those values related to temporal or geographical trends,
    polluted sites and known sources of copper.

         The largest release of copper is to  land; the major sources of
    release are mining operations, agriculture, solid waste, and sludge
    from sewage treatment works.  Mining and milling contribute most of
    the solid wastes.  Copper is released to  water as a result of
    natural weathering of soil, discharges from industries and sewage
    treatment plants, and from antifouling paints.  Copper compounds may
    also be intentionally applied to water to kill algae.  Copper is
    emitted to the  air naturally from windblown dust and volcanoes;
    however, anthropogenic sources contribute more to modern atmospheric
    levels from activities such as primary copper smelters, ore processing
    facilities and incineration (ATSDR, 1990).

    5.1.1  Air

         Hong et al. (1996) measured copper concentrations in Greenland
    ice samples.  The results revealed that anthropogenic sources of
    atmospheric copper first occurred in the Bronze Age, and that peaks of
    pollution occurred 2000 years ago due to the Romans and 900 years ago
    due to the Sung dynasty in China, before rapidly rising over the last
    century with some evidence of decline in recent years.

         The concentrations of copper in air depend on the proximity of
    the site to major sources such as smelters, power plants, and
    incinerators.  Average concentrations are in the range 5-50 ng Cu/m3
    in rural areas and 30-200 ng Cu/m3 in urban locations (Nriagu,
    1979b).  Evans et al. (1984) reported on the US EPA's national
    surveillance network for the years 1977, 1978 and 1979.  Copper levels
    in air were 133, 138 and 96 ng/m3, respectively, for urban samples
    and 120, 179 and 76 ng/m3 for non-urban samples.  In the study 10 769
    urban and 1402 non-urban air samples collected for 24 h were analysed.
    The maximum urban and non-urban copper concentrations were 4625 and
    4003 ng/m3, respectively.

         Atmospheric copper concentrations at the South Pole were found to
    range from 25 to 64 pg/m3 with a mean value of 36 pg/m3 (Zoller et
    al., 1974).  Copper concentrations in Atlantic aerosols were collected
    during 1980-1982.  Mean concentrations ranged from 1.0 to 4.5 ng/m3
    for the North Atlantic and from 0.29 to 0.31 ng/m3 for the South
    Atlantic.  In remote areas of the Atlantic, where the influence of
    continental sources is less, oceanic copper can make up over half of
    the total copper in the aerosol (Chester & Murphy, 1986).

         Sweet et al. (1993) analysed airborne particulate matter in
    southeast Chicago and East St Louis, USA.  Copper concentrations
    ranged from < 0.1 to 1610 ng/m3 in fine particles (< 1-2.5 µm), and
    from < 0.1 to 224 ng/m3 in coarse particles (2.5-10 µm).  Concen
    trations were found to be higher in samples from St Louis; these
    higher levels of copper in both fine and coarse fractions occurred in
    winds from the direction of several nonferrous metal smelters.

         Anderson et al. (1988) analysed atmospheric aerosols collected in
    Chandler, Arizona, USA in 1982.  Several major copper smelters are
    located approximately 120 km southeast of the sampling point.  The
    most abundant copper-bearing particle (particles containing > 0.5%
    copper), representing 74% of the total, was associated with sulfur,
    16% was associated with silicon and 4% was associated with chloride.
    Germani et al. (1981) reported that mean copper levels in particulate
    matter were found to be 2800 and 6800 ng Cu/m3 in the plumes of two
    copper smelters in Arizona, USA.  Mean concentrations ranging from
    2000 to 9500 ng Cu/m3 were reported for the first 8 km of plumes from
    five copper smelters (Small et al., 1981).  Atmospheric particulate
    aerosol samples were collected at sites along the normal plume pathway
    at distances ranging from 2.5 to 8.0 km from a copper smelter (western
    Poland).  Copper concentrations were inversely correlated to distance
    with levels of 165, 89 and 51 ng Cu/m3 (2.6, 1.4 and 0.8 nmol/m3) at
    distances of 2.5, 5.0 and 8.0 km, respectively (Zwozdziak et al.,

         Romo-Kröger & Llona (1993) analysed aerosols in the Chilean
    central Los Andes mountain range at varying distances from a copper
    mine.  Copper concentrations in fine (< 0.4 µm) particles ranged from
    414 ng/m3 (5 km from the mine) to 22 ng/m3 at > 25 km from the
    mine.  A similar correlation between distance from the mine and copper
    levels was found for coarse (> 8.0 µm) samples although levels were
    lower, ranging from 40 to 101 ng Cu/m3.  Romo-Kröger et al. (1994)
    found that copper levels were related to mining operations.  Sampling
    at 13 km from the mine revealed copper concentrations of 66 and 131
    ng/m3 for fine (< 2.5 µm) and coarse (2.5-15 µm) particles,
    respectively, during mining operations.  Sampling during strike
    periods gave levels of 22 and 50 ng Cu/m3, respectively.

         Johnson et al. (1987) reported elevated levels of copper in fog
    water 3 km downwind of a refuse incinerator in Switzerland.  Highest
    copper concentrations were associated with lower pHs.  The maximum
    concentration was 673 µg Cu/litre (10.6 µmol/litre) at pH 1.94, with
    levels > 127 µg Cu/litre being associated with pH values < 3.6.

         The annual average concentrations of copper in aerosols < 10 µm
    in the Netherlands varied between 11 and 25 ng/m3.  None of the eight
    sites was directly affected by industrial sources (Slooff et al.,

    5.1.2  Water and sediment

         Copper is widely distributed in water because it is a naturally
    occurring element.  Nriagu (1979b) reported average copper levels in
    seawater ranging from 0.15 µg/litre in open ocean to 1.0 µg/litre in
    polluted near-shore waters; levels in fresh water were 1.0-20
    µg/litre.  Other reports indicate that copper concentrations in
    seawater are highly variable, ranging from 0.005 µg/litre in the Black
    Sea (Haraldsson & Westerlund, 1988) to 40 µg/litre in estuaries in
    southwest Spain (Cabrera et al., 1987).  Additional variation in
    copper concentrations is related to depth and the area in the ocean
    examined.  Surface concentration in the North Pacific Ocean drops from
    0.1 µg Cu/litre (1.2 nmol/kg) in the California Current to 0.03-0.04
    µg Cu/litre (0.4-0.5 nmol/kg) in the central oceanic region, and
    increases to 0.24 µg Cu/litre (3 nmol/kg) in deep waters (Boyle et
    al., 1977; Bruland, 1980).  In the North Atlantic Ocean surface waters
    display values of copper from 0.07 µg/litre (1.1 nmol/kg) to 0.11
    µg/litre (1.7 nmol/kg), whereas concentration of the metal increases
    to 0.13-0.26 µg/litre (2-4 nmol/kg) in deep waters (Moore, 1978).
    Similarly, in the Ligurian Sea, Italy, Fabiano et al. (1988) reported
    3.57-16.6 µg dissolved Cu/litre in the surface layer (0-50 m) and
    0.7-2 µg/litre in deeper waters (200-2000 m).  Bryan & Langston (1992)
    reported dissolved copper concentrations of up to 600 µg/litre for
    Restronguet creek, a branch of the Fal estuary, United Kingdom, which
    receives acidic drainage from past and present mining activity.

         Bubb & Lester (1994) found mean copper concentrations in total
    and soluble (filter size 0.2 µm) river water for the river Stour,
    United Kingdom, to be 5.8 (3.0-19.5) and 2.2 (1.0-5.5) µg/litre,
    respectively.  Background levels were 1.0 µg Cu/litre derived from an
    upper catchment control site.  Fourfold increases in copper
    concentrations were apparent downstream of a sewage treatment works.

         Dissolved copper was monitored for 11 months in four recreational
    marinas, a large harbour, two major river systems and a heavily used
    shipping canal in Chesapeake Bay, USA.  Mean copper concentrations
    were 9.1, 13.2, 17.8 and 18.2 µg/litre for the four marinas, 7.9
    µg/litre for the harbour, 6.4 and 11.9 µg/litre for the two river
    systems and 9.6 µg/litre for the shipping canal. Copper concentrations
    ranged from < 10-80 µg/litre for the marinas to 10-14 µg/litre for
    the harbour and 10-20 µg/litre for the river systems and the shipping
    canal. The authors concluded that the likely source of the highest
    copper concentrations was from antifouling paints used on boats in the
    marinas (Hall et al., 1988).  An evaluation of dissolved copper
    concentrations at three sampling stations in 1989 showed that mean
    concentrations from biweekly sampling for four months were 2.7, 7.8
    and 10 µg Cu/litre. Copper concentrations decreased with distance from
    marinas, and at all three stations were significantly lower in 1989
    than in 1988 (Hall et al., 1992).

         Parrish & Uchrin (1990) sampled Lakes Bay, near Atlantic City,
    USA during the summer of 1986.  Dry weather concentrations of copper
    were found to be typical of those found in natural waters, but higher

    levels were recorded during storm events.  Significant amounts of
    copper were found to originate from a major stormwater sewer which
    discharges into the bay.  Total copper in runoff from a car park near
    Portland, Oregon, USA varied among different storm events over a wide
    range of concentrations (< 2-33 µg/litre).  Copper levels in a
    detention pond ranged from 5 to 12 µg/litre.  Copper was found to be
    deposited in pond sediments in a small highly concentrated plume (up
    to 130 mg/kg) extending from the runoff inlet pipe (Mesuere & Fish,

         Hurley et al. (1996) measured the concentration of copper and
    several other metals in 11 tributaries (rivers) feeding Lake Michigan,
    USA using low-level techniques.  They reported dissolved and total
    copper concentrations ranging from 0.2 to 2.0 and 0.4 to 5.5 µg/litre,

         Shiller & Boyle (1987) measured dissolved concentrations of
    copper in the lower Mississippi river, USA seven times.  The
    Mississippi was chosen because it is the most heavily industrialized
    of the 10 largest rivers in the world.  The authors concluded that the
    levels of copper and several other metals do not appear to be
    significantly higher than in several other less industrialized and
    disturbed rivers.  Dissolved copper concentrations ranged from 1.16 to
    1.96 µg/litre.  Samples from the Yangtze, Amazon and Orinoco rivers
    were analysed for comparison.  Dissolved concentrations of 1.24, 1.52
    and 1.20 µg/litre were determined, similar to levels in the
    Mississippi river.

         Ouseph (1992) reported that dissolved and particulate copper
    concentrations in the unpolluted zone of the river Periyar, India,
    were 0.8-10.0 µg/litre and 48-140 mg/kg, respectively, in 1985-1986.
    The Cochin estuary is subjected to various types of effluents from the
    Eloor and Chitrapuzha industrial belts.  Levels in the estuary ranged
    from 2.2 to 22.2 µg/litre for dissolved copper and from 44 to 298
    mg/kg for particulate copper.  Copper concentrations showed high
    seasonal variations, with the lowest levels being detected during the
    monsoon season.

         Filipek et al. (1987) found that dissolved copper concentrations
    reflected the acidity of waters affected by acid mine drainage of West
    Squaw Creek, California, USA.  At pH > 5, copper concentrations were
    generally below the detection limit (< 0.01 mg/litre).  Dissolved
    copper concentrations ranged from 0.12 to 13.5 µg/litre at pH 3-4, and
    at pH 2.4 a concentration of 190 µg/litre was found.  Hĺkansson et al.
    (1989) found that the transfer of copper from the aqueous to the solid
    particulate phase is significant at pH 3-3.5 and increases with pH.
    Copper concentrations in suspended solids were 2.7, 2.0 and 0.5 mg/kg
    at pH levels of 4.5, 5.4 and 6.5, respectively, in a drainage stream
    for a mine waste deposit.  Camusso et al. (1989) monitored seasonal
    variations in copper in suspended particulate matter in the north
    basin of the acidic (pH 4.4) Lake Orta, Italy, between 1985 and 1987.

    Copper in the lake occurred mainly in the dissolved form (94%) and
    levels are still high (32-34 µg/litre) because of past industrial

         Sediment is an important sink and reservoir for copper.
    Background levels of copper in natural river sediments range from 16
    to 5000 mg/kg (dry weight) (Förstner & Wittmann, 1981).  Copper levels
    in marine sediments range from 2 to 740 mg/kg (dry weight) (Nriagu,
    1979b).  Bryan & Langston (1992) reported that sediment copper levels
    in United Kingdom estuaries range from 10 to > 2000 mg/kg (dry
    weight), the highest values being for Restronguet creek which receives
    acidic drainage from mining activity.  In the creek, adsorption of
    most of the dissolved copper by flocculated oxides of iron and
    associated humic substances during estuarine mixing leads to very high
    sediment concentrations.

         Bubb et al. (1991) found that copper loadings for fluvial
    sediments from the river Yare, United Kingdom, ranged from 5 to 375
    mg/kg.  Levels displayed the profile of a pollution plume originating
    from a point source.  A peak located at 1-2 km from a sewage treatment
    works outlet was recorded.  Bubb & Lester (1994) found copper
    concentrations at 24.2 and 39.0 mg/kg above and below a sewage
    treatment works, respectively.  Background levels from a control site
    were 6.17 mg Cu/kg.

         Palanques & Díaz (1994) found that the surface sediments of the
    continental shelf off Barcelona, Spain, are greatly influenced by
    anthropogenic contamination of heavy metals discharged by the littoral
    sewers and the Besos river.  Copper concentrations ranged from 300 to
    400 mg/kg at the mouth of the Besos river and declined at increasing
    distances from the shoreline.

         A large gold and copper mining project began in 1984 on the Ok
    Tedi river, a tributary of the Fly river, Papua New Guinea.  Baker et
    al. (1990) analysed suspended sediment samples from the Torres Strait
    near the mouth of the Fly river system in 1989.  Mean copper
    concentrations ranged from 1.4 to 13.3 µg/kg.  The highest levels of
    copper were found at stations closest to the Fly river.  Sediments of
    the Ok Tedi river are enriched with copper.  Approximately 60% of the
    input has a particle size of < 100 µm and is transported as a
    suspended load throughout the entire length of the river (> 1000 km).
    Copper concentrations in the fraction < 2 µm reaches levels of 6000
    mg/kg (Salomons & Eagle, 1990).  Mean copper concentrations in the
    surficial sediments of the Fly river delta and the Torres Strait were
    28 and 8.2 mg/kg, respectively (Baker & Harris, 1991).

         Copper contamination of sediment samples in northern Sweden was
    correlated with distance from the Ronnskar smelter.  Concentrations
    ranged from 1556 mg Cu/kg at a distance of 3 km to 37 mg Cu/kg at 80
    km (Johnson et al., 1992).  Ünlü & Gümgüm (1993) analysed sediment
    samples from the Tigris river, Turkey, in the vicinity of the Ergani
    copper plant.  Copper concentrations were 641 mg/kg 5 km upstream of
    the plant, 3433 mg/kg at the outflow and around 900 mg/kg downstream.

    5.1.3  Soil

         Median total copper concentrations in uncontaminated soil were
    reported to be 30 mg/kg (range 2-250 mg/kg) (Bowen, 1985).  Shacklette
    & Boerngen (1984) analysed soil samples from various locations in the
    USA, finding that copper concentrations ranged from < 1 to 700 mg/kg
    with an average of 25 mg/kg.  Kabata-Pendias & Pendias (1984) reviewed
    the worldwide literature on copper in uncontaminated surface soils and
    report mean concentrations ranging from 6 to 80 mg Cu/kg (dry weight).
    Much higher levels were associated with mining activity,
    metal-processing industries and fertilizer and fungicide application.

         Copper can accumulate in soils from the long-term application of
    fertilizers or fungicides.  Reuther & Smith (1952) analysed soils from
    mature Florida citrus groves and found that copper oxide levels in the
    topsoil increased with grove age.  Copper oxide levels of 247 and 93
    mg/kg (dry weight) were measured at depths of 0-8 cm and 8-15 cm,
    respectively.  At depths of > 15 cm copper oxide levels of ć 18 mg/kg
    were measured. Copper oxide levels in adjacent untreated soil ranged
    from 1 to 2 mg/kg.  Christie & Beattie (1989) reported an accumulation
    of copper in soil from the application of pig slurry (50-200 m3/ha
    per year).  EDTA-extractable copper concentrations of up to 85.2 mg/kg
    were recorded; levels in control soils ranged from 4.4 to 5.4 mg/kg.
    Paoletti et al. (1988) found that in Italy vineyard soil to which
    copper-containing fungicide had been applied contained mean copper
    concentrations of 89.8 mg/kg (dry weight).  Soils from other locations
    contained mean levels ranging from 44.0 to 52.1 mg/kg.  Holmgren et
    al. (1993) analysed surface soil samples from agricultural regions
    throughout the USA.  Copper concentrations ranged from 0.3 to 495
    mg/kg (dry weight).  Copper levels were higher in the organic soil
    areas of Florida, Oregon and the Great Lakes, reflecting the use of
    copper fertilizers and fungicides.

         Fjeldstad et al. (1988) found that levels of copper in surface
    peat showed a negative correlation with distance from a nickel
    smelting factory in Kristiansand, Norway.  Dumontet et al. (1990)
    monitored copper in acidic peat located along two transects from a
    smelter plant in the Noranda region of Quebec, Canada and found that
    copper concentrations in surface samples (0-15 cm) ranged from 5525
    mg/kg at a distance of 1 km to 28 mg/kg at 42.5 km.  The majority of
    the deposited copper remained in the upper 15 cm of the soil profile.
    Soil samples taken in the vicinity of a copper smelter at Legnica in
    southern Poland contained copper levels of 7400 mg/kg (Helios Rybicka
    et al., 1994).  Wu & Bradshaw (1972) reported that soil copper levels
    in the vicinity of a metal refinery (southwest Lancashire, United
    Kingdom) established in 1900 contained total copper concentrations
    ranging from 1930 to 4830 mg/kg.  Hunter et al. (1987a) reported mean
    surface soil copper concentrations of 15.1, 543 and 11 000 mg/kg at a
    control site, 1 km from a copper refinery (Merseyside, United Kingdom)
    and at the refinery, respectively.  Beyer et al. (1985) monitored
    soils 10 km upwind and 2 km downwind of zinc smelters in eastern
    Pennsylvania, USA.  Copper concentrations ranged from 12 to 34 mg/kg

    and from 9.9 to 440 mg/kg (dry weight) for the two sites,
    respectively.  Almost all of the copper contamination was held at the
    surface of the mineral soil.

    5.1.4  Biota  Aquatic

         The levels of copper in marine algae vary from 0.64 µg/g in
     Laminaria religiosa from Japan (Suzuki et al., 1987) to 407 µg/g in
     Jania rubens from Antikyra Gulf, Greece (Malea et al., 1994).  An
    important source of variation in the copper content in algae is the
    part of the plant analysed, generally being higher in older parts than
    in fast growing, younger apices.

         Freshwater mussels  (Unio pictorum) in the area of a sailing
    boat harbour (Lake Balaton, Hungary) contained significantly higher
    levels of copper than those from open water areas.  Mean gill and
    adductor muscle copper concentrations were, respectively, 203 and 221
    mg/kg (dry weight) in the harbour and < 20 mg/kg in open water
    (V-Balogh, 1988).  Batley et al. (1992) analysed Sydney rock oysters
     (Saccostrea commercialis) from the Georges river, New South Wales,
    Australia.  Mean copper concentrations ranged from 12 to 95 mg/kg (wet
    weight) in 1988 and from 19 to 89 mg/kg in 1991, and the authors state
    that overall copper concentrations in oysters have fallen since the
    banning of tributyltin.  Claisse & Alzieu (1993) found an increase in
    copper concentrations in oysters collected between 1979 and 1991 in
    the bay of Arcachon, France.  Annual mean copper concentrations have
    increased from 48.3-81.1 mg/kg (dry weight) in 1979 to 74.6-135 mg/kg
    in 1991.  Data collected from 1977 to 1990 by the California mussel
    watch programme were analysed for long-term trends in copper.  Copper
    showed a steady increase over time at 5 of the 20 sampling stations.
    The authors suggest that the increases in copper may be related to
    increased vessel traffic and the increased use of copolymer copper
    antifouling paints (Stephenson & Leonard, 1994).

         Rainbow et al. (1989) monitored the copper concentrations in
    several species of talitrid amphipod at several sites in the United
    Kingdom.   Orchestia gammarellus was found to be the most suitable
    biomonitor of copper in British coastal waters.  Weeks (1992a) found
    the talitrid amphipod  Platorchetsia platensis to be a good indicator
    species in Danish waters.  Samples with significantly higher copper
    burdens, for example, 110 mg Cu/kg (dry weight) compared to 32 mg
    Cu/kg, were associated with local sources of metal enrichment, due to
    anthropogenic inputs (antifouling paint leachates) or geological
    conditions.  Negligible quantities of copper were found in cast exuvia
    of talitrid amphipods during the moult cycle (Weeks et al., 1992b).
    Moore et al. (1991) found the beach-hopper  (Orchestia gammarellus) 
    to be a very convenient and sensitive biomonitoring species for copper
    levels along the North Sea coasts.  Typical background concentrations
    were approximately 70 mg Cu/kg (dry weight); samples with higher

    concentrations (up to 218 mg Cu/kg) were associated with local sources
    of contamination such as antifouling paints or the metal-rich

         Alikhan et al. (1990) measured the concentration of copper in
    crayfish  (Cambarus bartoni) trapped from increasing distances, up to
    150 km from a nickel-copper smelter (Canada). Their results indicate
    that the concentrations in the crayfish decreased with increasing
    distance from the source; the highest concentration (1986 µg Cu/g) was
    measured in the hepatopancreas.

         Schmitt & Brumbaugh (1990) analysed freshwater fish from
    throughout the USA in 1984-1985. A mean copper concentration of 0.65
    mg/kg (wet weight) and a maximum copper level of 23.1 mg/kg were
    recorded.  No significant change in the mean concentration of copper
    was found when compared with monitoring results from 1976.

         Lee & Stuebing (1990) analysed liver tissue from river toads
     (Bufo juxtasper) near a copper mine in east Malaysia.  Mean copper
    concentrations in toads downstream of the mine and from a control area
    were 438 mg/kg (dry weight) and 46 mg/kg, respectively.  Copper levels
    of 117 and 273 mg/kg were recorded in toads collected from areas known
    to be rich in minerals.  Terrestrial

         Stewart et al. (1991) sampled tree ring wood from kahikatea trees
    in urban Christchurch and the west coast of South Island, New Zealand.
    For the urban ring wood cores copper levels showed an elevation over
    baseline levels with an approximately threefold increase beginning
    around 1940.  This was probably due to increased industrial emissions.

         Kalac et al. (1996) measured the concentrations of copper in
    edible mushrooms in the vicinity of mercury and copper smelters in
    eastern Slovakia.  Copper concentrations up to 236 mg/kg and 231 mg/kg
    (dry weight) were measured in  Lepiota procera and  Lepisia nuda, 

         The metalliferous hillocks of the Shaba Province in southwest
    Zaire have soil copper concentrations of up to 30 g/kg (Malaisse et
    al., 1979).  The region supports an extremely unusual endemic flora,
    composed mainly of herbs and grasses, that can tolerate concentrations
    of copper in excess of 1% in the soil.  Terrestrial higher plants
    which accumulate copper concentrations in excess of 1000 mg/kg (0.1%)
    (dry matter) are known as "hyperaccumulators" (Brooks et al., 1977).
    Brooks et al. (1980) reported hyperaccumulation of copper in 24 taxa
    from the Shaban region.  The most unusual of these is
     Aeollanthus biformifolium which can contain as much as 13.7 g/kg
    (1.37%) (dry weight) in the whole plant (Malaisse et al., 1978).

         The first workers to present data indicating hyperaccumulation of
    copper were Duvigneau & Denaeyer-De Smet (1963) who reported values of
    1200, 1660 and 1960 mg Cu/kg (dry weight) for
     Ascolepis metallorum, Silene cobalticola and
     Haumaniastrum robertii, respectively.

         The labiate (mint family)  Becium homblei occurs on copper
    deposits in Zaire, Zimbabwe and Zambia.  Reilly (1967) and Reilly &
    Reilly (1973) described  B. homblei as a cuprophile, tolerant
    to > 70 g Cu/kg (dry weight) in soil, and accumulating up to 17% of
    copper in the leaves, organically bound to the cell walls.  They also
    noted that some other species of  Becium in the same area had no
    special ability to accumulate copper.

         Hunter et al. (1987a) reported annual mean copper concentrations
    in the dominant plant species growing near a metal refinery in the
    United Kingdom  (Agrostis stolonifera, Festuca rubra,
     Equisetum arvense and  Tussilago farfara).  Mean copper
    concentrations ranged from 7.6 to 18.6, 22.8 to 25.8 and 73.3 to 260
    mg/kg (dry weight) at a control site, 1 km from a metal refinery and
    at the refinery respectively.  Vegetation levels of copper showed
    marked seasonal variations at contaminated sites with peak values
    during the winter months.  The increased levels were due to a
    combination of root absorption and accumulation of particles on
    external leaf surfaces.  Copper concentrations in grasshoppers
     (Chorthippus brunneus) ranged from 37.5 mg/kg (dry weight) at a
    control site to 380 mg/kg at the refinery (Hunter et al., 1987c).
    Hunter et al. (1987b) analysed invertebrates from both contaminated
    and semi-contaminated grasslands in the vicinity of a major copper
    refinery.  All species showed significant elevations of total body
    copper concentrations relative to controls.  Highest concentrations
    were found in isopoda species.  Detritivorous soil macrofauna showed
    accumulation of copper (2-4 times) with respect to concentrations in
    refinery site organic surface soil and plant litter.  Herbivorous
    invertebrates also showed body : diet concentration factors of 2-4
    times for copper.

         Ferns growing in the vicinity of ore smelters at Sudbury,
    Ontario, Canada, contained copper concentrations ranging from 27.2 to
    73.0 µg/g (dry weight).  Plants collected from control sites contained
    concentrations ranging from 7.4 to 11.5 mg Cu/kg (Burns & Parker,
    1988).  Analysis of lowbush blueberry  (Vaccinium angustifolium) at
    sampling sites ranging from 6.5 to 74 km from Sudbury smelting
    operations revealed a significant relationship between copper
    concentrations and distance from the smelter (Bagatto et al., 1993).
    Alikhan (1993) analysed terrestrial isopods  (Porcellio spinicornis) 
    2 km downwind of a primary smelting works (nickel) in Ontario, Canada.
    Mean copper concentrations in the isopods were 1137 mg/kg (dry weight)
    for the contaminated site and 685 mg/kg for a control site.  Leaf
    litter contained approximately 12 times more copper at the
    contaminated site than at the control site.

         Morgan & Morgan (1988) analysed earthworms  (Lumbricus rubellus 
    and  Dendrodrilus rubidus) from both contaminated (the vicinity of
    disused nonferrous metalliferous mines) and noncontaminated sites in
    Wales.  There were significant positive correlations between total
    copper concentrations in the earthworms and in the soil.  Copper
    concentrations in earthworms ranged from 8 and 9 mg/kg (dry weight) at
    uncontaminated sites to 104 and 34 mg/kg at contaminated sites for the
    two species.

         Ash & Lee (1980) analysed earthworms from roadside verges in the
    United Kingdom and found a relationship between traffic density and
    copper burden.  Mean copper concentrations ranged from 3.9 to 8.9
    mg/kg (dry weight) for heavy traffic, 2.3 to 6.6 mg/kg for
    intermediate traffic and 0.2 to 0.83 mg/kg for low levels of traffic.
    However, for the more contaminated sites other industrial sources of
    copper could not be ruled out.

         Wieser et al. (1976) found two species of isopods
     (Tracheoniscus rathkei  and  Oniscus asellus) to be good indicator
    species for copper.  Total copper concentrations in isopods ranged
    from 74 mg/kg (dry weight) for a spruce forest to 538 mg/kg for an
    overgrown slag heap of an old copper mine in the Tirol region of
    Austria.  Hopkin et al. (1993) proposed the isopod  Porcellio scaber 
    as an ideal candidate for biomonitoring the bioavailability of metals
    to soil and leaf litter invertebrates.  The authors provide a table of
    concentration ranges for this species related to degrees of
    contamination.  For example, isopod copper concentrations of < 250
    mg/kg (dry weight) would be classified as uncontaminated with medium
    contamination at 400-600 mg/kg and high contamination at 600-1000
    mg/kg.  Hopkin et al. (1986) analysed hepatopancreas and whole body of
    woodlice  (Porcellio scaber) collected from 89 sites in southwest
    England.  The main source of copper pollution was centred on
    Avonmouth, the site of a primary zinc, lead and cadmium smelting
    works.  The correlation coefficients between the concentrations of
    copper in woodlice and soil, and between woodlice and leaf litter,
    were positive and statistically significant.

         Rose & Parker (1983) reported concentrations of copper in tissues
    of ruffed grouse from a site near a copper-nickel smelter and a
    control, uncontaminated site near Sudbury, Ontario, Canada.  Mean
    copper concentrations in kidney, liver and breast muscle ranged from
    11.7 to 24.6, 12.6 to 16.3 and 1.5 to 2.3 mg/kg (dry weight),
    respectively.  Their results indicate no difference between the two

         Hunter & Johnson (1982) analysed small mammals in the vicinity of
    a copper refinery in the United Kingdom.  Liver concentrations were
    significantly elevated at the refinery in wood mouse
     (Apodemus sylvaticus) (23.7 mg Cu/kg dry weight) and common shrew
     (Sorex araneus) (56.1 mg Cu/kg) but not in short-tailed vole
     (Microtus agrestis) (13.5 mg Cu/kg).  However, even these
    significant accumulations were rather limited bearing in mind the soil
    copper levels of 2000-3000 mg/kg (dry weight) at the refinery site.

    At reference sites copper concentrations in whole-body samples of
    small mammals ranged from 8 to 13 mg/kg (dry weight) (Smith &
    Rongstad, 1982; Beyer et al., 1985).

    5.2  General population exposure

    5.2.1  Air

         Pulmonary exposure occurs through the inhalation of dusts, fumes,
    smoke and sprays that contain copper.

         Exposure to copper by inhalation is determined by air
    concentrations, particulate size and the respiratory rate.
    Concentrations of copper determined in over 3800 samples of ambient
    air at up to 29 sites in Canada over the period 1984-1993 averaged
    0.014 µg/m3.  The maximum value was 0.418 µg Cu/m3, detected in 66%
    of samples (Dann, 1994).  In the USA, air levels of copper vary
    between 96 and 138 ng/m3 in urban samples and 76 and 176 ng/m3 in
    non-urban settings (see section 5.1.1), though levels as high as 4629
    ng/m3 have also been recorded.

         Based on data collected in the province of Ontario, Canada,
    copper levels in ambient air have decreased over 70% in the last 10
    years, though some of this decrease is likely attributable to
    variations in sampling and analytical methods (OMME, 1992).

         Estimated mean intake, based on these data (22 m3 air/day)
    (ICRP, 1974) and the mean Canadian values, are less than 0.28 µg/day.

    5.2.2  Food and beverages

         The actual concentration of copper in food and beverages from
    various countries varies widely depending upon the food product, the
    growing conditions (soil, use of fertilizers high in copper, water,
    use of copper fungicides) and the type of processing used; in
    particular, pH levels and the use of copper vessels (Tanner et al.,
    1979; Muller et al., 1996).

         In some countries, it has been customary to prepare milk by
    boiling it in copper vessels.  Levels of copper in such milk have been
    reported as up to about 60 mg/litre (Muller et al., 1996).  Studies
    have shown that copper binds predominantly to casein, which is the
    main constituent of milk protein.  In acidic pH (as in gastric juice)
    casein liberates most of this bound copper as a copper ion, making it
    available for rapid absorption (O'Neill & Tanner, 1989). Calculations
    reveal that whereas total breast feeding would supply up to 0.9 µmol
    Cu/kg per day (60 µg/kg per day), feeding similar amounts of brassy
    milk would supply up to 14.6 µmol Cu/kg per day (930 µg/kg per day) or
    10-20 times the physiological intake per kg body weight per day.
    Traditional "tinning" of copper and brass vessels protects from such
    contamination by copper, yet it is a procedure often neglected because
    of cost and effort.

         Copper is widely distributed in foods, with organ meats (e.g.
    liver) and seafood having the highest concentrations (10-100 mg/kg)
    and dairy products having relatively low levels (Table 5).  High
    levels of copper have also been identified in wheat bran, beans and
    seeds, based on a recent, detailed investigation (Jorhem & Sundstrom,
    1993).  Baseline values have been reported as 0.2-0.3 µg Cu/litre for
    mother's milk and 0.7-1.1 µg Cu/kg for infant formula (Richmond et
    al., 1993).  Chocolate may contain more than 5 mg Cu/kg.  Values
    quoted for tea and coffee are highly variable but may exceed 10 mg
    Cu/kg (dry weight) (Slooff et al., 1989; ATSDR, 1990).  In general
    most other foods contain much less than 10 mg Cu/kg.

         Copper levels in common foodstuffs and beverages have been
    determined in many countries, including the USA (Pennington et al.,
    1986), Australia (NFA, 1992) and the Netherlands (Slooff et al.,
    1989).  Copper levels in representative foodstuffs in these three
    countries are given in Table 5.  From these market basket surveys,
    average daily intakes have been calculated (Pennington et al., 1986,
    1989; Slooff et al., 1989; NFA, 1992), or actual dietary surveys have
    been conducted to determine the daily intake from food and beverages
    (Pettersson & Sandström, 1995).

         Representative mean total daily intakes of copper from foods and
    beverages in several countries are given in Table 6.  As shown, the
    total daily intake of copper in adults varies between 0.9 and 2.2 mg.
    Intake in children has been estimated to be 0.6-0.8 mg/day (0.07-0.1
    mg/kg body weight per day).

         In relation to the intake of copper in food, the WHO (1996) noted
    the insufficiency of global data and concluded that:

         "The scarcity of adequately planned studies is again evident,
         with insufficient data from Africa, the Eastern Mediterranean and
         South-East Asia.  The apparently higher proportion of European
         studies suggesting undesirably low population mean intakes of
         copper needs to be investigated more closely to determine whether
         it is a truly characteristic feature of diets of the eastern
         German communities from which these particular samples were
         drawn.  Before it is concluded that intakes of copper are likely
         to be reasonably adequate in the Americas, the western Pacific
         fringe and the remainder of Europe, it must be strongly
         emphasized that none of the surveys covered were representative
         of those socially and nutritionally disadvantaged communities in
         which food preferences lead to the consumption of diets providing
         as little copper as those reported to induce clinical signs of
         deficiency elsewhere" (WHO, 1996).

         A summary of preliminary data from a global literature survey of
    dietary intakes by IAEA has been published (WHO, 1996).  When all the
    IAEA data are considered, approximately 10% of reported mean intakes
    are below the proposed minimum basal mean value for copper in adult
    males (1.2 mg/day) and approximately 25% are below the corresponding
    minimum normative mean population intake (1.4 mg/day).  Intakes five
    times higher than the basal minimum mean are observed in some
    population groups, but these are still well below the upper limit of
    the safe range of mean population intake (12 mg Cu/day for men) and
    there is no evidence from the IAEA database that the copper intake
    from diets for young children is sufficiently high to cause concern in
    the communities studied.

    5.2.3  Drinking-water  Organoleptic characteristics

         The taste of copper in drinking-water has been described as
    metallic, bitter and persistent. Taste thresholds have been reported
    between 0.8 and 5 mg Cu/litre, depending on the purity of the water
    (Cohen et al., 1960; Béguin-Bruhin et al., 1983).  Concentrations of
    copper greater than 5 mg/litre may render water unpalatable although
    individuals can adapt to such levels (Scheinberg & Sternlieb, 1994).
    Aesthetic considerations relating to copper levels in drinking-water
    include blue or green staining of plumbing fixtures, hair and laundry.  Copper concentrations in drinking-water

         Levels of copper in surface waters used for the production of
    drinking-water are presented in section 5.1.2. Copper is also
    introduced into drinking-water during distribution, owing to leaching
    from plumbing fixtures and copper piping.  Leaching is dependent upon
    a number of factors, including pH, temperature, hardness, carbon
    dioxide content of the water, the length of time in contact with the
    pipe or fixture and the age of the piping (Schock & Neff, 1988; Alam &
    Sadiq, 1989).  Some of these factors cannot easily be controlled; in
    particular, hard waters with high buffering capacity cannot have the
    pH raised sufficiently to moderate copper solvency (Dieter et al.,
    1991).  It is thus insufficient to ascribe all problems of copper
    solvency to soft, acidic waters with low buffering capacity and
    nonadjusted pH.

         In distributed water from 70 municipalities across Canada, median
    concentrations of copper ranged from < 0.02 mg/litre to 0.75
    mg/litre. In about 20% of the distributed water supplies, the level of
    copper was significantly higher than the corresponding treated water
    samples. Furthermore, the increase was higher in those areas where the
    water was soft and corrosive (Meranger et al., 1979).

    Table 5.  Levels of copper in foodstuffs (mg/kg wet weight)a

    Food stuff           Mean           Minimum      Maximum     n

         beef            0.8, 1.1       0.74         1.6         39
         pork            0.9, 1.4       0.44         7.22        150
         lamb            1.6            1.1          1.9         24

         beef            39             8.8          87          7
         pork            9.0            0.9          29          126
         lamb            97             28           195         32

         beef            3.7            2.8          4.2         6
         pork            6.1            2.9          15          75

         apples          0.25           0.21         0.31        6
         pears           0.81           0.48         2.7         24
         bananas         0.95, 0.96     0.70         1.2         12

         potatoes        0.72, 0.96     0.26         2.2         40
         carrots         0.40, 0.61     0.26         0.95        30
         lettuce         0.47, 0.72     0.20         1.4         40
         tomatoes        0.36, 0.55     0.29         1.1         26

         cod             0.19           0.12         0.28        5
         tuna            0.64           0.48         0.80        9

         flour           1.5            0.95         2.9         56
         bread (white)   1.5            0.89         2.2         32

         cow             0.06           trace        0.14        31
         human           0.54           0.22         0.90        28

    Cocoa powder         36.4           33.0         410         9

    a    Adapted from Jorhem & Sundstrom (1993) for Sweden
         and NFA (1993) for Australia

        Table 6.  Estimated average dietary intake of copper in various countries

    Country             Method of           Intake of copper    Reference
                        samplinga           (mg/day)

    Australia           MB (adult male)     1.9                 NFA (1992)
                        MB (adult female)   2.2
                        MB (2 years)        0.8

    Denmark             DD                  1.2                 Bro et al.

    Finland             TDb                 2.00                Kumpulainen
                                                                et al. (1987)

    Germany             DD                  0.95                Anke (1991)

    The Netherlandsc    MB                  1.5                 Slooff et al.

    Norway              DD                  1.0                 Pettersson &

    Sweden              MB                  1.20                Becker &

    United Kingdom      TD (adult male)     1.63                Gregory et al.
                        TD (adult female)   1.23                (1990)
                        TD (1.5-4.5 years)  0.5                 Gregory et al.

    USAc                MB (6-11 months)    0.47                Pennington
                        MB (2 years)        0.58                et al. (1986)
                        MB (adult male)     1.24
                        MB (adult female)   0.94

    a    MB = market basket survey; TD = total diet study;
         DD = duplicate diet study
    b    Total diet from food record
    c    In calculations of dietary intake of copper the USA
         and the Netherlands consider water as part of the diet

         In the USA, 85% of fully flushed tap water samples had copper
    levels below 0.06 mg/litre and 98% were below 0.46 mg/litre. Less than
    1% exceeded 1 mg Cu/litre and the maximum level measured was 2.37 mg
    Cu/litre (US EPA, 1991).

         The difference between samples of running water and those where
    water was standing for some time is evident from studies in several
    countries. Murphy (1993) measured copper levels in drinking-water
    fountains in 50 schools in New Jersey, USA. Median levels in
    first-draw water (0.26 mg Cu/litre) decreased significantly after 10
    min of flushing (0.068 mg Cu/litre), but increased by lunchtime to
    0.12 mg Cu/litre after normal use of fountains. In Canada, copper
    levels in running water from private wells were extremely low, but 53%
    of the samples from standing water exceeded 1 mg Cu/litre (Maessen et
    al., 1985).  In a study in one US city (Seattle), mean copper levels
    in running and standing water were reported as 0.16 and 0.45 mg/litre,
    respectively, with 24% of standing water samples exceeding 1.0
    mg/litre (Dangel, 1975).  In the Netherlands, values between 0.2 and
    3.8 mg Cu/litre were reported in water standing 16 h.  This compares
    to the level of 3.0 mg/litre in water standing 16 h, which is the
    maximum permissible level for copper in drinking-water in the
    Netherlands (Slooff et al., 1989).  These same authors report average
    copper levels between 0.04 and 0.69 mg/litre in other municipalities.

          Pettersson & Sandström (1995) reported that in a study of 400
    children aged 9-21 months the daily intake of copper from
    drinking-water ranged between 0.01 and 3.2 mg, with a mean of 0.3 mg.
    The study was conducted in two cities where it was suspected that
    levels of copper in drinking-water were high.  In these cities, the
    mean copper levels in standing water were 0.7 mg/litre with a 90th
    percentile of 2.1; in water for consumption, the mean was 0.6 mg/litre
    with a 90th percentile of 1.6 mg/litre.

         From the data available, and assuming a daily intake of
    drinking-water of 1.4 litres (IPCS, 1994), daily intakes of copper
    from drinking-water by adults will vary between less than 0.01 mg to
    over a few mg per day, with highest intake in areas with corrosive
    water using copper piping.

    5.2.4  Miscellaneous exposures

         In addition to airborne copper and copper in foods and beverages,
    the general population may be exposed to this metal from a variety of
    other sources.  It is extremely difficult to quantify such exposures
    and in most cases they make only a minor contribution to the daily
    intake of copper by the general population when compared to the major
    source of copper which is food and drinking-water (1-3 mg Cu/day).
    Intake of dietary supplements containing copper will also contribute
    to total exposure.

         In a study of the metal content of tobacco, the copper content in
    cigarette tobacco was found to vary between 9 and 66 µg Cu/g with a
    mean value of 15.6 µg Cu/g (Mussalo-Rauhamaa et al., 1986).
    Approximately 0.2% of this copper was detected in mainstream smoke

    (about 0.05 µg Cu/cigarette).  This would result in a daily exposure
    of about 1 µg Cu from 20 cigarettes (Mussalo-Rauhamaa et al., 1986).

         Dermal exposure to copper can result from the use of consumer
    products containing copper pigments, through the use of copper as an
    algicide in swimming pools and the use of copper jewellery.  No
    quantitative exposure levels could be found.

         Excluding the use of copper IUDs, the use of copper in medical
    applications has been replaced with other treatment regimens.
    However, in rare cases, notably the treatment of burns with copper
    sulfate, increased copper absorption has occurred with resulting
    toxicities observed (Eldad et al., 1995).  The use of copper IUDs may
    result in exposure to as much as 80 µg Cu per day (Kjaer et al., 1993)
    with decreasing levels after the first few weeks after insertion.

         Copper is a component of many amalgams used in dentistry,
    including mercury amalgams.  The loss of copper from these sources has
    been reported as minimal (Johansson & Moberg, 1991; Lussi et al.,

    5.3  Occupational exposures

         There is a wide range of industrial activities in which workers
    can be exposed to copper and copper compounds. Copper exposures in
    occupational settings are to particulates to which the metal or metal
    compound is adsorbed or to metal fumes (aerosols).

         In the mining industry, workers (miners and millers) are exposed
    to dusts both from rocks and from the ore itself, containing 0.05-5%
    of copper (Weant, 1985).  Multiple exposures occur, as the ore may
    contain high levels of nickel, arsenic and silica (McLaughlin et al.,
    1992).  Exposure to copper fumes and to a lesser extent dusts is a
    feature of smelting operations but can occur through brassing,
    welding, cutting or polishing of copper and brass and in joinery shops
    where preserved woods are used.  Other occupations in which exposures
    to copper and compounds occur are agriculture (fungicides), wood
    working, textiles, munitions and pyrotechnics, electrical, paint,
    paper and tyre manufacturing (Fisher, 1992).

         Very little published data could be found on copper
    concentrations in air within occupational settings.  Although dust and
    fume levels may be measured regularly, they are normally reported in
    terms of concentrations of other elements of greater toxicological
    significance (e.g. arsenic, lead, acid mist).  The bias towards
    reporting these contaminants explains the difficulty of relating any
    health effects noted in these environments to copper.  Most countries
    have set exposure standards for copper containing dust in the range
    0.5-1 mg Cu/m3 and for copper fumes between 0.1 and 0.2 mg Cu/m3
    (ILO, 1991).

         Some sense of the relationship between air copper and serum
    copper levels can be obtained from a study of copper milling and
    sanding operations in which exposures were reported as 0.01 and 0.68
    mg Cu/m3, respectively: plasma copper levels in these workers ranged
    from 660 to 1260 µg Cu/litre, all below the upper level reported for
    adults of 1300 µg Cu/litre (NIOSH, 1981a).  In another study (NIOSH,
    1981b), personal sampling of smelter workers in the blast and
    converter furnaces and in the sampling area had a mean copper fume
    concentration of 0.39 Cu/m3 with a range from 0.12 to 0.99 mg Cu/m3,
    while personal samples for workers exposed to copper dust during the
    cleaning of waste heat boilers and mertz furnace tear-down had average
    exposures ranging from 1.2 to 17.6 mg Cu/m3. Serum copper values in
    these workers were unrelated to occupational exposure levels.
    Particle size distribution for the dust exposures were not given,
    which may partly explain the lack of a relationship. Exposures during
    welding of brassware ranged from 0.027 to 0.89 µg Cu/m3 with a mean
    of 0.36 µg Cu/m3 (Rastogi et al., 1992).

    5.4  Total human intake of copper from all environmental pathways

         For healthy, non-occupationally-exposed humans the major route of
    exposure to copper is oral.  As shown in Table 6, the total daily
    intake of copper in adults ranges between 0.9 and 2.2 mg.  A majority
    of studies have found intakes to be at the lower end of that range.
    The variation reflects different dietary habits as well as different
    agricultural and food processing practices used worldwide.  In some
    cases, drinking-water may make a substantial additional contribution
    to the total daily intake of copper, particularly in households where
    corrosive waters have stood in copper pipes.  In areas without copper
    piping copper intake from drinking-water will seldom exceed 0.1
    mg/day, although intakes greater than a few mg per day can result from
    corrosive water distributed through copper pipes.  In general, total
    daily oral intakes of copper will be between 1 and 2 mg/day, although
    they may occasionally exceed 5 mg/day.

         All other intakes of copper (inhalation and dermal) are
    insignificant in comparison to the oral route.  Inhalation adds
    0.3-2.0 µg/day from dusts and smoke.  Even women using copper IUDs
    will be exposed to only 80 µg or less of copper per day in addition to
    their oral intake of between 1 and 3 mg.


         Copper is an essential trace element involved in a variety of
    critical metabolic processes. However, as with other essential trace
    elements such as iron and zinc, excessive exposure may be toxic. All
    mammals have metabolic mechanisms that maintain homoeostasis (a
    balance between metabolic requirements and prevention against toxic
    accumulation). Special populations with genetic defects or
    abnormalities in the metabolism of copper may be sensitive to levels
    of exposure that are nontoxic to persons without these defects. This
    chapter provides an overview of the metabolic mechanisms that provide
    copper homoeostasis in mammalian systems.

         An organism, or cells within an organism, will seek to maintain
    copper levels within a range that avoids both deficiency and excess.
    The mechanisms for absorption and storage of copper are relatively
    little studied but include biological chelators, specific receptors,
    sequestering peptides and proteins and uptake pumps.  Likewise, the
    defence mechanisms to prevent or limit copper toxicity include
    extracellular chelators, sequestering peptides and proteins, export
    pumps and disposal of the metal into vesicles.  Many of the peptides
    and proteins that are involved in these events have been characterized
    and their metabolic roles investigated.  The regulation of copper
    metabolism is not fully understood, although a great deal is being
    learned from simple model systems.

         Critical to the metabolism of copper is the chemical behaviour of
    the element and its complexes because this behaviour controls its
    interaction with other elements in processes such as absorption,
    transport, distribution and toxicity.  The general metabolism of
    copper is described in the following sections.  The bulk of the
    studies related here are derived from animal and other model systems.
    Where appropriate, sections will highlight human studies.

    6.1  Essentiality

         The essentiality of copper was not recognized until 1928 when
    Hart et al. (1928) showed copper to be essential for erythropoiesis in
    rats fed a milk-based diet. He was able to correct the anaemia by the
    addition to the diet of ash from animal or vegetable sources. He went
    on to demonstrate that the hydrogen sulfide precipitate from the ash,
    containing copper sulfide, was responsible for the recovery. Similar
    findings in humans established the basis for essentiality (Mills,
    1930; Josephs, 1931).

         Copper is also essential for the utilization of iron in the
    formation of haemoglobin (Friberg et al., 1979) and in the maturation
    of neutrophils (Percival, 1995).

         The essentiality of copper arises from its specific incorporation
    into a large number of enzymatic and structural proteins.  The role of
    copper in oxidation/reduction enzyme activities is a consequence of
    its ability to function as an electron transfer intermediate. Thus
    copper is present in enzymes involved in cellular respiration, free

    radical defence, neurotransmitter function, connective tissue
    biosynthesis and cellular iron metabolism. In some of them, copper is
    required as a cofactor, e.g. superoxidase dismutase 1 (SOD1),
    cytochrome oxidase and ceruloplasmin.  Moreover, the oxidase
    activities of ceruloplasmin and SOD1 have been shown to specifically
    require copper. In other cases, copper appears to be involved as an
    allosteric component of enzymes, conferring an appropriate structure
    for their catalytic activity.  No other element can substitute into
    these proteins to provide the redox properties that copper provides.
    These enzymes serve critical functions in their respective organisms
    (Hartmann & Evenson, 1992; Linder & Hazegh-Azam, 1996).  An
    illustrative selected list of the enzymes that rely on the redox
    properties of copper for catalysis is shown in Table 7.

         Copper plays an important role in the activation and repression
    of gene transcription.  Studies of copper-regulated transcription in
    yeast have advanced the identification of the mechanisms of action of
    copper-regulated transcription factors in eukaryotes.  ACE1 (Dameron
    et al., 1991) and AMT (Zhou & Theil, 1991) are homologous copper-DNA
    binding proteins that regulate the synthesis of the metallothionein
    message through specific fungal promoter elements in, respectively,
     Saccharomyces cerevisiae and  C. glabrata.  The  S. cerevisiae SOD
    is also regulated by ACE1 (Gralla et al., 1991; Carry et al., 1991).
    Metal responsive elements (MREs), 13-15 base pair repeats, have been
    found in the metallothionein promoters of all higher eukaryotes, but
    the metal-regulated transcription factors have not been characterized.
    Mac1 has been found to regulate the transcription of FRE1 (encoding a
    plama membrane protein associated with both Cu(II) and Fe(III)
    reduction) and CTT1 (encoding the cytosolic catalase) (Jungmann et
    al., 1993).

         Despite the obvious differences in physical form, at a
    metabolic/biochemical level animals have very similar molecular
    requirements for copper.  The deficiencies, therefore, are very
    similar to those described for copper deficiencies in humans.  The
    copper-dependent enzyme lysyl oxidase, for instance, has been
    associated with connective tissue disorders involving cardiovascular
    lesions, bone formation and eggshell development. Cardiovascular
    lesions associated with copper deficiencies have been found in mice
    (Rowe et al., 1977), rats (Petering et al., 1986), rabbits (Hunt &
    Carlton, 1965; Hunt et al., 1970), pigs (Ganezer et al., 1976;
    Schoenemann et al., 1990), and cattle (Mills et al., 1976). In
    chickens and mice the lesions have been linked to decreases in lysyl
    oxidase (Rowe et al., 1977).  Similarly rats (Alfaro & Heaton, 1973),
    cattle (Mills et al., 1976) and chicks (Rucker et al., 1969) manifest
    bone formation defects in copper deficiencies. Copper-deficient hens
    lay eggs with weak or no shells as a result of the failure of lysyl
    oxidase in the oviduct (Harris et al., 1980).  Animals also show
    evidence of hair discolouration and brittleness and flaccid skin, as
    seen in humans (Blakley & Hamilton, 1985).

        Table 7.  Copper metalloenzymes and proteinsa

    Enzyme                                  Function

    Amino acid oxidase                      amino acid metabolism
    Ascorbate oxidase                       terminal oxidase in plants
    Azurin                                  electron transfer
    Benzylamine oxidase                     oxidation of amines
    Ceramide galactosyl transferase         myelin synthesis
    Ceruloplasmin                           copper transport, oxidation
    Cytochrome c oxidase                    terminal oxidase in animals
    Diamine oxidase                         amine metabolism
    Dopamine-ß-hydroxylase                  norepinephrine (noradrenalin) synthesis
    Galactose oxidase                       carbohydrate metabolism
    Haemerythrin                            oxygen transport
    Haemocyanin                             oxygen transport
    Indole 2,3-dioxygenase                  amine metabolism
    Laccase                                 terminal oxidase, plants
    Lysyl oxidase                           collagen, elastin cross-linking
    Plastocyanin                            electron transfer in plants
    Polyphenyl oxidase                      quinone biosynthesis
    Prostaglandin reductase                 prostaglandin biosynthesis
    Rusticyanin                             electron transfer in fungi
    Stellacyanin                            electron transfer in fungi
    Superoxide dismutase                    superoxide radical destruction, dismutation
    Tyrosinase                              amino acid metabolism, pigment formation
    Uricase                                 nucleic acid metabolism
    Spermine oxidase                        amine metabolism
    Tryptophan 2,3-dioxygenase              amino acid metabolism
    Monoamine oxidasea                      neurotransmitter synthesis

    a  Linder & Hazegh-Azam (1996)

    6.2  Homoeostasis

    6.2.1  Cellular basis of homoeostasis

         An interpretation of the intracellular homoeostasis of copper in
    an human hepatocyte (the pathway and regulation of the importation,
    utilization, detoxification and export of copper) is illustrated in
    Fig. 1.  Copper itself has a major role in the regulation of the
    mechanisms that control its cellular homoeostasis.

         Copper as Cu(II) entering into hepatocytes is initially reduced
    and complexed by glutathione, prior to binding and induction of
    metallothionein (Freedman, 1989).  Alternatively, copper entering the
    cell may be exported by a copper ATPase translocase.

         Metallothionein, the main intracellular copper-binding protein,
    is a protein with 62 amino acids and two domains, rich in cysteine
    (30%), which can bind up to 12 Cu(I) atoms.  The metallothioneins are
    involved in the detoxification and possibly storage of excess copper
    (Bremner, 1987).  All metallothioneins are transcriptionally regulated
    by metals, except two newly isolated metallothioneins that may have
    specialized functions (Hammer, 1986; Palmiter, 1993; Palmiter et al.,
    1993).  A wide variety of metals have been shown to induce the
    synthesis of metallothioneins.  The mammalian transcription factor is
    a complex of proteins activated by a wide range of metals (Palmiter,
    1993).   When copper binds to the transcription factor complex, its
    affinity for metal regulatory elements in the promoter of the
    metallothionein gene is enhanced.  The resulting increased level of
    metallothionein sequesters the excess copper, preventing toxicity.

         Copper ions are exported from liver cells by a P-type copper ATP
    translocase (Cox, 1995).  The copper translocases in liver are located
    in the Golgi, endoplasmic reticulum and plasma membrane and are
    responsible for copper transport.  A mutation of this gene is
    responsible for Wilson disease.  Copper is poorly incorporated into
    ceruloplasmin when the translocase is defective (Cox, 1995). Metal
    ions are also sequestered into lysosomes, especially in conditions of
    copper overload (Mohan et al., 1995).

    6.2.2  Absorption in animals and humans

         Foods rather than water contribute virtually all of the copper
    consumed, and the copper content of different foods varies
    considerably.  Absorption of copper occurs primarily through the
    gastrointestinal tract although small amounts can be incorporated
    through inhalation and skin contact.  The intestinal absorption
    process is affected by numerous physiological and dietary factors as
    described in section 6.4.

         Radioisotope studies in experimental animals suggest that copper
    is absorbed from the stomach to some extent, but that the major site
    of absorption is the duodenum (Van Campen & Mitchell, 1965).  The pH
    of the stomach is such that many weak copper complexes will
    dissociate.  Enzymatic degradation of proteins and dietary fibres
    should also make the metal more available.  It also appears likely
    that low molecular weight substances (e.g. amino acids) in

    FIGURE 2

    gastrointestinal secretions such as saliva, gastric and pancreatic
    juice, bind copper and thereby maintain the metal in solution in the
    alkaline milieu of the upper small intestine (Gollan & Dellor, 1973).
    Moreover, it has been suggested that copper is primarily absorbed in
    the form of amino acid complexes (Marceau et al., 1970). Limited
    absorption of copper also occurs at the distal part of the small
    intestine. Absorption of copper across the brush border into the cells
    of the intestinal mucosa and its subsequent transfer across the
    basolateral membrane into interstitial fluid and blood occur by
    different mechanisms. Transfer across the mucosal barrier probably
    occurs by non-energy-dependent diffusion. With the levels of copper
    normally ingested, transfer of copper across the basolateral membrane
    appears to be rate-limiting and is mediated by a saturable,
    energy-dependent mechanism. At higher intakes, additional diffusional
    or carrier-mediated systems in the basolateral membrane come into
    play, and it seems likely that these are the sites where competition
    for absorption between copper and other transition metal ions takes
    place (Linder, 1991).

         Turnlund et al. (1989) have used stable isotope methodology to
    study copper absorption in adults.  Diets were labelled extrinsically
    with 65Cu and isotope mass ratios were analysed in the diets and
    stools by thermal ionization mass spectrometry. Copper absorption was
    dependent on the amount of copper in the diet; when a low copper diet
    (0.78 mg Cu/day) was given, absorption was 55.6%, whereas it was 36.3%
    from the same diet with copper added to an adequate level (1.68 mg
    Cu/day) and 12.4% from the same diet but with high copper content
    (7.53 mg Cu/day). Thus, it appears that copper absorption in adults is
    saturable and that the percentage absorbed decreases with the level of
    dietary copper. However, total retention of copper increased with the
    level of dietary copper. Balances were positive even at the lower
    copper level studied, suggesting that copper intakes of approximately
    0.8 mg/day are adequate to sustain balance.

         Early balance studies in preterm infants by Cavell & Widdowson
    (1964) and Dauncey et al. (1977) showed negative balances of copper
    for several months after birth.  Most of the copper was found in the
    stool, suggesting ineffective absorption or poor retention mechanisms.
    Negative copper balance was also found in 40% of infants studied by
    Tyrala (1986) despite feeding a formula with a copper concentration of
    2.1 mg/litre. More recent studies in "healthy" preterm infants fed
    modern artificial formula or unpasteurized human milk using combined
    chemical balance and stable isotope tracer (65Cu) determinations
    indicate that they absorb sufficient copper to meet the requirements

    imposed by growth. Twelve infants fed preterm human milk absorbed
    40-60% of intake while 33 receiving premature formula absorbed only
    15%. The absolute retention of copper in infants fed human milk (40-50
    µg/kg per day) approached the expected retention based on  in utero 
    accretion data. This study demonstrates that infants respond to a
    higher copper intake in a similar way to adults, by increasing fecal
    losses and decreasing percentage absorption (Ehrenkrnaz et al., 1989).

         A portion of the absorbed copper is lost during the turnover of
    the intestinal cells and is subsequently lost in the faeces.  Copper
    absorbed into the intestinal endothelial cells can be sequestered by
    metallothionein or may pass into the portal circulation.
    Metallothionein may be an intermediate through which all or part of
    the absorbed copper passes in route to the circulation (Felix et al.,
    1990). Most of the copper transfer across the serosal membrane appears
    to be done by the copper translocase.  This mechanism operates in
    animals and humans, and homologous proteins have been identified in
    yeasts (Rad et al., 1994) and bacteria (Odermatt et al., 1993; Solioz
    et al., 1994).  Intestinal metallothionein may be acting as a
    temporary metal-storage protein and be involved in the detoxification
    of excess copper.

         Pulmonary absorption occurs through the inhalation of dusts,
    fumes, smoke and sprays.  Persistent exposure to copper in sprays,
    such as Bordeaux mixture, can lead to increased absorption and
    accumulation (Pimentel & Marques, 1969; Pimentel & Menezes, 1975;
    Viren & Silvers, 1994).

         Topical use of copper compounds, as treatment for or prevention
    of microbial infections, can lead to increased copper absorption
    (Eldad et al., 1995).

    6.2.3  Transport, distribution and storage

         The liver is the major organ for the distribution of copper in
    mammals.  The liver sequesters the newly absorbed copper, routing it
    through the blood to other tissues (Owen, 1965; Evans, 1973; Marceau &
    Aspin, 1973a; Sternlieb, 1980).  In blood, copper is distributed into
    a nonexchangeable red cell pool, a plasma pool associated with
    proteins, and a labile pool of low molecular weight complexes.  In
    humans, approximately 80-90% of the plasma copper is tightly bound
    ceruloplasmin while the rest is bound to albumin and amino acids.

         In rats, ingested copper (64Cu) appears first in the blood
    complexed to albumin; a small portion of newly absorbed copper was
    later shown to complex with amino acids in the serum (Neumann &
    Sass-Kortsak, 1967).  Albumin is a 68 kDa protein, found in serum and
    in the interstitial spaces, which has copper binding sites.
    Approximately 50% of the copper in whole blood is in erythrocyte SOD
    and small peptide complexes.  Erythrocyte copper does not play a role

    in the transport of newly absorbed copper from the gut to the liver
    (Gubler et al., 1953). Ceruloplasmin does not have a role in transport
    of copper from gut to the liver, which is principally carried out by
    albumin and amino acid complexes.  Recently  in vivo NMR analysis of
    whole blood has confirmed in humans that copper in the portal
    circulation is bound to albumin (Bligh et al., 1992) adding weight to
    the earlier studies (Bearn & Kunkel, 1964).

         Transport from the liver to peripheral tissues is one of the most
    widely debated issues in the field of copper metabolism, but it is
    thought to involve ceruloplasmin, albumin, transcuprien or amino
    acids.  Metallothionein has been suggested to play an important role
    in the transport of copper in fetal blood. Its concentration is
    elevated in the plasma and there appears to be little copper bound to
    ceruloplasmin and albumin (Bremner, 1987). The proposal that
    metallothionein is involved in the fetal copper transport has been
    questioned, as mouse mutants lacking metallothionein develop normally
    (Michalska & Choo, 1993; Masters et al., 1994).

         Transport of copper from the liver to the peripheral tissues is
    presumed to require either ceruloplasmin or serum albumin.  The
    available studies can neither exclude or prove the possibility that
    one of these proteins is an obligatory copper transporter (Linder et
    al., 1998).  The peripheral tissues of humans with little or no
    ceruloplasmin are not copper deficient (Frommer, 1981).  Radioisotope
    studies (Owen, 1965; Marceau & Aspin, 1973a,b), in which an isotope of
    copper (64Cu or 67Cu) is used to trace the transfer of copper from
    one metabolic pool to another, are more supportive of ceruloplasmin's
    role in copper transport.  Its role is also supported by nutritional
    studies (DiSilvestro & Harris, 1981; Harris & DiSilvestro, 1981) and
    combined isotopic and nutritional studies (Dameron & Harris, 1987a,b;
    Percival & Harris, 1990, 1991; Steinkuhler et al., 1991).  The
    conflicting observations could be reconciled if there is redundancy in
    the transport process, as might be expected for a critical process
    like the delivery of copper.

         Receptors for ceruloplasmin have been tentatively identified in
    the plasma membrane fractions of chick aorta and heart (Stevens et
    al., 1984), rat erythrocytes (Stern & Frieden, 1993), rat liver
    (Kataoka & Tavassoli, 1984; Tavassoli et al., 1986; Omoto & Tavassoli,
    1990) and rat brain (Mash et al., 1990).  Membrane receptors for
    ceruloplasmin have also been described in human erythrocytes (Barnes &
    Frieden, 1984) and leukocytes (Kataoka & Tavassoli, 1985), and K562
    cells (Percival & Harris, 1988, 1990).  The studies by Percival &
    Harris (1990) imply that the copper may be removed from ceruloplasmin
    after reduction and that the protein may not be internalized.

         A carrier-mediated facilitated diffusion system for uptake of
    copper complexes, amino acids and small peptides, into rat
    hypothalamus has been identified (Hartter & Barnea, 1988).  The system
    has a broad ligand specificity with respect to amino acids (histidine,
    cysteine, threonine, glycine) and polypeptides (Gly-His-Lys,
    glutathione) but will not transport albumin-bound copper.

         Absorbed copper is primarily incorporated into the soluble
    fraction of the liver and is associated with three main liver
    fractions in the cytosol: a high molecular weight pool that has not
    been completely identified, a 30 000 kDa pool which appears to be SOD
    and a 10 000 kDa pool composed mostly of metallothionein.  In chicks
    and other animals, newly absorbed copper appears to be initially
    incorporated into SOD and metallothionein (Balthrop et al., 1982), the
    amount incorporated into each varying with the amount of copper
    absorbed and the route of administration (Prins & van den Hamer,
    1981).  Some of the copper that enters the liver is not retained in or
    does not enter the protein fractions and is instead excreted through
    the bile.  Copper bound to metallothionein may be targeted for
    excretion through the bile, but may be used in the synthesis of other
    copper proteins (Bremner, 1987).  The role of metallothionein in the
    cellular detoxification of copper, and possible roles for this protein
    in the uptake, storage and transport of copper, have been reviewed by
    Bremner (1987).

         The liver synthesizes and regulates the plasma levels of
    ceruloplasmin, the major copper-binding protein in serum and
    cerebrospinal fluid.  Some other tissues also synthesize
    ceruloplasmin, or isoforms produced from alternative splice sites
    (Yang et al., 1990).

         Ceruloplasmin (ferroxidase) is a 160 kDa, blue, heavily
    glycosylated, alpha2-globulin, with 6-8 tightly bound Cu(II) atoms
    (Owen, 1982).  It is an acute-phase plasma protein, increasing in
    concentration in a variety of non-specific diseases.  It also has
    ferroxidase activity and facilitates the oxidation of Fe2+ to Fe3+
    (Frieden & Hsieh, 1976).

         Copper-deficient diets lower total liver copper, metallothionein
    copper (Balthrop, 1982), and copper-zinc SOD activity (Dreosti &
    Record, 1978; Bettger et al., 1978).  Synthesis of fully active
    ceruloplasmin by the liver is decreased or eliminated in
    copper-deficient animals (Owen, 1965; Harris & DiSilvestro, 1981) and
    in humans with Wilson disease.  In contrast, deficient diets can lower
    the copper enzyme levels in some tissues even when the tissue copper
    level is constant.  Aortic lysyl oxidase, an extracellular enzyme,
    decreases in chicks on a copper-deficient diet (Harris et al., 1974),
    even though the tissue copper level does not decrease (Balthrop et
    al., 1982).

         Copper balance and tissue distribution in typical adult humans is
    summarized in Fig. 1.  Liver copper content accounts for close to 20%;
    this is the only true storage site that can be mobilized in case of
    negative copper balance. Muscle accounts for nearly 40% of total body
    copper and brain close to 20%.  Connective tissue, blood and kidney
    each accounts for 8%.

         The fetus is fully dependent on copper uptake from the maternal
    circulation. The transport of copper through the placenta is mediated
    by a specific carrier copper transport from ceruloplasmin (McArdle &
    Erlich, 1991; Lee et al., 1993). Other copper-binding complexes such
    as albumin, or histidine-bound copper, can also contribute to the
    fetal supply (Wirth & Linder, 1985). The fetus accumulates copper at a
    mean rate of close to 50 µg/kg per day, principally over the later
    half of pregnancy; over half of the copper is stored in the liver,
    mainly in the form of metallothionein (Widdowson et al., 1974). The
    increase in fetal liver store is due to both increased liver size and
    higher concentration per unit of liver weight. The brain is the second
    site for copper in fetal life; by the end of gestation the fetus will
    have accumulated close to 15 mg of copper, of which 9 mg will be in
    the liver.  After birth the concentration of copper in the liver drops
    during the initial months of life, reaching adult levels by 6 months.
    Copper saturation of metallothionein is high during the first 6 months
    of life (up to 50%), dropping quickly thereafter (Klein et al., 1991).
    Biliary secretion is extremely low  in utero and rises progressively

         Pregnancy is associated with increase copper retention: this may
    be due in part to decreased biliary excretion induced by hormonal
    changes typical of pregnancy. Serum copper and ceruloplasmin rise
    significantly during the last trimester (McArdle, 1995). Maternal
    plasma copper concentrations during the latter half of gestation are
    5-7 times higher than levels measured in the cord blood.

    6.2.4  Excretion

         Bile constitutes the major route of excretion of liver copper in
    mammals, and thus represents the most important homoeostatic mechanism
    determining the hepatocellular levels of the metal (Cousins, 1985;
    Winge & Mehra, 1990). Approximately 80% of the copper leaving the
    liver is excreted via the bile (Winge & Mehra, 1990).  The urinary
    excretion of copper is quantitatively unimportant and only 30-60 µg of
    copper is eliminated through this route per day in adult human
    (Harris, 1991).

         Several pathways have been proposed to explain copper transport
    into the bile (Kressner et al., 1984). Kinetic studies using
    radioisotopes of copper have revealed that the intracellular source of
    copper to be excreted in the bile is in a different compartment from
    the copper destined for incorporation into ceruloplasmin (Dunn et al.,
    1991).  The existence of at least two transcellular pathways via the
    hepatocytes has been proposed. Copper transport into bile takes place
    in association with the biliary excretion of glutathione (Freedman et
    al., 1989).  It has been suggested that glutathione is involved in the
    final step of copper excretion from the hepatocyte into the bile
    (Alexander & Aaseth, 1980). The coordinated release of copper and
    lysosomal enzymes into the bile of normal and copper-loaded rats
    suggests that biliary copper may be largely derived from lysosomes
    (Gross et al., 1989) and thus biliary copper excretion may be related
    also to the hepatocellular content of metallothionein.

         Copper is found bound to a range of unidentified components of
    both high and low molecular weight, which may consist of protein,
    micelles, bile salts, peptides and amino acids, depending on the
    species and on the degree of copper loading (Bremner, 1987).  However,
    none of the major forms can be related to copper complexes identified
    in the liver, although small amounts of ceruloplasmin, metallothionein
    and glutathione or their degradation products may be present (Sato &
    Bremner, 1984; Bremner et al., 1987).

         In rats, net biliary copper excretion is relatively low in the
    first week of life and is independent of metallothionein and
    glutathione secretion.  Excretion increases significantly as
    glutathione output increases (Mohan et al., 1995).  Studies with human
    hepatic and gallbladder bile have documented the presence of a major
    high molecular weight glycoprotein, which avidly binds copper (Gollan
    & Dellor, 1973).  A low molecular weight component(s) is also present
    in both rat and human bile (Gollan & Dellor, 1973).  Both the high and
    low molecular weight components await characterization.  Copper bound
    to the macromolecular component in bile undergoes minimal intestinal
    reabsorption.  Thus, biliary copper does not appear to undergo
    significant enterohepatic circulation (Gollan & Dellor, 1973), with
    most being recovered in the faeces (Winge & Mehra, 1990).

         In sheep, biliary excretion of copper does not represent the
    major elimination pathway. However, this route of copper excretion can
    be enhanced by the administration of tetrathiomolybdate (Winge &
    Mehra, 1990). In addition to an elevation in biliary excretion of
    copper, the hepatic copper levels are also reduced in treated sheep
    (Gooneratne et al., 1989). The limited biliary excretion of copper in
    sheep may partly account for the susceptibility of sheep to
    copper-associated toxicity (Winge & Mehra, 1990).

         Animals that tolerate copper well exhibit an enhanced biliary
    excretion of copper.  Copper-loaded rats, with hepatic copper levels
    up to 8-fold greater than controls, have shown a 10-fold increase in
    biliary copper output (Gross et al., 1989).  Biliary obstruction

    induced by deliberate ligation or pathological lesions, or due to a
    particular metabolic state of the animal, leads to significant hepatic
    copper retention as well as some increase in urinary copper excretion
    (Gross et al., 1989). Retention of hepatic copper also occurs in
    pregnant rats correlating with diminishing biliary excretion (Winge &
    Mehra, 1990).

         At least three genetic disorders associated with defective
    hepatobiliary copper transport and accumulation of copper in the liver
    have been described: Wilson disease (hepatolenticular degeneration) in
    human and copper toxicosis in Bedlington terriers and Long-Evans
    cinnamon rats (Sternlieb, 1980; Schilsky & Sternlieb, 1993; Mori et
    al., 1994). These disorders are characterized by a decreased biliary
    copper excretion, but differ from each other in the hepatic
    distribution of the retained copper.

         Minimal amounts of copper are lost in human sweat.  The loss is
    not believed to be sufficient to disturb the normal copper balance
    (Turnlund et al., 1990).

    6.3  Methods of studying homoeostasis

         The purpose of this section is to highlight appropriate clinical
    and biochemical methods that can be used to assess the copper status
    of laboratory animals and humans.  The goal is not to provide a
    compendium of methods and analytical techniques but to offer an
    overview of how to conduct these studies.

    6.3.1  Analytical methods

         A detailed discussion of analytical methods for the determination
    of copper in solids and dilute liquids is given in chapter 2 of this
    monograph and in WHO (1996).  In general, solid samples require an
    acid digestion prior to flame AAS.  Low concentration samples require
    more sensitive methods such as GF-AAS.  Radioactive copper isotopes
    64Cu and 67Cu (chapter 2) have been widely used in experimental
    animals and cell culture studies to follow the uptake and distribution
    of the metal (Petris et al., 1996).  The short half-lives of these
    isotopes and safety considerations make them less suitable for human
    studies.  The stable isotope 65Cu is now widely available and
    relatively inexpensive. Determination of the enrichment of the
    65Cu/63Cu ratio in human body fluids and excreta after a bolus dose
    of 65Cu can be measured either by thermal ionization mass
    spectrometry (TIMS) (Turnlund et al., 1989) or by ICP-MS (Lyon & Fell,
    1990; Lyon et al., 1995, 1996).

    6.3.2  Intake

         The principle purpose of dietary intake analysis is to determine
    the adequacy of copper supply and bioavailability for the general
    population or sub-populations.  Dietary analysis requires the
    determination of copper in food and liquids that are consumed.

    6.3.3  Diet

         The preferred procedure for assessment of copper intake is the
    use of "duplicate diet studies" in which a duplicate portion of all
    food normally consumed by the test subject is collected, and the total
    copper content determined.  A secondary method is to estimate the
    copper intake through dietary surveys using food composition from
    tables.  Descriptions of methods for dietary assessment of the trace
    elements have been published by WHO (1996).

         There is a need for standardized sampling and analytical
    procedures for the determination of dietary copper.  There is also a
    great need for standardized sampling and analytical procedures for the
    analysis of copper in drinking-water.  Where appropriate, the copper
    content of foods such as infant formulae prepared using drinking-water
    should also be measured.

    6.3.4  Balance studies

         The difference between the total copper input (diet and water)
    and the total output (faeces and urine) is the  copper balance. 
    Balance data provide an estimate of whether the body is losing or
    gaining copper. Copper balance can be used to estimate the amount
    required to prevent deficit, since a negative balance in the long run
    will give rise to clinical signs of deficit; conversely, a positive
    balance, except during growth, will give rise to potential problems
    once reserves are replete. In order to achieve copper balance children
    require 0.1-0.15 mg Cu/kg body weight per day; adults need 0.02-0.05
    mg Cu/kg body weight (1-3 mg/day).  In general the percentage of
    copper absorbed from the intestinal tract decreases as copper intake

         Estimation of copper excretion is primarily made by the
    determination of fecal copper loss. Healthy subjects are in
    equilibrium; that is, dietary intake equals fecal copper output (see
    Fig. 1 on page 78). The duration of faecal collection should be at
    least 3-5 days for children and appropriate inert markers should be
    used to ensure completeness of collection.  Longer periods may be
    necessary for adequate balance studies in adult humans.  Fecal output
    represents both the copper that is not absorbed from the gut and also
    any excreted through the bile.

         Urinary copper is a minor pathway for excretion (see Fig. 1) but
    should be measured to assure completeness of any balance study.
    Urinary copper is increased when renal tubular function is
    compromised. It can also be increased in copper overload (O'Donohue et
    al., 1993).  Sequential measurement of urinary copper excretion can be
    used to monitor chelation therapy in Wilson disease.

         The balance data from chemically defined diets are used to
    develop an understanding of the bioavailability and percentage
    retention using different copper intakes.  Such data can be used to
    estimate the amount of copper required to prevent deficit and give

    some information on the functional and clinical effects of excess
    intakes.  Some balance studies are summarized in Table 8.

         The use of copper tracers, radioisotopes and stable isotopes
    provides kinetic information to complement the balance studies. The
    results from such studies can be mathematically modelled to provide
    estimates of whole body and specific tissue compartments, such as
    liver stores. True absorption and endogenous losses can be directly
    measured from the copper isotope ratios in stool and diet (Turnlund et
    al., 1991).

         The reference interval for serum copper for normal adult males is
    in the range 800-1200 µg/litre (WHO, 1996).  Values for women are
    about 10% higher.  Serum copper is reduced in moderate to severe
    symptomatic copper deficiency.  However, serum copper concentration is
    not a sensitive marker of recent onset of deficiency (Milne et al.,
    1990; Turnlund et al., 1990; Milne & Johnson, 1993). Other conditions
    which modify these laboratory parameters include inflammation or
    infection, neoplasms and anticonvulsant or oestrogen therapy
    (Solomons, 1979; Fischer et al., 1990; Jain & Mohan, 1991; Nielsen et
    al., 1992; Milne & Johnson, 1993).

         In copper-deficient infants, it is mainly the ceruloplasmin-bound
    fraction of serum copper that is decreased (Holtzman et al., 1970).
    The non-ceruloplasmin fraction of serum copper is much less affected
    and is more rapidly restored when copper supplementation is initiated.
    Apo-ceruloplasmin cannot be detected in human serum during copper
    deficiency, suggesting that even if the apo-form may accumulate in the
    liver (Holtzman et al., 1970), ceruloplasmin is not released until the
    holo-form can be formed.  However, even if apo-ceruloplasmin cannot be
    detected in its completely unsaturated form, low ceruloplasmin enzyme
    activity, concomitant with normal immunoreactive ceruloplasmin levels,
    has been observed in copper-deficient human adults.  In fact, it has
    been suggested that the ratio between ceruloplasmin oxidase activity
    and its mass concentration determined by immunological methods may be
    used as an indicator of copper status (Milne & Johnson, 1993).  Recent
    studies by one group, in which the enzymatic activity and
    concentration of ceruloplasmin have been measured, show that in copper
    deficiency there is a reduction of enzymatic activity of ceruloplasmin
    and the ceruloplasmin protein concentration is conserved (Johnson &
    Murphy, 1988).  Therefore, the enzymatic activity/concentration
    ceruloplasmin ratio may be a better indicator of copper status, with
    the additional advantage that it is not influenced by factors such as
    hormones and gender (Vohra et al., 1965).

         Plasma copper will be elevated (up to three times the upper
    reference value) in acute copper toxicity.  In such circumstances,
    signs of intravascular haemolysis may be present.  However, in chronic
    copper overload, plasma copper and ceruloplamin concentrations are not
    elevated (O'Donohue et al., 1993).

        Table 8.  Daily copper intake and copper balance studies

    Subjects           Methods                            Results                                                                    Reference

    4 patients aged    metabolic balances were            mean copper total excretion and retention were 1.39 and 0.34               Thorn et
    between 0.36       performed on subjects who          µmol/kg per day  at a mean copper intake of 1.73 µmol/kg per day           al. (1978)
    and 1.53 years     had been on a comminuted           (110 µg/kg body weight per day) increasing to 1.72 and  0.51 µmol/kg
                       chicken diet mixed with a          per day, respectively, at a mean copper intake of 2.23 µmol/kg per
                       trace element supplement           day (142 µg/kg body weight per day)
                       for at least 3 weeks

    11 girls,          the effect of feeding two          copper excretion in the feces was significantly increased when             Greger
    12.5-14.2 years    different levels of zinc           subjects consumed the diet with the higher level of zinc. The copper       et al.
                       (11.32 mg and 11.64 mg/day)        fecal losses and apparent retention of the girls when fed 11.64 mg of      (1978)
                       on copper balance was              zinc daily were 30.60 ± 6.50 ng/day and -0.97 ± 6.09 mg/day,
                       determined during a 30-day         respectively. The corresponding figures for girls when fed 11.32 mg/day
                       period                             of zinc were 27.99 ± 1.67 ng/day and 1.40 ± 1.56 mg/day, respectively

    11 men aged        subjects were confined to a        absorption and retention averaged 36.3 ± 1.3% and 0.17 mg/day,             Turnlund
    22-35 years        metabolic research unit for 90     respectively, with an adequate-copper diet (1.68 mg/day). Absorption       et al.
                       days to determine the effect of    averaged 55.6 ± 0.9% and retention averaged -0.316 mg/day for 6 days       (1989)
                       the level of dietary copper on     and 0.093 mg/day for the next 36 days of a low-copper diet
                       absorption and retention           (0.785 mg/day).
                                                          Absorption averaged 12.4 ± 0.9% with a high-copper diet (7.53 mg/day)
                                                          and retention was strongly positive at first, decreasing linearly with
                                                          time. In conclusion: copper absorption is strongly dependent on dietary
                                                          copper level and copper balance can be achieved by most young men from
                                                          a diet of 0.8 mg of copper daily

    10 obese men       balance studies were               the mean daily intakes of zinc and copper in the soy group were 6.81       Lowy et
                       conducted over 40 days. Two        and 3.1 mg/day, respectively, and in the collagen group these figures      al. (1986)
                       diets providing, 400 kcal          were 0.32 and 0.54 mg/day, respectively. Copper balances were
                       (1.7 MJ) and 100 g of protein      determined during eight 5-day periods. During each period copper
                       daily were administered; to        balance was markedly positive in the soy-diet group and negative in
                       five subjects, a collagen diet     the collagen-diet group
                       that was severely deficient
                       in both zinc and copper,

    Table 8.  (continued)

    Subjects           Methods                            Results                                                                    Reference
    10 obese           and another five subjects,
    men                a soy diet that provided a
                       marginal intake of zinc and
                       an adequate intake of copper

    24 men aged        subjects received one of two       apparent copper balance was significantly greater when the subjects        Reiser
    21-57 years        diets low in copper (1.03 mg       consumed the fructose diet (copper intake 1.11 ± 0.02 mg, balance          et al.
                       per day and 2850 kcal, 12 MJ)      0.17 ± 0.08 mg)  as compared to the starch diet (copper intake             (1985)
                       and containing 20% of the          0.94 ± 0.04 mg, balance -0.08 ± 0.08 mg)
                       calories as either fructose or

         SOD is a copper-containing enzyme found in the cytosol of
    virtually all cells, including the erythrocyte.  Reduced SOD activity
    has been demonstrated in copper-deficient animals and in humans (Uauy
    et al., 1985).  This decrease is proportional to the magnitude of the
    deficiency of this mineral (Harris & Percival, 1991). Studies in
    humans have shown decreased activity of erythrocyte SOD in
    copper-deficient patients or in subjects receiving a low copper intake
    (Disilvestro & Harris, 1981; Van der Berg & Beynen, 1992). SOD
    activity was restored to a normal level when the subjects' diet or
    drinking-water was supplemented with copper (Vohra et al., 1965; Van
    der Berg & Beynen, 1992).

         It has also been shown in humans that cytochrome c oxidase
    activity of leukocyte and platelets is reduced in copper deficiency
    (Johnson & Murphy, 1988).  This decrease occurs before the appearance
    of a reduction of SOD activity (Johnson & Murphy, 1988).  If
    confirmed, this finding suggests that cytochrome c oxidase activity in
    leukocytes or platelets could be a sensitive indicator of copper
    status.  Although there is no single specific indicator of copper
    deficiency (WHO, 1996), evidence of deficiency can be based on
    observing the rate of disappearance of copper-dependent enzymic
    activities and their subsequent return to normal levels with copper
    supplementation.  Deficiency studies are very valuable because
    specific proteins can be singled out and studied with little
    interference from other cuproenzymes.  For instance, extracellular
    lysyl oxidase, intracellular SOD and mitochondrial cytochrome oxidase
    can be assayed, and changes over time following copper repletion
    experiments can be used to trace the movement of copper through the
    cellular compartments. To be a sensitive tool in nutritional studies,
    an enzyme must respond reversibly to a copper deficiency, be easily
    quantitated and have a short half-life so the change in activity can
    be measured rapidly. Unfortunately, the copper enzymes used in many
    studies are difficult to quantitate, hard to purify and have long
    half-lives.  The sensitivity of deficiency studies can be enhanced by
    using copper isotopes to label the target proteins, which can then be
    identified and quantitated enzymatically, immunochemically or by both
    procedures.  The major requirement in such experiments is that the
    turnover, synthesis or activation of the enzyme must be rapid so the
    isotope can be incorporated into the target protein and measured in a
    reasonably short period of time.

         Excessive copper accumulation in the liver can be determined by
    needle biopsy. This requires an adequate sample taken under controlled
    conditions in order to avoid contamination.  Analysis must be carried
    out in a specialized laboratory.  This is the preferred method for
    measurement of copper excess and should be included in the evaluation
    of children and adults with liver disease of unknown aetiology.  The
    reference value for liver copper is 20-40 µg/g (dry weight) but is
    significantly higher in the newborn.  Nonspecific copper accumulation
    occurs in a variety of cholestatic liver disease without a specific
    pathological effect.  Liver copper in excess of 250 µg/g (dry weight)
    in the presence of other biochemical and clinical evidence is

    indicative of Wilson disease, ICC or ICT (see chapter 8).  Copper
    accumulation in other tissues can be assessed only by postmortem

    6.4  Biochemical basis of copper toxicity

         The requirement for copper in various organs or systems within
    the body is effectively regulated by homoeostatic control mechanisms.
    Toxicity is likely to occur only when such homoeostatic control within
    any particular compartment is overwhelmed and/or basic cellular
    defence or repair mechanisms are impaired.

         The essentiality and potential toxicity of copper in biological
    systems relies basically on the specific electron configuration,
    particularly of the outer electron shells.  Accordingly, the cuprous
    (Cu+) ion is highly polarizable and binds mainly to nitrogen- and
    sulfur-containing ligands by sharing their electronic orbitals. Cupric
    (Cu2+) ions, on the other hand, are able to form both coordination
    complexes with oxygen-containing ligands and partly covalent bonds
    with nitrogen- and sulfur-containing centres. Therefore, copper has to
    be considered fairly reactive and able to bind strongly to many types
    of electron-rich structures. The affinity of copper ions towards a
    particular ligand, however, is also influenced by the polarizability
    of the ligand itself (Nriagu, 1979).

         Toxicity of copper may arise when excess copper provokes the
    following adverse reactions:

    *    Structural impairment of essential metal binding sites by
         displace ment of metals resulting, for example, in membrane
         changes such as depolarization and impairment of receptors or
         transporter molecules (Alt et al., 1990).

    *    Functional impairment by binding of copper to crucial sites in
         such macromolecules as DNA or enzymes particularly containing
         sulfhydryls, carboxylates or imidazoles (Alt et al., 1990). This
         will lead to direct protein damage, or oxidative DNA changes
         leading to various functional changes, because of the large
         number of enzymes dependent upon copper and the possible
         misreading of genetic codes.

    *    Cellular injury due to the production of oxyradicals by the
         Fenton reaction (Goldstein & Czapsky, 1986):

               Cu+  +  H2O2  -->  Cu2+  +  OH*  +  OH-

         The excessive production of such radicals will initiate a cascade
    of oxidation-reduction reactions (oxidative stress) finally leading to
    the loss of cellular integrity. The causes of injury considered
    include increased cytosolic calcium levels, ATP depletion, thiol
    oxidation, lipid peroxidation, DNA damage and critical damage to
    organelles such as mitochondria and lysosomes.

         Threshold levels for copper toxicity have not yet been
    established, although the main intracellular binding site for copper,
    metallothionein, appears to become saturated with copper before the
    occurrence of any toxic effects. Metallothionein also has been
    suggested to act as an intracellular antioxidant, thereby protecting
    cells by the direct scavenging of reactive oxygen species.  In vitro 
    metallothionein exhibits a very high reaction constant for hydroxyl
    radicals (Thornalley & Vasak, 1985) and according to recent
    experiments, mouse cells lacking metallothionein were more sensitive
    to oxidative stress (Liu et al., 1995).

    6.5  Interactions with other dietary components

         The absorption of copper is inhibited by the presence of some
    other essential and nonessential trace metals (e.g. zinc, iron,
    molybdenum, lead and cadmium) (WHO, 1996).  The absorption of copper
    is also influenced by a number of other dietary and endogenous
    factors.  Easily digested proteins may enhance copper absorption; for
    example, proteins in human milk are more easily digested than proteins
    in cow's milk and lend to enhance copper absorption.  Citrate,
    phosphate and glutamate all form complexes with copper that facilitate
    absorption.  Phytate, dietary cellulose fibre and ascorbic acid
    decrease copper absorption (Cousins, 1985).

    6.5.1  Protein and amino acids

         Animal protein enhances copper absorption (Turnlund et al.,
    1983). Copper absorption was higher from an animal protein diet (41%)
    than from a plant protein diet (34%). Different milk proteins have
    been shown to have varying effects on copper status: whey protein had
    a negative effect on copper absorption (Lynch & Strain, 1990).  Soy
    protein isolates, as used in infant formula, reduce copper
    bioavailability (Lo et al., 1984; Greger & Mulvaney, 1985). Specific
    amino acids are known to form complexes with divalent cations such as
    copper. Histidine chelates copper with a greater affinity than it does
    zinc (Ashmead et al., 1985). Copper accumulation in the mucosal tissue
    was higher when an excess of histidine to copper and zinc was used
    (Wapnir & Balkman, 1992). It is possible that a copper-histidine
    complex may be an effective way to provide bioavailable copper. In
    contrast, cysteine has an inhibitory effect on copper utilization
    (Robbins & Baker, 1980; Baker & Czarnecki-Maulden, 1987). This effect
    on copper absorption is evident at both deficient and excess copper
    levels in the diet (Aoyagi & Baker, 1994).

    6.5.2  Phytate and fibre

         Turnlund et al. (1984) used stable isotopes to study the effect
    of copper on the absorption of phytate and alpha-cellulose in young
    men. They found no effect of either component in human subjects and
    suggested that high levels of phytate or fibre do not decrease copper
    absorption. The authors proposed that zinc-phytate complexes
    precipitate at the pH of the gastrointestinal tract, whereas
    copper-phytate complexes do not. Since phytate in the soluble

    copper-phytate complex can easily be replaced by other chelators, such
    as amino acids (Jacobsen & Slotfeldt-Ellingsen, 1983), there may be no
    inhibitory effect of phytate on copper absorption. A study on cereal
    products supports this hypothesis (Lyon, 1984); zinc solubilized from
    cereal by the addition of acid precipitated completely when the pH was
    raised to 7, whereas copper remained in solution.

    6.5.3  Ascorbic acid

         Van den Berg & Beynen (1992) suggested that the primary effect of
    high dietary ascorbic acid was to reduce intestinal absorption of
    copper, but that it also increased hepatic uptake and biliary
    excretion of 64Cu. The effect of ascorbic acid on copper metabolism
    was more pronounced in copper-deficient than in copper-adequate

         Finley & Cerklewski (1983) found decreased ceruloplasmin oxidase
    activity and lower serum copper in young adult men after 64 days of
    1500 mg ascorbic acid/day (values were determined after the vitamin
    was discontinued).  However, this effect could be independent of lower
    copper absorption, as Jacob et al. (1987) found no difference in
    copper absorption in young men given different levels of ascorbic
    acid. Ascorbic acid may promote the dissociation of copper from
    ceruloplasmin, thus lowering its oxidase activity. This was supported
    by the finding that immunological quantitation of ceruloplasmin showed
    no change in apoprotein levels. A clinical study on low birth weight
    (LBW) infants fed formula supplemented with ascorbic acid (50 mg/day)
    did not show any negative effects on copper balance (Stack et al.,
    1990).  However, the LBW infants were largely in negative copper
    balance and thus may have been copper deficient. It is possible that
    ascorbic acid under these conditions may not exert overall negative
    effects on copper utilization as observed in copper-deficient rats
    (Van den Berg et al., 1994).

    6.5.4  Zinc

         High levels of dietary zinc have a negative effect on copper
    absorption. Since supplemental zinc is often used in infants, children
    and pregnant women in order to avoid possible zinc deficiency, the
    possible interference with copper absorption needs to be considered.
    High doses of zinc (40-50 mg/day) have been used successfully to treat
    patients with Wilson disease (Brewer et al., 1983; Hoogenraad & van
    den Hamer, 1983). Zinc limits the amount of copper absorbed (Lyons et
    al., 1995), possibly by increasing intestinal metallothionein
    concentrations and, therefore, slowing the progression of the disease
    (Fischer et al., 1983; Oestreicher & Cousins, 1985).  However, high
    intakes of zinc should be viewed with some concern since copper
    deficiency may be induced.  Conversely, copper supplementation may
    interfere with zinc absorption (Salim et al., 1986).

         Human subjects fed diets with different zinc/copper ratios have
    not exhibited a significant effect on copper absorption. August et al.
    (1989) used a stable isotope of copper to study copper absorption in

    young adults and elderly subjects. They used zinc/copper ratios of
    2 : 1, 5 : 1 and 15 : 1, finding no significant effects of these
    ratios on copper absorption.

    6.5.5  Iron

         Copper absorption may also be affected by high levels of dietary
    iron. Haschke et al. (1986) studied the effect of two levels of iron
    fortification of infant formula on copper balance in full-term
    infants. They found that the higher level of iron (10.8 mg/litre)
    resulted in lower copper balance than when the lower iron level was
    used (1.8 mg/litre). Barclay et al. (1991) have shown reduced SOD
    levels in premature infants given iron supplements. Earlier studies in
    experimental animals had shown a reduction in liver copper
    concentrations when dietary iron was increased 10-fold (Smith &
    Bidlack, 1980). However, modest supplements of iron did not appear to
    affect serum copper levels in older infants (Yip et al., 1985).
    Several studies suggest that high dietary iron only affects copper
    absorption when copper status is low or marginal (Cohen et al.,
    1985a,b; Johnson & Murphy, 1988).

         High intakes of iron and ascorbate may act together to adversely
    affect copper status.  Johnson & Murphy (1988) found that high iron
    with ascorbic acid caused severe anaemia in copper-deficient rats and
    decreased plasma ceruloplasmin by 44% in copper-adequate rats. Since
    iron and ascorbate are commonly used together in nutritional
    supplements for humans, the possibility of a negative effect on copper
    metabolism should be considered.

    6.5.6  Carbohydrates

         In rats, dietary fructose worsens the effects of copper
    deficiency (Fields et al., 1984; Reiser et al., 1985) in that fecal
    and urinary excretion of copper are elevated when the rats are fed
    fructose as compared to starch. Data from humans do not support these
    findings (Reiser et al., 1985; Holbrook et al., 1989).

    6.5.7  Infant diets

         Studies on full term infants fed on breast or cow's milk formula
    suggest that copper is better absorbed from human milk than from a
    cow's milk formula (Dörner et al., 1989). Studies using stable
    isotopes of copper support this finding (Ehrenkranz et al., 1989).
    Studies in suckling rats have revealed slightly higher copper
    bioavailability (estimated from uptake of 64Cu by 6 h post-dosing)
    from human milk than from cow's milk formula (Lonnerdal et al., 1985).
    A more recent study, using the same rat pup model, evaluated several
    varieties of infant formula (Lonnerdal et al., 1994). In general,
    copper absorption was relatively high from milk formulae but lower
    from soy formulae. The lower copper bioavailability from cow's milk
    combined with its low copper content most likely explains the copper
    deficiency found in some premature infants fed cow's milk formulae.

    6.5.8  Other interactions (molybdenum, manganese, selenium)

         Dietary molybdenum, in the presence of sulfate, forms insoluble
    complexes with copper thereby decreasing the availability of copper
    for absorption.  Thus, high levels of molybdenum in the diet may
    induce or aggravate copper deficiency (Ladefoged & Sturup, 1995).  The
    addition of copper to diets of rats decreases tissue manganese levels,
    suggesting that copper impairs manganese absorption.  Manganese
    absorption is greatest in animals that are deficient in copper and
    manganese (Johnson & Korynta, 1992).  Research efforts on
    copper-selenium interactions have not been revealing, except for
    showing the complementarity in antioxidant protection of copper SOD
    and selenium-containing glutathione peroxidase (Fischer et al., 1992;
    Olin et al., 1994).


         The effects of exposure of experimental animals to common
    inorganic salts of copper have been summarized in Tables 9-12.  These
    studies represent the better-quality and better-documented studies in
    each toxicological area.  Studies in which the compound was
    administered by injection have generally not been included, owing to
    their uncertain relevance to environmental or occupational exposures.
    The results of such studies have, however, been included in the table,
    when no information was available for more relevant routes of

         In this section and the associated tables, information on dosage
    with respect to body weight was obtained from the original papers
    wherever possible.  When doses were not expressed in this way by the
    investigators and could not be calculated from the data provided,
    approximate doses have been estimated based on data presented in
    standard sources (IAT, 1963; FDO, 1965; Gold et al., 1984).

    7.1  Single exposure

    7.1.1  Oral

         The acute oral toxicity of various copper salts is summarized in
    Table 9.  A wide range of LD50 values has been reported, with the
    most soluble salts (e.g. copper(II) sulfate and copper(II) chloride)
    generally being more acutely toxic than those with lower solubility
    (e.g. copper(II) hydroxide and copper(I) oxide).  From the available
    information on copper(II) sulfate, rats appear to be less susceptible
    to copper than domestic animals; this pattern is also evident in
    studies involving repeated exposure (section 7.2).  In the various
    acute studies, as the lethal oral dose is approached, signs of copper
    toxicity include excessive salivation, vomiting, diarrhoea, gastric
    haemorrhage, increased heart rate, hypotension, haemolytic crisis,
    convulsions and paralysis.

    7.1.2  Dermal

         In the only dermal studies identified, LD50 values of > 1124
    and > 2058 mg Cu/kg body weight per day were reported, the first for
    rats exposed to copper(II) oxysulfate (NIOSH, 1993) and the second for
    rabbits exposed to copper(II) hydroxide (Tomlin, 1994).

    7.1.3  Inhalation

         The LC50 value for inhalation exposure of rabbits to copper(II)
    hydroxide (physical form and duration unspecified) was > 1303 mg
    Cu/m3 (Tomlin, 1994).  Intratracheal instillation in rats of
    copper(II) oxide at 222 mg Cu/kg body weight was lethal (NIOSH, 1993).

        Table 9.  Toxicity of copper compounds after a single oral exposure

    Salt             Species          LD50 value       Equivalent       Reference
                                      (mg/kg body      copper dose
                                      weight)          (mg Cu/kg
                                                       body weight)

    Copper(II)       rat              595              208              NIOSH (1993)
     acetate         rat              710a             226              Smyth et al.
                     mouse            1600a            509              Schafer &
                                      (lethal dose)                     Bowles (1985)

    Copper(II)       rat              159              82               Lehman (1951)
     carbonate       mouse            320              165              Schafer &
                                      (lethal dose)                     Bowles (1985)

    Copper(II)       rat (male)       1350             388              Hasegawa et
     carbonate       rat (female)     1495             430              al. (1989)
     hydroxide       rabbit           317              91               NIPHEP (1989)

    Copper(II)       rat              140              66               Lehman (1951)
     chloride        mouse            190              90               NIPHEP (1989)
                     guinea-pig       32               15               NIPHEP (1989)

    Copper(II)       rat              1000             651              Pesticide
     hydroxide                                                          Manual (1991)

    Copper(II)       rat              940b             247              Smyth et al.
     nitrate                                                            (1969)

    Copper(I)        rat              470              417              Smyth et al.
     oxide                                                              (1969)

    Copper(II)       rat              700-800          417-476          Tomlin (1994)
     oxychloride     rat              1440             857              NIPHEP (1989)

    Copper(II)       rat              300              120              Lehman (1951)
     sulfate         rat              960c             244              Smyth et al.
                     mouse            50 (LD100)       20               Venugopal &
                                                                        Luckey (1978)
                     rabbit           125              50               Eden & Green

    a  Monohydrate
    b  Trihydrate
    c  Pentahydrate

         Guinea-pigs exposed to copper(II) oxide aerosol at 1.6 mg/m3
    (1.3 mg Cu/m3, as particles with a count median diameter
    approximately 0.03 µm) for 1 h showed significant reductions
    ( P <0.05) in tidal volume, minute volume and lung compliance, both
    during and after exposure, while respiratory frequency was slightly
    but not significantly increased (Chen et al., 1991).

         In two studies involving the intratracheal instillation in rats
    of copper(II) oxide (Hirano et al., 1993) or copper(II) sulfate
    pentahydrate (Hirano et al., 1990) at doses of up to 0.1 or 0.05 mg
    Cu/rat, respectively (roughly 0.36 or 0.18 mg Cu/kg body weight),
    acute inflammatory changes were evident in the lungs from 0.018 mg
    Cu/kg body weight with the soluble sulfate salt and from 0.073 mg
    Cu/kg body weight with the insoluble oxide.

    7.2  Short-term exposure

         There have been numerous studies of the effects of short-term
    exposure to copper compounds. In rats exposed by the oral route to
    approximately 30-50 mg Cu/kg body weight per day as copper(II)
    sulfate, the most common compound-related effects observed have
    included those on the liver, kidney and lungs, as well as alterations
    in haematology (particularly anaemia) and in blood biochemistry.
    Effects are qualitatively similar with other copper compounds, and in
    other species.  However, pigs and especially sheep are more
    susceptible to the toxic effects of copper compounds; exposure of
    sheep to doses of 1.5-7.5 mg Cu/kg body weight per day in diet as
    copper(II) sulfate or copper(II) acetate was associated with
    progressive liver damage, followed by a haemolytic crisis and
    ultimately death.  In inhalation studies, morphological changes were
    induced in the tracheal epithelium and in the alveoli by short-term
    inhalation of 0.06 mg Cu/m3 copper(II) sulfate in mice, but not in

    7.2.1  Oral

         The most comprehensive studies of short-term toxicity in rats and
    mice were conducted by Hébert et al. (1993).  In a 15-day feeding
    study in rats involving the administration of up to 16 000 mg/kg
    copper(II) sulfate pentahydrate in the diet (estimated intakes up to
    305 mg Cu/kg body weight per day), weight gain was reduced from 194 mg
    Cu/kg body weight per day, but there were no other overt signs of
    toxicity.  Effects on the forestomach were evident from 45 mg Cu/kg
    body weight per day, on the kidneys from 93 mg Cu/kg body weight per
    day, and on the liver and bone marrow from 194 mg Cu/kg body weight
    per day.  The NOEL in this study was 23 mg Cu/kg body weight per day
    (Hébert et al., 1993).  When the same investigators administered
    copper(II) sulfate to rats in the drinking-water for 15 days at up to
    30 000 mg/kg (estimated intakes up to 97 mg Cu/kg body weight per
    day), the various clinical signs of toxicity and deaths that were
    evident from around 31 mg Cu/kg body weight per day were attributed to
    dehydration, as a result of the poor palatability of the

    drinking-water.  The NOEL in females was 26 mg Cu/kg body weight per
    day, while in males there was evidence of kidney damage from the
    lowest dose tested of 10 mg Cu/kg body weight per day (Hébert et al.,
    1993).  (Effects on gastric mucosa have only been observed in rodent
    studies in which copper(II) sulfate was administered in the diet, and
    not in the drinking-water studies.  It is likely that these effects
    are due to irritation, particularly as copper(II) sulfate may
    dissociate to form sulfuric acid in the stomach.)

         From the evidence of one 15-day feeding study (Hébert et al.,
    1993), mice appear to be less sensitive than rats to the toxic effects
    of copper.  When copper(II) sulfate pentahydrate was administered at
    up to 16 000 mg/kg in the feed, weight gain was reduced only in
    females at the top dose (estimated intake 781 mg Cu/kg body weight per
    day), while the only effects observed on microscopic examination of
    the liver, kidneys and forestomach were hyperplasia and hyperkeratosis
    in the forestomach from 197 (males) or 216 (females) mg Cu/kg body
    weight per day.  The NOEL in this study was 92 mg Cu/kg body weight
    per day in males and 104 mg Cu/kg body weight per day in females.  In
    the equivalent drinking-water study, the findings (reduced water
    consumption, body weight, clinical signs at doses of 58-62 mg Cu/kg
    body weight per day and higher) were again, as in the rats, thought to
    be confounded by dehydration of the treated animals (Hébert et al.,

         Other studies summarized in more extensive reviews on copper
    (Slooff et al., 1989; ATSDR, 1990) have deficiencies in design and/or
    level of experimental details and results, which make it impossible to
    utilize in any dose-response evaluation.  They are, therefore, not
    considered here.

    7.2.2  Inhalation  Copper(II) sulfate

         When unspecified numbers of mice and hamsters were exposed by
    inhalation to copper(II) sulfate aerosol at 0.06 mg Cu/m3 for 3
    h/day, 5 days/week for 1 or 2 weeks, the tracheal epithelium and the
    alveoli of mice were altered in appearance, whereas hamsters showed no
    treatment-related effects on the tracheal epithelium or on ciliary
    activity (Drummond et al., 1986).  Copper chloride

         In an inhalation study, repeated exposure of rabbits (group sizes
    not specified) to copper(II) chloride aerosol at 0.6 ± 0.3 mg Cu/m3
    for 6 h/day, 5 days/week for 4-6 weeks did not produce any
    histological lesions in the lungs, and alveolar macrophage activity
    appeared to be unaffected despite some morphological changes
    (Johansson et al., 1983, 1984; Lundborg & Camner, 1984).

    7.3  Repeated exposure: subchronic toxicity

         There are a limited number of studies of the subchronic toxicity
    of copper compounds to animals.  In comprehensive studies in rats,
    there were histopathological effects on the forestomach and
    indications of anaemia at 34 mg Cu/kg body weight per day as
    copper(II) sulfate in diet.  Higher doses elicited degenerative
    changes in the liver and kidney in rats in this and several other
    studies, with recovery observed in some of these.  As was observed in
    the short-term studies (section 7.2), mice are markedly less sensitive
    than rats to the toxicity of copper(II) sulfate.  Other copper
    compounds have not been well studied, although exposure of rats to
    approximately 10 mg Cu/kg body weight per day as copper(I) chloride
    induced transient reductions in the activities of glutathione
     S-transferases, and the same dose as copper(II) carbonate increased
    systolic blood pressure and haemoglobin levels.

    7.3.1  Oral  Copper(II) sulfate

         The critical study is that of Hébert et al. (1993) which is
    described here.  Details of other experiments of repeated long-term
    exposures of copper are given in Table 10.

         In comprehensive 90-day studies in both rats and mice (Hébert et
    al., 1993), in which copper(II) sulfate pentahydrate was administered
    in the feed at up to 8000 mg/kg in rats (up to 138 mg Cu/kg body
    weight per day) and up to 16 000 mg/kg in mice (up to around 1000 mg
    Cu/kg body weight per day), there were no overt signs of toxicity
    other than a dose-related reduction in growth (statistically
    significant in male and female rats from 67 and 138 mg Cu/kg body
    weight per day, respectively, and in male and female mice from 97 and
    267 mg Cu/kg body weight per day).  Microscopic examination of the
    tissues revealed hyperplasia and hyperkeratosis in the forestomach in
    both species (from 34 mg Cu/kg body weight per day in rats and from
    187-267 mg Cu/kg body weight per day in mice), and liver and kidney
    effects in the rats only (from 67 mg Cu/kg body weight per day).  In
    the rats, iron levels were reduced in the spleen, and haematological
    changes indicative of microcytic anaemia were observed at 34 mg Cu/kg
    body weight per day and higher.  The NOEL was 17 mg Cu/kg body weight
    per day in rats, and 44 and 126 mg Cu/kg body weight per day in male
    and female mice, respectively.  The liver and kidney effects observed
    in the rats in this study included inflammation of the liver and
    degeneration of the kidney tubule epithelium, and were similar to
    those found at higher doses (> 100 mg Cu/kg body weight per day) in
    more limited studies in rats (Haywood, 1980, 1985; Haywood & Loughran,

        Table 10.  Toxicity of copper after repeated oral doses

    Species          Protocol                              Results                                                       Effect level   Reference

    Copper(II)       copper sulfate pentahydrate given     Survival was unaffected. Body weight gain was significantly   NOEL: 17 mg    Hébert
    sulfate          in the feed for 92 days at levels of  depressed in the males at 4000 mg copper sulfate/kg diet      Cu/kg body     et al.
    Rats (F344/N,    0, 500, 1000, 2000, 4000 and 8000     (P < 0.05) and in both sexes at 8000 mg copper sulfate/kg     weight per     (1993)
    groups of 10     mg/kg diet. Estimated intakes were    diet (P < 0.01). Average feed consumption was reduced         day
    males and 10     0, 8, 17, 34, 67 or 138 mg Cu/kg      in both sexes at 8000 mg copper sulfate/kg diet. There
    females,         body weight per day.                  were no other clinical signs of toxicity in the treated rats  LOEL: 34 mg
    additional       Comprehensive microscopic                                                                           Cu/kg body
    groups of 10     examinations carried out at the       Gross and microscopic lesions of the forestomach              weight per
    males & 10       top dose level, in the controls,      (hyperplasia and hyperkeratosis of the limiting ridge) were   day
    females for      and in the animals that died early.   seen at 2000 mg copper sulfate/kg diet and above.
    pathology        Liver, kidney and forestomach         Inflammation of the liver was seen in all rats at 8000 mg
    studies at       examinations were carried out to      copper sulfate per kg diet, all males and 6/10 females at
    intermediate     establish a NOEL. Intermediate        4000 mg copper sulfate/kg diet and one male at 2000 mg
    time points)     haematology and clinical chemistry    copper sulfate/kg diet. In the kidneys, cytoplasmic protein
                     evaluations carried out on            droplets were evident, particularly at the top two doses,
                     days 5 and 21, and urinalysis         and minimal nuclear enlargement of, and degeneration in, the
                     on day 19. These tests also           tubule epithelium were seen at the top dose. From 2000 mg
                     carried out at termination            copper sulfate per kg diet, iron levels were reduced in the
                     of the study                          spleen (both sexes) and haematological changes indicative of
                                                           microcytic anaemia were seen on day 21 and at the end of the
                                                           study. Significant increases in red bloodcells and
                                                           reticulocytes were seen in the high-dose males at the end of
                                                           the study. A number of other clinical chemistry and urinalysis
                                                           parameters were affected at the top two dose levels

    Rats (Wistar,    Rats fed diets containing 0 or 3000   In group not supplemented with copper for first 15 weeks      only one dose  Haywood
    groups of 16     mg Cu/kg as copper sulfate for        of experiment, clinical effects (lethargy, ruffled coats)     tested         &
    males)           15 weeks (equivalent to 270 mg        seen on administration of 6000 mg Cu/kg diet. No such effect  (effects at    Loughran
                     Cu/kg body weight/day). Four rats     seen in 'copper-primed' group. Livers of rats given 3000      100mg Cu/kg    (1985)
                     per group killed and livers removed   mg Cu/kg diet for 15 weeks showed only mild effects           body weight
                     for examination, remaining rats       (believed to indicate ongoing recovery from damage that       per day)
                     then fed diets containing 6000 mg     was assumed to have occurred in the earlier weeks) at 15
                     Cu/kg as copper sulfate for a         weeks, and feeding of 6000 mg Cu/kg diet for a further 3
                     further 3 weeks                       weeks had no significant hepatotoxic effects. The unprimed

    Table 10.  (continued)

    Species          Protocol                              Results                                                       Effect level   Reference
                                                           group suffered hepatocellular necrosis and inflammation
                                                           after the 3-week exposure to 6000 mg/kg

    Rats (strain     Rats fed diet containing 2000 mg      Inflammation and extensive necrosis of the liver and bile     only one dose   Haywood
    unspecified,     Cu/kg diet as copper sulfate          duct hyperplasia were evident by week 6. By week 15 there     tested          (1980)
    groups of 24     (equivalent to about 100 mg Cu/kg     was considerable recovery, although some fibrosis and less    (effects at
    treated and      body weight/day). Groups of 4         marked hyperplasia of the bile duct could still be seen       100 mg Cu/kg
    12 control       treated and 2 control rats killed                                                                   body weight
    males)           after 1, 2, 3, 6, 9 and 15 weeks      Greenish discolouration of the kidneys was seen in some       per day)
                     and their liver and kidneys           rats at week 6. Microscopic effects (eosinophilic droplets
                     examined histologically               in the cytoplasm of cells in the proximal convoluted tubules
                                                           and desquamation of these cells into the lumen) first
                                                           appeared at week 3, and were more severe at 6 weeks.
                                                           Regeneration was almost complete at 15 weeks

                                                           The investigators concluded that repeated copper dosing
                                                           elicits a similar response in the kidneys and the liver, both
                                                           organs adapting to the excess copper, resulting in the
                                                           development of tolerance in the treated rats

                     Blood was taken from the above        Alanine aminotransferase activity was significantly increased                Haywood
                     rats prior to sacrifice and           (P < 0.05) at week 1 (indicative of liver damage), rose to a                 &
                     analysed for enzyme activity          maximum around weeks 6-9, and remained at that level to                      Comerford
                                                           the end of the study. Ceruloplasmin activity was elevated                    (1980)
                                                           (P < 0.05) from week 6 until the end of the study. Alkaline
                                                           phosphatase activity and bilirubin levels were unaffected
                                                           by copper treatment

    Rats (Wistar,    Rats fed diets containing 0,          Rats receiving 6000 mg Cu/kg diet did not grow and were       LOEL: 270      Haywood
    groups of 28     3000, 4000, 5000 or 6000 mg           in poor condition. Two died at 2 weeks. At 6 weeks the        mg Cu/kg       (1985);
    males)           Cu/kg diet as copper sulfate for      survivors developed diarrhoea, began to lose weight and       body weight    Haywood
                     up to 15 weeks (equivalent to 0,      were killed. At 3000-5000 mg/kg of copper sulfate, the        per day        &
                     270, 360, 450 and 540 mg Cu/kg        animals showed clinical signs of toxicity (poor growth,                      Loughran
                     body weight per day based on          ruffled fur) at around 3-5 weeks, but their condition                        (1985)
                     the mean final weight of the rats     subsequently improved; by week 15 they appeared sleek
                     fed 3000 mg Cu/kg diet). Four         and active, but were only half the weight of controls

    Table 10.  (continued)

    Species          Protocol                              Results                                                       Effect level   Reference
                     rats at each dose level killed at
                     1, 2, 3, 4, 5, 6 and 15 weeks.        Microscopic changes were evident in the liver (necrosis,
                     Liver and kidneys removed for         inflammation, hepatocytic hypertrophy, nuclear
                     histological examination              enlargement) within 1-2 weeks, depending on the dose, but
                                                           began to subside from week 6 onwards, with regeneration by
                                                           week 15 (except at 6000 mg Cu/kg diet where the effects
                                                           persisted). Microscopic effects on the kidneys (an increase
                                                           in eosinophilic cytoplasmic droplets in cells of the
                                                           proximal tubules followed by extrusion of the droplets and
                                                           exfoliation of the cells, degenerative changes to proximal
                                                           tubules) were seen at 2-5 weeks at all dose levels, with
                                                           recovery from weeks 6-15

    Mice (B6C3F1,    Copper sulfate pentahydrate given     Survival was unaffected. A dose-related depression in         NOEL: 44       Hébert
    groups of 10     in the feed for 92 days at levels of  body weight gain was observed in both sexes                   and 126 mg     et al.
    males and        0, 1000, 2000, 4000, 8000 and         (statistically significant from 2000 mg copper sulfate/kg     Cu/kg body     (1993)
    10 females)      16 000 mg/kg diet. Estimated          diet in males and 4000 mg copper sulfate/kg diet in females,  weight per
                     intakes were 0, 44, 97, 187, 398      P < 0.05), although average feed consumption was              day in males
                     and 815 mg Cu/kg body weight          similar in treated and control mice. No other clinical        and females
                     per day in males and 0, 52, 126,      signs of toxicity were observed                               respectively
                     267, 536 and 1058 mg Cu/kg
                     body weight per day in females.       Gross and microscopic lesions of the forestomach              LOEL: 97
                     Comprehensive microscopic             (hyperplasia and hyperkeratosis of the limiting ridge)        and 267 mg
                     examinations carried out at the       were seen at 4000 mg copper sulfate/kg diet and above         Cu/kg body
                     top dose level, in the controls,                                                                    weight per
                     and in the animals that died early.   There were no reported effects on the liver or kidneys,       day in males
                     Liver, kidney and forestomach         and iron levels in the spleen were normal                     and females
                     examined to establish a NOEL                                                                        respectively

    Copper(I)        Rats given drinking-water             Activity of glutathione S-epoxide transferase was             only one       Freundt
    chloride         containing 0 or 100 mg CuCl/litre     significantly inhibited (P < 0.05) after treatment            dose tested    &
    Rats (Sprague-   (equivalent to 0 or 10 mg Cu/kg       for 15 days (-29% compared with controls) but not             (effects seen  Ibrahim
    Dawley, groups   body weight per day). Livers          after 30 or 90 days. Glutathione S-aryl transferase           at 10 mg       (1991)
    of 5 females)    removed after 15, 30 or 90 days       activity was unaffected after 15 days, was                    Cu/kg body
                     of treatment for determination of     significantly inhibited (P < 0.05) after 30 days              weight per
                     activity of glutathione S-epoxide     (-7%), and was still slightly but not significantly           day)

    Table 10.  (continued)

    Species          Protocol                              Results                                                       Effect level   Reference
                     transferase and glutathione           reduced after 90 days (-6%). (These enzymes catalyse
                     S-aryl transferase                    the metabolic inactivation of reactive substances)

    Copper(II)       Rats given 18 or 100 mg Cu/kg         Body weight, urine output and feed and water intakes did      only one       Liu &
    carbonate        diet as copper carbonate for          not differ with copper intake. High-dose rats showed          dose tested    Medeiros
    Rats (Wistar or  15 weeks (equivalent to about         increased systolic blood pressure compared with low-dose      (effects seen  (1986)
    spontaneously    1.7 and 9.6 mg Cu/kg body             rats, particularly in the Wistar strain (Wistar P < 0.05,     at 9.6 mg
    hypertensive     weight per day). Blood pressure       SHR P < 0.01 at week 15). Haemoglobin levels were increased   Cu/kg body
    rats (SHR),      measured 3 times/week                 at high copper intake (P < 0.05), while total cholesterol,    weight per
    groups of 10                                           triglycerides and glucose levels in the blood were            day)
    males of                                               unaffected
    each strain)
                                                                                                                                               Copper chloride

         The task group was aware of an ongoing study in guinea-pigs which
    were orally dosed from their first day of life with milk formula
    containing copper(II) chloride (10, 15, 30 mg Cu/kg body weight per
    day) for 28 days in order to study the effect of exposure to copper in
    early life on copper homoeostasis and toxicity (Summer & Dieter,
    personal communication, 1996).

    7.4  Long-term exposure chronic toxicity or carcinogenicity

         The chronic toxicity/carcinogenicity of copper compounds has not
    been well characterized (see Table 11).  Increased mortality and
    growth retardation or effects on the liver, kidneys or stomach have
    been observed in rats following long-term ingestion of 27-150 mg Cu/kg
    body weight per day as copper(II) sulfate, or 44-45 mg Cu/kg body
    weight per day as copper(II) acetate, in several limited studies.
    Long-term ingestion of copper(II) sulfate at 10 mg Cu/kg body weight
    per day induced marked hepatotoxicity in rabbits.  An oral study in
    dogs did not show significant toxic effects at the highest dose of 8.4
    mg Cu/kg per day, given as copper gluconate (Shanaman et al., 1972).

         The available studies of the carcinogenicity of copper compounds
    in rats and mice have given no indication that copper salts are
    carcinogenic.  However, the short duration or low level of exposure,
    the small group sizes employed, the limited extent of
    histopathological examination, or inadequate reporting limits the
    conclusions which can be drawn from such studies.  The studies
    summarized in Table 11 are, therefore, inadequate to test the
    carcinogenic potential of copper compounds with any degree of
    certainty.  In several studies, administration of copper compounds
    inhibited the development of tumours induced by known carcinogens (see
    Table 11).

    7.5  Reproductive and developmental toxicity

         As shown in Table 12, there is some limited evidence that
    exposure to copper compounds can affect reproduction in animals.  In
    some studies of rats exposed by the oral route, the weights and/or
    histology of the testes, seminal vesicles, uterus or ovaries have been
    affected by chronic intakes of 27-120 mg Cu/kg body weight per day as
    copper(II) sulfate, acetate, or gluconate, although the results are
    inconsistent between studies and the reporting of some studies is
    deficient.  In mice, there were no effects on male or female
    reproductive organs at 398-537 mg Cu/kg body weight per day as
    copper(II) sulfate in the diet.  In a single study of rats inhaling
    copper(II) chloride aerosol, there were effects on sperm, testis
    weight and circulating levels of reproductive hormones.

        Table 11.  Chronic toxicity or carcinogenicity after long-term exposure

                     Protocol                              Results                                               Effect level            Reference

    Copper(II)       Rats fed diets containing 0, 530      Body weight gain was retarded at 1600 mg Cu/kg        LOEL (non-neoplastic    Harrisson
    sulfate          or 1600 mg Cu/kg diet as copper       diet in the males. Stomachs of the high-dose          effects):               et al.
    Oral             sulfate for 40-44 weeks (approx.      females were enlarged. Other findings at the high     27 mg Cu/kg body        (1954)
    Rats             0, 27 or 80 mg Cu/kg body weight      dose were 'bronzed' kidneys, 'bronzed' or yellowish   weight per day in
    (Sprague-Dawley, per day in males and 0, 40 or 120     livers, hypertrophied ridges between cardiac          males, 40 mg
    groups           mg Cu/kg body weight per day in       and peptic portions of stomach, and blood in the      Cu/kg body weight
    of 25 males      females). (Reduced amounts fed        intestinal tract. Microscopic effects (not further    per day in females
    and 25           for the first month of the            described) were seen in the kidneys in the high-dose
    females)         experiment.) Microscopic              group (presumably in both males and females), and
                     examination of limited number of      effects on the liver were seen in both males and
                     organs Study inadequately described   females, presumably in both dose groups

    Rats             Rats given diets containing           Excess copper caused decreased body weight gain       Toxic effects at        Carlton
    (Sprague-Dawley, deficient (1 mg Cu/kg diet) or        and increased mortality with or without DMN or AAF    40 mg Cu/kg body        & Price
    groups of 50     excess (800 mg Cu/kg diet) levels     treatment. The only effects reported in the rats not  weight per day          (1973)
    or 58 males,     of copper (as copper sulfate) for 9   exposed to these two carcinogens were liver
    additional       months (equivalent to about 0.05      necrosis and transitional nodules in the liver in     Exposure too short
    groups of        or 40 mgCu/kg body weight per         3/32 and 1/32 animals, respectively at 800 mg Cu/kg   and group size
    55-102 males     day). Within each treatment group,    diet (none at 1 mg Cu/kg diet), and 1 kidney tumour   inadequate to
    also given       separate groups given DMN in          the low-copper group (42 rats)                        assess the
    dimethyl         the drinking-water (50 mg Cu/kg                                                             carcinogenic potential
    nitrosamine      diet) or AAF in the diet (0.06%),     Both DMN and AAF exposure markedly increased          of copper sulfate
    (DMN) or         in both cases for 4 days in every     the incidence of liver necrosis and transitional      itself, but the data
    acetylamino-     8 for 6 months, or no further         nodules and each induced a similar incidence of       suggest it may
    fluorene         treatment. Five rats per group        liver tumours in rats fed excess copper or            have an inhibitory
    (AAF)            killed after 90 days, and an          copper-deficient diets. There were no kidney          effect on
                     additional 5/group killed every       neoplasms in the AAF-treated groups, but 57% of       DMN-induced kidney
                     30 days thereafter. Limited           the rats in the DMN group on a copper-deficient       tumours and
                     range of organs examined              diet (17/30) had kidney neoplasms compared with       AAF-induced
                     microscopically                       0% (0/29) on the higher copper diet                   extra-hepatic tumours

    Table 11.  (continued)

                     Protocol                              Results                                               Effect level            Reference
                                                           The incidence of AAF-induced extrahepatic
                                                           neoplasms was apparently reduced by the excess
                                                           copper diet (5/30 vs 11/27 in the low copper group)

    Mice             Copper sulfate pentahydrate           The numbers of mice with ovarian tumours were         Exposure too short      Burki &
    (C57BL/6J,       supplied in the drinking-water at     0/10, 0/12, 11/11 and 6/11 in the untreated           and group size          Okita
    groups of        198 mg/litre for 46 weeks             controls, copper-treated mice, DMBA-treated mice and  inadequate to           (1969)
    10-12            (equivalent to about 10 mg Cu/kg      DMBA + copper-treated mice respectively,              assess the
    females)         body weight per day). One group       suggesting that copper sulfate may inhibit tumour     carcinogenic potential
                     received copper sulfate treatment     development to some extent. The corresponding         of copper sulfate
                     alone, a second was given an          figures for lymphomas were 1/10, 2/12, 3/11           itself, but the data
                     intravenous injection of DMBA         and 3/11                                              suggest it may
                     (a known carcinogen) 2 weeks                                                                inhibit the
                     after copper treatment began, and                                                           development of
                     two further groups were untreated                                                           DMBA-induced ovarian
                     or received DMBA treatment only.                                                            tumours
                     Mice killed at 46 weeks and a
                     limited range of organs studied

    Rabbits          10 ml of a 1% solution of copper      Effects on the liver included degeneration and        Only one dose           Tachibana
    (strain and      sulfate (equivalent to about 10 mg    vacuolation of the hepatocytes, granule formation     tested (effects         (1952)
    numbers          Cu/kg body weight) given to           in the cytoplasm, morphological changes in the        at 10 mg Cu/kg
    unspecified)     rabbits daily or on alternate days    nuclei, and atrophy and compensatory hypertrophy      body weight
                     "for up to 400 days and over".        "in the late stage".  Marked infiltration of round    per day)
                     Rabbits evidently killed at           cells (mainly lymphocytes) into "interhepatic
                     various time intervals, some          tissues" was seen after 200 days (and to a lesser
                     as early as 33 days. Liver            extent after shorter periods of administration).
                     examined macroscopically              Proliferation of the interstitial connective
                     and histologically                    tissues was also evident after 200 days, and became
                                                           much more marked after 300 days, "with a resulting
                                                           picture of liver cirrhosis". Haemorrhage and
                                                           necrosis of the liver occurred in some animals

    Table 11.  (continued)
                     Protocol                              Results                                               Effect level            Reference

                                                           A dysfunction in sugar metabolism was evident after
                                                           30-60 days of copper administration, with temporary
                                                           recovery after 90 days but further impairment after
                                                           120-150 days. There were no effects on serum
                                                           bilirubin or total serum proteins.

    Copper           Rats fed diets containing 0 or        Mortality was increased, and food intake and body     Only one dose           Harrisson
    gluconate        1600 mg Cu/kg diet as copper          weight gain were retarded by 1600 mg Cu/kg diet in    tested (effects at      et al.
    Oral             gluconate for 40-44 weeks             both sexes. Stomachs enlarged in both sexes, while    80 mg Cu/kg             (1954)
    Rats             (equivalent to about 0 or 80 mg       hypertrophy of the uteri, ovaries, or seminal         body weight per
    (Sprague-Dawley, body weight per day in males,         vesicles was observed. Other findings were "bronzed"  day in males,
    groups of 25     and 0 or 120 mg Cu/kg body            kidneys, "bronzed" or yellowish livers,               and 120 mg Cu/kg
    males and        weight per day in females).           hypertrophied ridges between cardiac and peptic       body weight per
    25 females)      (reduced amounts fed for the first    portions of stomach, and blood in the intestinal      day in females)
                     month of the experiment).             tract. Microscopic effects (not further described)
                     Microscopic examination of a          were seen in the kidneys of copper-exposed rats
                     limited number of organs. Study       (presumably in both sexes), and effects on the liver
                     inadequately described                were seen on both males and females. Levels of
                                                           copper in liver were nearly twice as high as in rats
                                                           receiving an equivalent dose of copper as
                                                           copper(II) sulfate, corresponding to their
                                                           relative toxicities

    Dogs (Beagle,    Dogs fed diet containing 0,           No effect on mortality or body weight gain. Physical  Elevated SGPT           Shanaman
    groups of        0.012%, 0.06% and 0.24% copper        examinations, haematology, urinalysis and most        in 2 of 12 dogs         (1972)
    6-8 males        gluconate for 6-12 months             blood biochemical analysis revealed no effect of the  on 8.4 mg Cu/kg
    and 6-8          (equivalent to 0, 0.42, 2.1           compound except in two of the 12 dogs on the          body weight per
    females)         and 8.4 mg Cu/kg per day). Detailed   highest dose which showed elevated levels of serum    day evaluated by
                     study of haematological biochemical   GPT; this was reversible. No compound related         the Task Group
                     and urinalysis parameters, and        gross on microscopic pathologic lesions or changes    as not
                     tissue copper concentrations in       in organ weight were seen. At 6 and 12 months,        toxicologically
                     kidney, liver and spleen. Detailed    there was a gross-dependent increase in copper        significant
                     necropsy, histopathology and          level in kidney, liver and spleen. Liver biopsy from
                     organ weight information provided     4 animals at 0, 4 and 12 weeks after withdrawal of
                                                           12 months dosing (0.24% copper gluconate) showed
                                                           some reversibility of liver copper level

    Table 11.  (continued)

                     Protocol                              Results                                               Effect level            Reference

    Copper(II)       Rats fed diets containing 0 or 0.5%   Rats in all groups were reported to consume the       Study inadequate        Howell
    acetate          copper acetate (approximately 87      same amounts of food. In one experiment, of           for assessing the       (1958)
    Oral             mg Cu/kg body weight per day)         animals treated with DMAB alone, 17/20                carcinogenic
    Rats (various    throughout their lifetimes. Second    developed tumours, compared with 4/16 in those        potential of copper
    strains,         set treated in the same way,          exposed to both DMAB and copper acetate.              acetate itself, but
    groups of        except 0.09%                          Comparable incidences for a subsequent                the data suggest
    5 males          p-dimethylamino-benzene (DMAB),       experiment were 7/8 and 0/8, respectively             it has an inhibitory
    and 5            a known liver carcinogen, included                                                          effect on
    females)         in the diet for the entire period.                                                          DMAB-induced tumours
                     Liver, spleen and grossly abnormal
                     tissues were examined

    Rats             Control group fed meal containing     Growth was reduced by 23% in the treated rats.        Only one dose           Llewellyn
    (Holtzman,       18 mg Cu/kg diet, treated group       Weights of the heart, spleen, lung and kidney were    tested                  et al.
    groups of        fed meal supplemented with 2600       unchanged, while testis weights were increased.       (non-neoplastic         (1985)
    10 males)        mg Cu/kg diet copper acetate          Effects on liver weight are unclear from the          effects at 45 mg
                     (approximately 45 mg Cu/kg body       information provided                                  Cu/kg body weight per
                     weight per day) for 21 weeks.                                                               day)
                     Limited number of organs              Examination of the bones revealed no qualitative
                     weighed. Long bones                   (osteoporosis, osteomalacia, modelling defects) or
                     radiographed and measured             quantitative effects, although femur length was
                                                           decreased relative to controls (P < 0.05)

    Intraperitoneal  Injection of copper acetate 3 times   Only 5/20 mice survived at the top dose. The          Inadequate group        Stoner
    injection        per week for 8 weeks at total         numbers of mice with lung tumours were 4/15 (27%),    size to determine       et al.
    Mice (Strain     doses of 36, 90 or 180 mg/kg body     9/18 (50%) and 3/5 (60%) for the 36, 90 and 180       whether copper          (1976)
    A/strong,        weight (roughly 12, 31 or 63 mg       mg/kg body weight groups respectively, compared       acetate increases
    groups of 10     Cu/kg body weight). Control mice      with 7/19 (37%) in the control group. The average     the spontaneous
    males and 10     received vehicle alone (0.85%         number of lung tumours per mouse (0.40, 0.56 and      lung tumour
    females)         NaCl). Mice sacrificed 22 weeks       2.00 tumours per mouse in the low-dose, mid-dose      incidence in this
                     after the last injection. Microscopic and high-dose groups, respectively) increased         susceptible strain
                     examination limited to the lungs      dose-dependently but was not statistically            of mice

    Table 11.  (continued)

                     Protocol                              Results                                               Effect level            Reference
                     and any tissues that appeared         significantly different from the control incidence
                     abnormal on gross examination         (0.42) at any dose level. No other tumours were
                     of a small number of organs           identified in a limited range of tissues

    Copper(II)       Mice given 0 or 1000 mg copper        Study results inadequately reported. Survival was     The group sizes         Bionetics
    8-hydroxy-       8-hydroxyquinoline/kg body weight     apparently unaffected by the treatment                were too small          Research
    quinoline        (roughly 0 or 180 mg Cu/kg body                                                             and an inadequate       Labs.
    Oral             weight) by gavage (in 0.5%            No statistically significant increases in tumour      number of doses         (1968)
    Mice (B6C3F1     gelatine) on days 7-28 of age,        incidences were observed in either strain of mice     were tested to
    and B6AKF1,      and then fed diets containing         compared with controls                                assess the
    groups of 18     2800 mg compound/kg diet                                                                    carcinogenic
    males and 18     (providing about 60 mg Cu/kg                                                                potential of copper
    females per      body weight per day) for remainder                                                          8-hydroxyquinoline
    strain)          of the 18-month study. Extent
                     of microscopic examination
                     unclear, but certainly very limited

    Unspecified      Rats maintained on diets              No colonic tumours occurred in rats treated only      Carcinogenic            Greene
    copper salts     containing 0.6, 25 or 100 mg          with copper, while all DMH-treated rats had tumours.  potential of            et al.
    Oral             Cu/kg diet copper (equivalent to      There was a significant increase (P < 0.001) in       copper cannot           (1987)
    Rats             0.03, 1.25 or 5 mg Cu/kg body         colonic tumours (3.14 ± 0.39 tumours/cm colon) in     be assessed
    (Sprague-Dawley, weight per day) for 25 weeks and      rats fed the copper-deficient diet (0.6 mg Cu/kg      from this
    groups of        then killed. A second series also     diet) and treated with DMH, compared with rats fed    study
    10 males)        received 16 weekly doses of a         diets containing normal or high copper levels and
                     carcinogen (1,2-dimethylhydrazine,    treated with DMH (0.74 ± 0.07 and 0.76 ± 0.08
                     DMH, 20 mg/kg body weight)            tumours per cm colon, respectively). A greater
                                                           proportion of these tumours were malignant
                                                           (P < 0.01) in the copper-deficient group (92%
                                                           compared with 70 and 76% in the normal and high
                                                           copper groups)

    Table 12.  Reproductive and developmental toxicity of copper
    Species          Protocol                                Results                                                  Effect level     Reference

    Copper(II)       Copper sulfate pentahydrate given       No effects were seen on testis, epididymis or cauda      No effects       Hébert
    sulfate          in the diet for 92 days at              epididymis weight, spermatid counts or sperm motility    observed at 67   et al.
    Oral             concentrations of 0, 500, 2000 or 4000  in males of either species, at any tested dose. The      mg Cu/kg body    (1993)
    Rats (F344/N,    mg/kg. Estimated intakes 0, 8, 34       length of the oestrous cycle in females was              weight per day
    groups of 10     or 67 mg Cu/kg body weight per          unaffected. A slight dose-related decrease was seen
    males and        day. Sperm morphology and               in the percentage of the oestrous cycle spent in
    10 females)      vaginal cytology evaluated              oestrus but this effect did not achieve statistical
                                                             significance (P > 0.05)

    Mice             Males and females given 0, 0.5, 1,      Developmental malformations (including                   NOEL: 53 mg      Lecyk
    (C57BL and       1.5, 2, 3 or 4 g copper sulfate/kg      hydrocephalus, encephalocoeles, and abnormalities of     Cu/kg body       (1980)
    DBA, groups      feed (approximately 0, 27, 53, 80,      the ribs and vertebrae) occurred in groups of both       weight per
    of 7-22          106, 159 or 213 mg Cu/kg body           strains given > 3 g/kg feed. C57BL stock had             day
    females,         weight per day) for 1 month prior       abnormalities in 1/55 and 3/35 live fetuses and DBA
    unspecified      to mating. Treatment presumably         stock in 2/56 and 4/45, in the 3 and 4 g/kg feed         LOEL: 80 mg
    number of        continued in females until sacrifice    groups respectively. No abnormalities were found         Cu/kg body
    males)           on day 19 of pregnancy                  in controls (65 live C57BL fetuses, 76 live DBA          weight per
                                                             fetuses). Mean values for litter size, live fetuses      day
                                                             and mean fetal weight were reduced in groups of both
                                                             strains given > 1.5 g/kg feed. Statistical
                                                             significance not reported, but reductions appear to
                                                             have been dose-related in some cases

    Mice             Mice given 0 or 6 mg Cu/kg per litre    No data were presented on litter size or the incidence   One dose group   Kasama
    (C3H/HeN         as copper sulfate in drinking-water     of abnormalities. Copper administration during           only (effects    & Tanaka
    and C3H/HeJ,     from day 13 of pregnancy to delivery    pregnancy alone did not affect body weight or organ      observed at      (1988)
    females,         (approximately 1.6 mg Cu/kg body        weights (cerebrum, liver and kidney) of the offspring    1.3-1.6 mg
    numbers          weight per day). Half of the            within 24 h after birth, but continued copper            Cu/kg body
    unspecified)     copper-treated animals then received    administration during lactation resulted in significant  weight per
                     5 mg Cu/kg per litre as copper sulfate  reductions in neonatal body weight at 7-13 days of       day)
                     in the drinking-water during lactation  age (P < 0.05) and in the weight and protein content
                     (approximately 1.3 mg Cu/kg body        of the cerebrum, liver and kidney of neonates at
                     weight per day) while the remainder     13 days of age (P < 0.05). The offspring of the
                     received tap water alone. Neonates      copper-treated animals showed various changes
                     sacrificed and examined at 13 days      in enzyme activity in these organs
                     of age

    Table 12.  (continued)

    Species          Protocol                                Results                                                  Effect level     Reference
    Mice             Copper sulfate pentahydrate given       No effects were seen on testis, epididymis or cauda      No effects       Hébert
    (B6C3F1,         in the diet for 92 days at              epididymis weight, spermatid counts or sperm             observed at      (1993);
    groups of 10     concentrations of 0, 1000, 4000 or      motility in males at any tested dose. The length of      398 mg Cu/kg     Hébert
    males and        8000 mg/kg diet. Estimated intakes      the oestrous cycle in females was unaffected             body weight per  et al.
    10 females)      0, 44, 187 or 398 mg Cu/kg body weight                                                           day in males,    (1993)
                     per day in males and 0, 52, 267 or                                                               537 mg Cu/kg
                     537 mg Cu/kg body weight per day                                                                 body weight per
                     in females. Sperm morphology and                                                                 day in females
                     vaginal cytology evaluated

    Mink             Males and females given 0, 25, 50,      There were no overt toxic effects in the                 NOEL: 6 mg       Aulerich
    (standard        100, 200 mg Cu/kg diet as copper        copper-treated adults. No information was provided on    Cu/kg body       et al.
    dark, groups     sulfate pentahydrate                    developmental malformations. Kit weight at 4 weeks       weight per       (1982)
    of 4 males       (approximately 3, 6, 12 or 24 mg Cu/kg  (but not at birth) was significantly reduced in the 100  day
    and 12           body weight per day), for               mg/kg group (P < 0.05). No such effect was evident
    females)         9 months before mating and for          at 200 mg/kg. Kit mortality (birth to 4 weeks) in the    LOEL: 12 mg
                     3 months after mating                   100 and 200 mg/kg groups appeared to be increased        Cu/kg body
                                                             (38% and 32% compared to 12% in controls                 weight per
                                                             (statistical significance not reported), and in all      day
                                                             treated groups litter mass (at weaning) was
                                                             reduced (statistical significance not reported), with
                                                             some evidence of a dose-related effect. An adverse
                                                             effect of copper on lactation was suggested

    Copper(II)       Rats given 0 or 2600 mg/kg copper       An increase in relative testis weight was seen           One dose group   Llewellyn
    acetate          acetate in the diet (approximately      in treated rats. No data were presented to support       only (effect     et al.
    Oral             45 mg Cu/kg body weight per day)        this statement                                           observed at 45   (1985)
    Rats             for 21 weeks followed by sacrifice.                                                              mg Cu/kg body
    (Holtzman,       The control diet contained 18                                                                    weight per day)
    groups of        mg/kg copper (roughly 1 mg Cu/kg
    10 males)        body weight per day). Testis
                     weights examined at termination

    Table 12.  (continued)

    Species          Protocol                                Results                                                  Effect level     Reference
    Rats             An increasing concentration (up to      There were no overt signs of toxicity in the treated     Only one dose    Haddad
    (Wistar albino,  0.185%) of copper acetate               females. In the groups that continued to normal          group (effects   et al.
    groups of 14     administered in the drinking-water      delivery or were sacrificed at 21.5 days of pregnancy,   observed at      (1991)
    treated and 6    for 7 weeks immediately prior to        the number of offspring per litter and the mean fetal    65 mg Cu/kg
    or 7 control     mating (up to approximately 65 mg       weight were similar to the values in the control groups. body weight
    females for      Cu/kg body weight per day). Groups      External examination and serial sectioning revealed      per day)
    each of the      sacrificed at 11.5 or 21.5 days of      no malformations. Examination of the 11.5 day old
    three times      pregnancy, or after delivery. (It is    embryos revealed significant reductions (P < 0.005)
    of sacrifice)    not clear whether copper acetate        in mean yolk sac diameter, crown to rump length and
                     exposure continued during               mean somite number. In the 21.5 day old fetuses
                     pregnancy)                              there was a significant reduction in ossification in 6
                                                             of the 7 ossification centres examined, while in
                                                             newborn rats only 3 centres (cervical vertebrae,
                                                             caudal vertebrae and hindlimb phalanges) showed
                                                             a similar reduction (P < 0.025)

    Copper(II)       0, 1600 mg Cu/kg as copper              The authors reported hypertrophy of the uteri, ovaries   One dose group   Harrisson
    gluconate        gluconate in the diet (approximately    and seminal vesicles. However, in the tabled data, it    only (effects    et al.
    Oral             0 or 82 mg Cu/kg body weight per        appears that the weight of the uterus and ovaries is     observed at 82   (1954)
    Rats             day in males and 0 or 120 mg Cu/kg      reduced in females, and that the weight of the testes    mg Cu/kg body
    (Sprague-Dawley, body weight per day in females) for     is reduced, while that of the seminal vesicles is        weight per day
                     40-44 weeks. (Reduced amount            unaffected in males. The histopathology of these         in males, 120
    groups of 25     fed for the first month of the          tissues was evidently unremarkable. Levels of copper     mg Cu/kg body
    males and        experiment.) Microscopic examination    in liver were nearly twice as high as in rats receiving  weight per day
    25 females)      of a limited number of organs.          an equivalent dose of copper as copper(II) sulfate       in females)
                     Study inadequately described

    Copper(II)       Exposure to aerosols containing         The rats exposed at 19.6 mg Cu/m3 showed overt           LOEL: 2.5 mg     Gabuchyan
    chloride         5.2 or 41.4 mg copper chloride/m3       signs of toxicity (not further described). Both          Cu/m3            (1987)
    Inhalation       (approximately 2.5 or 19.6 mg           concentrations significantly increased the incidence
    Rats (white,     Cu/m3) for 4 months. Functional         of dead and abnormal sperm (P < 0.05) in comparison
    groups of 11     state and morphology of gonads          with untreated controls. Sperm motility, testis weight
    or 12 exposed    assessed after 2.5 and 4 months         and testosterone and oestradiol levels were all
    and 12           of exposure                             reduced in a dose-related manner, although statistical
    control                                                  significance (P < 0.05) was reached only at the
    males)                                                   higher concentration. Significant reductions in the

    Table 12.  (continued)

    Species          Protocol                                Results                                                  Effect level     Reference
                                                             levels of luteinizing hormone, follicle-stimulating
                                                             hormone and prolactin were evident at the lower
                                                             concentration (P < 0.05), but no dose-response
                                                             relationship was apparent

         In a limited number of studies, oral exposure of rodents to
    copper compounds during gestation induced embryo/fetotoxic effects and
    (at higher doses) developmental effects.  Exposure to copper(II)
    sulfate induced effects on neonatal body weight, and on organ weights
    and biochemistry in mice at 1.3-1.6 mg Cu/kg body weight per day,
    while higher doses were embryolethal to mice (at 80 mg Cu/kg body
    weight per day) and to mink (at 12 mg/kg body weight per day).
    Developmental effects, including delayed ossification, were induced in
    rats exposed to 65 mg Cu/kg body weight per day as copper(II) acetate,
    and terata were induced in mice at 159 mg Cu/kg body weight per day as
    copper(II) sulfate.

    7.6  Mutagenicity and related end-points

    7.6.1  Copper sulfate  In vitro

         The genotoxicity of most copper compounds has not been
    extensively studied.

         Copper (II) sulfate, when studied in strains T98, T100 and TA102
    of  Salmonella typhimurium with and without metabolic activity, even
    at cytotoxic concentrations or the limit of solubility, did not
    exhibit mutagenic activity (Moriya et al., 1983; Marzin & Phi, 1985).
    A similar lack of activity was reported, at up to cytotoxic
    concentrations, in the absence of a metabolic activation system in the
    SOS Chromotest with  Escherichia coli PQ37 (Olivier & Marzin, 1987),
    in a test for reversion to streptomycin independence in  E. coli 
    Sd4-73 (Iyer & Szybalski, 1958), in the rec-assay with
     Bacillus subtilis H17 and M45 (Matsui, 1980) and in tests for
    penicillin and/or streptomycin resistance in  Micrococcus aureus 
    FDA209 (Clark, 1953).

         When rat hepatocytes were incubated for 20 h with 7.9, 15.7, 31.4
    or 78.5 µmol/litre copper(II) sulfate solution (the highest
    concentration being moderately cytotoxic), there was a significant
    increase in unscheduled DNA synthesis at each concentration in a
    roughly dose-related manner.  Copper was shown to have accumulated in
    the nucleus at these dose levels (Denizeau & Marion, 1989).  In vivo

         A single intraperitoneal injection of copper(II) sulfate
    pentahydrate in mice induced a dose-related increase in the incidence
    of chromatid type chromosome aberrations in the bone marrow 6 h after
    dosing between 0.28 and 1.7 mg Cu/kg body weight (Agarwal et al.,
    1990).  Only at the highest dose tested (1.7 mg Cu/kg body weight)
    were chromosomal breaks enhanced significantly. In the micronucleus
    test no evidence of genotoxic activity was found in mice given a
    single injection of copper(II) sulfate pentahydrate at 1.7, 3.4 and
    5.1 mg Cu/kg body weight (Tinwell & Ashby, 1990). Bhunya & Pati (1987)

    reported a significant dose-related increase in the incidence of
    micronuclei after two injections at doses between 1.3 and 5 mg Cu/kg
    body weight per injection; however, this study did not utilize a
    positive control and is thus difficult to interpret.

    7.6.2  Other copper compounds  In vitro

         Copper(II) chloride also showed no evidence of mutagenic activity
    in  Salmonella typhimurium strains TA98, TA102, TA1535 and TA1537 in
    the presence or absence of a metabolic activation system when studied
    at concentrations up to those causing cytotoxicity (Wong, 1988).  It
    was similarly inactive in the rec-assay with  Bacillus subtilis H17
    and M45, as was copper(I) chloride (Nishioka, 1975; Kanematsu et al.,

         Copper(II) 8-hydroxyquinoline showed evidence of weak mutagenic
    activity in one strain (TA100) of  S. typhimurium in the presence,
    but not in the absence, of a metabolic activation system.  No activity
    was evident in four other  Salmonella strains, nor in
     Escherichia coli WP2 hcr, in either the presence or the absence of a
    metabolizing system (Moriya et al., 1983).  An earlier study reported
    negative results in strains TA98, TA100, TA1535 and TA1537, with or
    without metabolic activation, but the maximum concentration tested was
    very low (Räsänen et al., 1977).

         In Chinese hamster V79 cells, copper(II) nitrate produced
    dose-related increases in the mutation frequency (resistance to
    8-azaguanine) at 0.01 and 0.1 mmol/litre and in the frequency of
    sister chromatid exchanges at 0.01-0.5 mmol/litre (Sideris et al.,
    1988).  The investigators reported an increase in the molecular weight
    of DNA isolated from the cells, which was attributed to binding of the
    copper ions to the DNA.

    7.7  Other studies

    7.7.1  Neurotoxicity

         There are few studies of the neurological effects of copper
    compounds.  In rats, oral exposure to copper(II) sulfate in two
    studies did not affect the results of behavioural tests, but did alter
    brain neurochemistry.  Injection of copper(II) chloride altered levels
    of neurotransmitters in the brain of rats.  Copper sulfate

         Dietary administration of 250 mg/kg Cu (as copper(II) sulfate
    pentahydrate) to groups of six male rats for 30 days, providing 5 mg
    Cu/rat per day (equivalent to about 20 mg Cu/kg body weight per day)
    did not affect their locomotor activity, learning ability or

    relearning capacity and memory (Murthy et al., 1981). Analysis of
    biogenic amines in the brain revealed a significant increase in
    dopamine and norepinephrine (noradrenaline) levels  (P < 0.02).

         In another study using rats loaded with copper through
    administration of 0.125% copper(II) sulfate in the drinking-water for
    11 months (equivalent to about 46 mg Cu/kg body weight per day), there
    were no overt effects on the behaviour of the eight treated females
    (de Vries et al., 1986).  Neurological effects in the brain included a
    disturbance in striatal dopamine metabolism (reduced levels of the
    dopamine metabolite, 3,4-dihydroxyphenylacetic acid), a three-fold
    increase in the affinity of D2-dopamine receptors and a 50% reduction
    in the number of these receptors.  Brain levels of dopamine and
    noradrenaline, and that of the noradrenaline metabolite,
    3,4-dihydroxyphenylethylene glycol, were unaffected in copper-loaded
    animals (de Vries et al., 1986).  Copper chloride

         Daily intraperitoneal injections of copper(II) chloride to 12
    male rats at a dose of 2 mg Cu/kg body weight per day for 21 days
    resulted in significant increases in dopamine and norepinephrine
    (noradrenaline) levels in the brain  (P < 0.05), while the level of
    5-hydroxytryptamine in the brain was similar to that in saline-treated
    controls (Malhotra et al., 1982).

    7.7.2  Immunotoxicity

         Only copper(II) sulfate has been tested for its immunomodulatory
    effect.  In studies summarized in this section, oral exposure of mice
    to this compound affected measures of both humoral and cell-mediated
    immune function, while inhalation adversely affected host resistance
    and pulmonary macrophage activity.  Copper(II) sulfate

         The administration of copper(II) sulfate in the drinking-water of
    mice at 50, 100 and 200 mg Cu/litre for up to 10 weeks resulted in the
    dose-related inhibition of a number of immune system parameters in two
    studies.  (These levels would normally be equivalent to 10, 20 or 40
    mg Cu/kg body weight per day, but water consumption decreased with
    increasing copper concentrations.  It was reported that total copper
    intake increased with increasing level, though no further detail was
    provided.)  At 50 mg Cu/litre, the lymphoproliferative response to
    lipopolysaccharide from  E. coli was depressed, while the production
    of autoantibodies against bromelain-treated mouse red blood cells was
    increased (Pocino et al., 1991).  These parameters were also affected
    at 100 and 200 mg Cu/litre, along with decreased lymphoproliferative
    response to concanavalin A, and decreased antibody response and
    delayed-type hypersensitivity response to sheep erythrocytes (Pocino
    et al., 1990, 1991).  A NOEL could not be established in these two

         In an inhalation study in mice, single or repeated 3 h exposures
    to copper(II) sulfate aerosol resulted in significant
    immunosuppressive effects, including reduced bactericidal activity of
    the alveolar macrophages to  Klebsiella pneumoniae and reduced
    resistance to infection by  Streptococcus zooepidemicus.  These
    effects were evident after a single exposure at 0.28 mg Cu/m3 and
    above and after 5 or 10 daily exposures at 0.06-0.07 mg Cu/m3.  A
    NOEL was not established in these studies (Drummond et al., 1986).

         In hamsters, a single 4 h exposure to copper(II) sulfate
    pentahydrate aerosol at 0.3-7.1 mg Cu/m3 resulted in reduced
    pulmonary macrophage activity and volume from 3.2 mg Cu/m3 within 1 h
    after exposure; no effect was observed at 0.3 mg Cu/m3 (Skornik &
    Brain, 1983).

    7.8  Biochemical mechanisms of toxicity

         The mechanism(s) by which copper may lead to cell injury are
    discussed in section 6.


    8.1  General population: copper deficiency and toxicity

         Copper is an essential element.  Most tissues therefore have
    measurable amounts of copper associated with them and, in general,
    cells, tissues and organisms have mechanisms to maintain its
    availability while limiting its toxicity (homoeostasis).

         In most situations, if we explore the indices of function
    affected by copper excess or deficit we will find altered indicators
    prior to the onset of clinical signs or symptoms. In some situations
    we can use the functional indicators instead of clinical signs, since
    they are closely associated. The least significant manifestations in
    terms of human health are the physiological changes that occur in
    response to high or low copper intakes.  Most of the changes observed
    in these situations represent adaptive or homoeostatic mechanisms to
    prevent deficit in response to low intake or prevent toxicity in
    response to high intake.

    8.2  Copper deficiency

         Characteristic clinical features of copper deficiencies in
    infants are anaemia refractory to iron, and low copper plasma levels
    (Sturgeon & Brubaker, 1956). Copper deficiency has been considered the
    likely cause of the anaemia, but it was not until the completion of a
    series of controlled case studies of copper deficit in infants
    recovering from malnutrition (Cordano et al., 1964) that the full
    spectrum of copper deficiency was demonstrated. Subsequent reports
    during the 1970s of acquired copper deficiency in low-birth-weight
    neonates and in infants and children receiving copper-free total
    parenteral nutrition, clarified the indispensable nature of copper as
    an essential nutrient for humans (Widdowson et al., 1974; Shaw, 1992).

    8.2.1  Clinical manifestations of copper deficiency

         Clinically evident copper deficiency occurs relatively
    infrequently in humans. The most consistent clinical manifestations of
    copper deficiency are anaemia, neutropenia and bone abnormalities
    including fractures. The haematological changes are characterized by
    the existence of a hypochromic, normocytic or macrocytic anaemia,
    accompanied by a reduced reticulocyte count, hypoferraemia,
    neutropenia and thrombocytopenia. In a small proportion of cases there
    is microcytic anaemia (Williams, 1983). Bone marrow cytological
    examination reveals megaloblastic changes and vacuolization of the
    erythroid and myeloid progenitors. There is also an arrest of the
    maturation of myeloid precursors and the appearance of ringed
    fibroblasts. These alterations are unresponsive to iron therapy but
    are readily corrected by copper supplementation (Schubert & Lahey,
    1959; Prohaska et al., 1985). The current prevailing view is that
    anaemia in copper deficiency is due to defective iron mobilization
    resulting from reduced ceruloplasmin (ferroxidase l) activity.

         A summary of some reports of clinical manifestations of copper
    deficiency in humans is given in Table 13.  As seen clearly from the
    table, many of the reports of deficiency originate in infants and
    young children, particularly those with low birth weight or
    malnourished after birth. Healthy infants receiving less than 0.1 mg
    Cu/kg body weight per day are at risk of deficit.  For those with low
    birth weight or affected by protein energy malnutrition the figure is
    close to 0.2 mg/kg per day. These latter conditions affect a sizeable
    proportion of children at a global level. It has been estimated that
    about 16% of live births or some 20 million infants per year are of
    low birth weight (< 2500 g) (WHO, 1990). The presence of bone
    abnormalities is very common in copper deficiency in low-birth-weight
    infants and in young children (Heller et al., 1978; Danks, 1988; Shaw,
    1992). These abnormalities, which mimic the changes observed in
    scurvy, include osteoporosis, fractures of the long bones and ribs,
    epiphyseal separation, fraying and cupping of the metaphyses with spur
    formation, and subperiosteal new bone formation (Danks, 1988; Shaw,
    1992). Less frequent manifestations of copper deficiency are
    hypopigmentation of the hair and hypotonia (Danks, 1988; Shaw, 1992),
    impaired growth (Castillo-Duran & Uauy, 1988), increased incidence of
    infections (Castillo-Duran et al., 1983), and alterations of
    phagocytic capacity of the neutrophils (Heresi et al., 1985). In
    addition, abnormalities of cholesterol and glucose metabolism have
    been reported, but are not so well established (Klevay et al., 1984,
    1986; Reiser et al., 1987). Prevalence of cardiovascular disease has
    been linked to high zinc and low copper in the diet but this
    hypothesis has not been validated (Lukaski et al., 1988).

         It has been shown that copper deficiency is associated with
    increased incidence of infection and impaired weight gain in infants
    recovering from malnutrition (Castillo-Duran et al., 1983;
    Castillo-Duran & Uauy, 1988). The initial randomized controlled trial
    included 27 infants recovering from protein energy malnutrition: 13
    received 80 µg/kg per day of copper supplement for 3 months while 14
    matched infants received a placebo. Plasma copper and ceruloplasmin
    dropped in the placebo group, 30% of whom had low copper plasma
    levels, while values rose in the supplemented group during the rapid
    growth phase of recovery. The mean number of upper respiratory
    infections, febrile days, and number of febrile episodes per child per
    month were similar in both groups. However, seven infants presented
    clinical evidence of severe lower respiratory infection (mainly
    pneumonia) in the placebo group versus only one subject in the copper
    supplemented group ( P < 0.025) (Castillo-Duran et al., 1983). In a
    separate case control study, 11 infants identified as
    copper-deficient, based on low plasma copper and low ceruloplasmin,
    and 10 matched copper-sufficient infants at a similar stage of their
    nutritional recovery, were supplemented with 80 µg Cu/kg, as copper
    sulfate, daily for 30 days. The daily weight gain and daily energy
    intake were significantly higher relative to controls in the
    copper-deficient group shortly after supplementation (Castillo-Duran &
    Uauy, 1988).

        Table 13.  Clinical copper deficiency

    Subjects               Study and results                                                                                      Reference

    11 copper-deficient    In a prospective case control, growth was evaluated 1 month before and 1 month after copper            Castillo
    infants (plasma        supplementation with 80 mg/kg body weight. Weight/age and weight/length indices increased              -Duran et
    copper < 70 µg/litre   significantly after supplementation in the copper-deficient group. Daily energy intake was             al. (1988)
    and ceruloplasmin      significantly higher in the copper-deficient group after supplementation than it was in the control
    < 200 mg/litre) and    group. Daily weight gain after supplementation increased significantly in the copper-deficient group
    10 control infants     and the value for daily weight gain after supplementation was significantly higher than that of the
                           control group for the equivalent amount of time

    24 males aged          The subjects received diets low in copper (1.03 mg/day per 2850 kcal [12 MJ]) and containing either    Reiser
    21-57 years            20% of the calories as fructose or cornstarch. During the course of feeding the diets for 11 weeks,    et al.
                           four of the subjects exhibited heart-related abnormalities and were removed from the study             (1985)
                           (1 myocardial infarction, 2 severe tachycardia and 1 a type II second-degree heart block). There
                           were no changes in serum copper and ceruloplasmin. However, fructose ingestion significantly
                           reduced erythrocytic SOD. Repletion of the subjects with 3 mg Cu/day for 3 weeks significantly
                           increased SOD levels in subjects previously fed fructose but not starch. These results suggest that
                           the type of dietary carbohydrate fed can differentially affect indices of copper status in humans.
                           Copper deficiency could play a role in human heart disease

    24 males aged          The subjects were fed an experimental diet inadequate in copper (0.36 mg/day per 1000 kcal             Reiser
    21-57 years            [4.18 MJ]) for 11 weeks showed significant increase in LDL cholesterol and significant decrease        et al.
                           in HDL cholesterol when compared to either their pretest self-selected diets (0.57 mg Cu/day per       (1987)
                           1000 kcal) or a repletion diet (1.41 mg Cu/day per 1000 kcal [4.18 MJ])

    8 men aged             The subjects were fed diets low in copper (0.89 ± 0.10 mg/day), for periods ranging from 105 to        Milne
    18-36 years            120 days. One man who was in a negative balance showed a significantly reduction in plasma             et al.
                           copper, immunoreactive ceruloplasmin and erythrocyte SOD. Serum cholesterol was                        (1990)
                           significantly elevated by the end of the 15 week depletion. Another two men presented a slightly
                           negative balance and a trend to lower plasma copper and SOD. Two of four subjects tested
                           had impaired glucose clearance during depletion. Conclusion: intakes of below 0.9 mg/day
                           apparently result in signs of copper depletion in healthy adults

    Table 13.  (continued)

    Subjects               Study and results                                                                                      Reference
    11 men aged            The effects of low-copper diets on indexes of immune response were examined in 11 subjects             Kelley
    21-32 years            during a 90 day metabolic study. Daily copper intake for the first 24 days, the next 42 days and the   et al.
                           last 24 days of the study was 0.66, 0.38 and 2.49 mg, respectively. Feeding the diet with              (1995)
                           0.38 mg/day was associated with a significant decrease in the proliferation of peripheral blood
                           mononuclear cells cultured with phytohemagglutinin, concavalin A, or pokeweed, and an increase
                           in the percentage of circulating B cells (CD 19+)

    3 month old infant     An infant with a birth weight of 1140 g fed an infant formula low in copper developed low plasma       Al-Rashid
                           copper and ceruloplasmin, anaemia, neutropenia, apnea, metaphyseal flaring and cupping.                & Spangler
                           These changes were reversed after copper supplementation                                               (1971)

    6 month old infant     An infant with a birthweight of 1140 g fed exclusively with cow's milk presented hypocupraemia,        Ashkenazi
                           low ceruloplasmin, sideroblastic anaemia, neutropenia, osteoporosis with blurring and cupping          et al.
                           of the metaphyses, depigmentation of skin, enlarged and distended blood vessels of the scalp,          (1973)
                           and hypotonia. Treatment with 3 mg Cu/day reversed these abnormalities

    7 month old infant     An infant receiving total parenteral nutrition (TPN) from birth to 7 months showed osteoporosis        Heller
                           and soft tissue calcifications. Plasma copper and ceruloplasmin levels were markedly reduced.          et al.
                           The infant died and postmortem examination showed a reduced liver copper content.                      (1978)
                           A 10 month preterm infant required TPN during the first 4 months of life because of bowel
                           resection at age 10 days presented hypocupraemia, anaemia, neutropenia, osteoporosis,
                           irregularity of the metaphyses and subperiosteal new bone formation. These changes were
                           reversed by the feeding of a formula containing 1 mg Cu/litre

    7 month old infant     A preterm infant (birth weight 2050 g) fed only powdered milk who presented a persistent               Tanaka
                           diarrhoea, developed hypocupraemia, neutropenia, and severe anaemia. Bone radiography showed           et al.
                           generalized osteoporosis, flaring and cupping of the metaphyses of the long bones and a fracture       (1980)
                           of the right fibula. All these abnormalities were alleviated after treatment with copper sulfate

    Two 6 month old        One infant fed only cow's milk since birth presented decreased serum copper and ceruloplasmin,         Levy
    infants                microcytic anaemia and neutropenia. Another infant fed a diet predominantly mainly of cow's milk,      et al.
                           presented reduced concentration of serum copper and ceruloplasmin, and microcytic anaemia.             (1985)
                           A radiological study showed increased density of the preparatory calcification areas with spur
                           formation at the proximal parts of the femurs. In both cases the abnormalities were recovered
                           after the addition of chicken, meat and vegetables

    Table 13.  (continued)

    Subjects               Study and results                                                                                      Reference

    30 year old woman      Following extensive bowel resection, a woman received parenteral nutrition not supplemented            Zidar
                           with copper. The patient developed hypocupraemia, subnormal ceruloplasmin levels, anaemia              et al.
                           and severe neutropenia. Following supplementation of the parenteral solution with 4 mg Cu/day          (1977)
                           an increase in reticulocyte count, haemoglobin and neutrophils was observed

         Copper deficiency is associated with altered immunity in humans
    (Prohaska & Failla, 1993). Heresi et al. (1985) studied 19
    hypocupraemic infants before and after 1 month of copper
    supplementation. The phagocytic activity of polymorphonuclear
    leukocytes increased by 30% after copper supplementation while
    immunoglobulins remained unchanged. Kelley et al. (1995) described a
    decrease in the proliferation of peripheral blood mononuclear cells
    cultured with different mitogens in 11 men receiving a low-copper

         An increased concentration of total cholesterol and low density
    lipoprotein (LDL) cholesterol and a reduction of high density
    lipoprotein (HDL) cholesterol concentration have been observed in
    subjects fed an experimental diet low in copper (Klevay et al., 1984).
    Low copper intake has also been demonstrated to diminish glucose
    tolerance (Klevay et al., 1986), alter cardiac rhythm and
    electrocardiogram, and modify the hypertensive response to a hand-grip
    test (Lukaski et al., 1988). However, other studies have not validated
    the results of changes in cholesterol and glucose metabolism.

         The role of copper deficit in altered neurodevelopment has been
    postulated on the basis of the high copper content of the brain,
    especially of the basal ganglia. The existence of a prenatal critical
    phase in central nervous system (CNS) development during which copper
    deficiency can cause CNS damage has been suggested (Danks, 1988). This
    could explain the severe mental deficiency associated to prenatal
    tissue deficit found in Menkes disease while postnatally acquired
    nutritional copper deficiency is not accompanied by neurological

    8.2.2  Biological indicators of copper deficiency: balance studies

         The determination of the levels of copper intake which will
    prevent deficiency without resulting in toxicity (homoeostasis) has
    been discussed fully in section 6.3.  Several of the most promising
    biological indicators for copper deficiency as well as toxicity, for
    example, cytochrome c oxidase, levels of LDL, ceruloplasmin and serum
    copper are also discussed in section 6.3.

         In view of the importance of this subject for the determination
    of human health risks (deficit and excess) from exposure to copper, it
    is repeated here for emphasis.

    8.3  Toxicity of copper in humans

    8.3.1  Single exposure

         Acute toxicity due to ingestion of copper is infrequent in humans
    and is usually a consequence of the contamination of beverages
    (including drinking-water) or from accidental or deliberate ingestion
    of high quantities of copper salts.

         Numerous case reports of single oral exposures to high levels of
    copper have been reported.  Such exposures, including suicide attempts
    with copper sulfate, have occurred in youths and adults at doses
    ranging from 0.4 to 100 g Cu (Chuttani et al., 1965; Mittal, 1972;
    Stein et al., 1976; Walsh et al., 1977; Chugh et al., 1977; Williams,
    1982; Jantsch et al., 1985).  Symptoms including vomiting, lethargy,
    acute haemolytic anaemia, renal and liver damage, neurotoxicity,
    increased blood pressure and respiratory rates.  In some cases, coma
    and death followed.  There are also a number of reports of high dose
    copper ingestion in beverages (35-200 mg/litre; Hopper & Adams 1958;
    Semple et al., 1960).

    8.3.2  Repeated oral exposures  Gastrointestinal and hepatic effects

         In case reports and cross-section studies, consumption of
    drinking-water contaminated with copper has been associated with
    nausea, abdominal pain, vomiting and diarrhoea (Table 14).  In none of
    these studies have the doses of copper ingested been well
    characterized. In addition, microbiological quality of the water
    supplies or other contributing factors were not assessed.  Also,
    symptoms may have been over-reported owing to lack of blinding of

         An often cited report is that of Wyllie (1957) in which acute
    gastrointestinal symptoms were reported in 10 people consuming a
    cocktail contaminated with copper from the cocktail shaker.  Owing to
    limitations in reporting and confounding, this study is considered
    inadequate to serve as a basis for characterization of concentrations
    of copper which results in adverse health effects.

         In a family in Vermont, USA, living  at the end of a copper main,
    there were recurrent episodes of gastrointestinal illness.  There were
    no symptoms in two other families of similar age and sex distribution
    on the same street exposed to lower levels (Spitalny et al., 1984).
    Symptoms ceased with a change of water source.

         Knobeloch et al. (1994) reported on five investigations of
    gastrointestinal upset associated with ingestion of
    copper-contaminated water.  Data were obtained from questionnaires on
    age, weight, water use habits, duration of exposure and symptoms.
    There was generally a higher incidence of intermittent or constant
    symptoms of diarrhoea, abdominal cramps or nausea in those who
    consumed first-draw water, in infants and young children and among
    residents of newly constructed or renovated houses.  In one study,
    gastrointestinal symptoms occurred in 8 of 14 people ingesting 0.6-3.8
    mg Cu/day from drinking-fountains (1.6-7.7 mg Cu/litre) compared with
    3/26 people ingesting < 0.55 mg Cu/day from drinking-water.

        Table 14.  Gastrointestinal effects associated with copper in potable water or beverages

    Observations                                                                          Comments                               Reference

    10 of 13 nurses experienced nausea, vomiting, diarrhoea, weakness, abdominal          owing to limitations in reporting      Wyllie
    cramps and headache following ingestion of an alcohol lemon cocktail from             and confounding (alcohol, fasted       (1957)
    cocktail shakers containing copper; reconstruction of the episode suggested           state); unknown whether 5.3 mg
    that copper ingestion varied between 5.3 and 32 mg                                    is a LOAEL or NOAEL; study
                                                                                          considered inadequate to establish
                                                                                          effect levels

    In three of four family members residing in Vermont at the end of a copper main,      well-conducted study that provides     Spitalny
    there were recurrent episodes over 1.5 years of gastrointestinal illness 5-20 min     useful information on levels of        et al.
    after drinking tap water in the morning (median level of copper in incoming           copper in water which induce           (1984)
    water, 3.1 mg/litre; single maximum level 7.8 mg/litre); no symptoms in two           acute effects
    other families of similar age and sex distribution on the same street exposed to
    lower levels (medians, 1.58 and 0.02 mg/litre); copper levels in hair significantly
    higher in symptomatic family; symptoms ceased with change of water source

    Three children (1-2.5 years old) with prolonged diarrhoea and weight loss             limited usefulness for risk            Stenhammar
    exposed to tap water containing 0.22-1 mg/litre. Symptoms disappeared when            assessment                             (1979)
    water replaced with that of lower copper content

    Association between the copper content in drinking water (0.35-6.5 mg/litre           viral or other microbiological         Berg &
    in first-draw water) at 7 new Swedish kindergartens and diarrhoea in attending        causes of diarrhoea were not           Lundh
    children < 3 years old. The symptoms disappeared when the children went               studied. Limited usefulness for        (1981)
    home for a few days but reappeared when they returned to the kindergarten             risk assessment

    Five different case reports of gastrointestinal illness in individuals, families or   data inadequate to establish           Knobeloch
    residents completing questionnaires. Higher incidence of gastrointestinal effects     effect levels                          et al.
    with first-draw water compared with flushed water                                                                            (1994)

         Micronodular cirrhosis and acute liver failure was described in a
    case report (O'Donohue et al., 1993). A 26-year-old male consumed
    copper tablets at 30 mg/day (tablet formulation unspecified) for 2
    years, followed by 60 mg/day for an unspecified period, before
    presenting with symptoms of liver failure.  The patient had
    Kayser-Fleisher rings; laboratory investigations revealed normal serum
    copper (22.6 mmol/litre) and serum ceruloplasmin (0.27 mmol/litre) but
    very high urinary excretion of copper (207 mmol/24 h) compared to the
    normal (< 1.2 µmol/24 h).  An emergency liver transplant was
    performed and the patient made a good recovery. The mean copper
    content of the removed liver was 3230 µg/g (normal 20-50 µg/g).
    Histology resembled that of Indian childhood cirrhosis and Wilson
    disease (see section 8.4).  Reproduction and development

         After adjusting for confounding variables, there was no
    association between the risk of spontaneous abortion in a population
    of Massachusetts women exposed to copper in drinking-water (> 1
    mg/litre) during 1976-1978 (Aschengrau et al., 1989).  In a small
    study of trace element status, there was a significant positive
    relationship between placental copper and birth weight, and a negative
    correlation between the copper/zinc ratio and birth weight (Mbofung &
    Subbarau, 1990).  These data are inadequate to assess the
    reproductive/developmental effects of copper in humans.  Cancer

         Epidemiological studies in which the association between copper
    intake and/or levels of copper in serum and cancer has been
    investigated are presented in Table 15.

         In geographical/ecological studies in China (Chen et al., 1992)
    and the USA (Schrauzer et al., 1977), associations between serum
    copper or copper intake and some cancers were reported. However, owing
    to the lack of consideration of individual exposure and confounding
    factors in such studies, they contribute little to assessment of the
    weight of evidence for carcinogenicity.

         Interpretation of the available analytical epidemiological
    (case-control or cohort) studies is complicated by the fact that
    increased serum concentrations of copper could be related to
    alterations in copper handling resulting from the disease state.
    Available analytical epidemiological studies in which concentrations
    of copper in serum were determined only following diagnosis of cancer
    (Çetinkaya et al., 1988; Cavallo et al., 1991; Prasad et al., 1992;
    Dabek et al., 1992) are uninformative, therefore, with respect to the
    possible aetiological role of cancer in the disease.  In prospective
    studies where concentrations of copper in serum have been determined
    prior to disease development, associations between serum copper levels
    generally greater than 1.25 mg/litre and either total or breast cancer
    have been observed, though there is no convincing evidence of a

        Table 15.  Epidemiological studies on cancer in the general population

    Study protocol                             Results                                               Comments                        Reference

    A nested, matched case-control study       The mean serum copper level in the                    When adjusted for other         Coates
    was conducted to compare the serum         control group was 115 ± 36 µg/dl,                     factors which might influence   et al.
    copper levels of 133 cancer cases          whereas the case group mean was                       both the serum copper levels    (1989)
    identified between 1974 and 1984           123 ± 37 µg/dl. The groups were split                 and the risk of all cancer
    among 5000 members of a North              into quartiles with copper serum levels               sites combined (i.e.
    West Washington State employee             corresponding to 43-92, 93-107,                       occupational status, family
    cohort, with 241 controls selected at      108-125 and 126-276 µg/dl. The                        history of cancer, cigarette
    random from the same initial cohort.       relative risk estimates of cancer, all                smoking, alcohol consumption
    Cases and controls were matched for        sites combined, by quartile levels of                 and use of exogenous
    age (in 5-year groupings), sex, race       serum copper, increased steadily, with                oestrogens), the relative risk
    (white/nonwhite) and year and season       that in the upper quartile reaching                   estimates did not differ
    of blood sampling. 48% of the study        statistical significance (RR=1.0, 1.1, 1.3            appreciably from the
    population was male and 97% was            and 2.4 for the quartiles and 95%                     unadjusted risk estimates
    white. Blood had been collected in the     CI=0.6-2.2, 0.7-2.7 and 1.1-5.1 for
    initial study in 1972-1974 (before         the 2nd-4th quartiles, respectively)

    A case-control study of 35                 There was no difference in the serum                  Numbers in the individual       Prasad
    early-diagnosed oesophageal cancer         copper levels of the cases compared                   tertiles were small; limited    et al.
    patients who had not received treatment    with the controls (1.29 ± 0.03 and                    control for confounders;        (1992)
    and were attending, for the first time,    1.24 ± 0.04 mg/litre, respectively).                  serum analyses for copper
    a cancer hospital in India. Dietary        When the cohorts were analysed                        after diagnosis; though
    habits over the preceding 6 months         according to blood copper levels                      more cases in highest
    and blood biochemical parameters           corresponding to 0.75-0.99,                           tertile based on serum
    were assessed and compared with            1.00-1.25 and > 1.25 mg/litre,                        copper, no difference
    35 control subjects matched for age,       more cases occurred in the highest                    between daily copper
    sex, socioeconomic status, rural/urban     group compared with the controls                      intake for cases and
    residence, and chewing, smoking            (20 and 13, respectively; P < 0.025).                 controls
    and drinking habits (minimal control       There was no difference between the
    for confounders)                           daily copper intake values for cases
                                               and controls (3.6 ± 0.64 and
                                               3.4 ± 0.43 mg)

    Table 15.  (continued)

    Study protocol                             Results                                               Comments                        Reference

    A 6-9  year prospective follow-up          The mean levels of serum copper                                                       Kok et al.
    study of an initial cohort of a Dutch      were not significantly increased in                                                   (1988)
    population of 10 532, aged 5 years         the cancer death patients over
    or more, was conducted to the end of       those in the controls (1.33 mg/litre
    December 1983. The serum copper            compared with 1.25 mg/litre; P=0.08).
    concentrations (sampled on initial         For subjects in the highest serum
    entry into the study) of 64 cancer         quintile (> 1.43 mg/litre), the relative
    death patients and 62 cardiovascular       risk, adjusted for various factors,
    death patients were compared with          of death from cancer, was 3.7
    those from randomly selected,              (95% CI=1.5-9.1) compared with
    sex- and age- (in 5 year intervals)        the adjusted relative risk pooled
    matched members of the original cohort,    from quintiles 2-4 (serum copper
    still alive on 31 December 1983. Each      range 1.05-1.43 mg/litre). For the
    case was matched with two controls.        lowest serum quintile (< 1.05 mg/litre),
    Cancer cases and their controls            the adjusted relative risk of death
    were matched for smoking status            from cancer was 1.8 (95% CI=0.7-4.7)

    A case-control study was conducted         The mean dietary intakes of copper                    Results essentially negative    Cavallo
    on 214 patients, first diagnosed for       in the control and case cohorts were                  but serum copper                et al.
    primary carcinoma of the breast and        estimated to be 2.8 ± 1.1 and                         concentrations determined       (1991)
    not previously undergoing therapy,         2.7 ± 1.1 mg/day, respectively. The                   after admission
    randomly selected among consecutive        correlation between copper intake
    admissions to a cancer institute in        and copper blood level was examined
    Milan, Italy, from May 1982 to June        and was found not to be significant.
    1985. Controls (N=215) were patients       Both groups were split into quartiles
    with a variety of diagnoses other than     of dietary copper intake for
    breast cancer. Dietary copper intakes      comparison. No significant trend in
    were estimated from dietary                the OR estimates for breast cancer
    questionnaires. Blood samples were         were found
    taken the day after admission and the
    serum copper levels determined

    Table 15.  (continued)

    Study protocol                             Results                                               Comments                        Reference

    A second set of 47 cases and 46            Mean serum copper levels were
    age-matched controls from Montpellier,     significantly decreased in the cases
    France, which represented a                when compared with the controls.
    sub-sample of a larger study concerning    The mean serum copper level was
    diet and breast cancer, was                found to be significantly higher in
    investigated. Controls consisted of        the cases than the controls
    patients admitted, for the first time,
    to neurology or neurosurgery wards.
    Blood samples were taken the day after
    admission and the serum copper levels

                                               When the results of the mean blood
                                               copper levels in the two areas were
                                               pooled, the difference between the
                                               cases and controls was found to be
                                               substantially less, but the mean level
                                               was still statistically higher in controls.
                                               When the groups were split into
                                               quartiles of serum copper level, the
                                               pooled ORs were not significantly
                                               different from each other nor was there
                                               any significant trend in values.
                                               Adjustment for dietary zinc, which
                                               competes in the absorption of copper,
                                               and other elements, in particular iron,
                                               vitamin C and raw fibre, did not allow
                                               the correlation between copper intake
                                               and blood level to reach significance

    Table 15.  (continued)

    Study protocol                             Results                                               Comments                        Reference
    Serum copper and zinc levels were          Mean serum copper levels increased                    Serum levels measured           Çetinkaya
    measured in 20 healthy women and           from control to benign to malignant                   after diagnosis. No control     et al.
    100 women with gynaecological              groups                                                for potential confounders       (1988)
    tumours. 70 patients had benign and
    30 had malignant genital tumours

    The plasma copper                          The breast cancer cases were diagnosed an             Adjustments were made           Overvad
    concentrations of a group of 46 women      average of 11 years (range 1-17 years) after          for possible confounding        et al.
    who developed breast cancer                entry into the study cohort. The mean initial         by known indicators of          (1993)
    between 1968 and 1985 were                 copper levels were 1.26 mg/litre in the control       breast cancer, i.e. family
    compared with an age-stratified            group and 1.31 mg/litre in the cases (95% Cl          history of breast cancer,
    random sample of 138 women.                for overall difference=-0.07-0.17). The groups        age, age at first live birth,
    Both groups were taken from an             were split into quartiles corresponding to            parity, weight and oral
    initial cohort of 5100 ostensibly          initial copper concentrations of < 1.03,              contraceptive use
    healthy women studied between              1.04-1.19, 1.20-1.33 and > 1.34 mg/litre,
    1968 and 1975, aged 28-75                  and the adjusted odds ratio for the 1.04-1.19         The authors suggest a
    years and living on the island             mg/litre quartile set at 1.0. The adjusted odds       U-shaped risk response
    of Guernsey, United Kingdom.               ratios were: 1.8 (95% CI=0.6-5.4), 1.6 (95%           although this is not
    Plasma samples were                        CI=0.5-5.4) and 3.2 (95% CI=1.1-9.4) for              supported by the reported
    collected at the start of the              the < 1.03, 1.20-1.33 and > 1.34 mg/litre             results
    study and on development of                quartiles, respectively, with only the last
    breast cancer, and the levels              group reaching statistical significance
    of copper analysed

    Total serum copper and                     The serum copper concentrations did not alter         The average estimated           Dabek
    cerulo-plasmin levels were determined      significantly with time during the study year. A      daily dietary copper intakes    et al.
    in 13 pre- and 10                          significantly higher serum copper level was noted     were apparently lower in        (1992)
    postmenopausal breast cancer patients      in the premenopausal breast cancer patients           the patients (1.46 mg/day)
    aged 39 ± 7 and 66 ± 6 years,              (mean = 18.7 ± 0.62 µmol/litre) when compared         than in the normal control
    respectively. The levels were              with the two premenopausal control groups             subjects (1.63 mg/day;
    compared with those in a group             (means = 16.5 ± 0.30 and 16.7 ± 0.43 µmol/litre,      difference P = 0.05) and
    of 14 pre- and 11 postmenopausal           respectively; P < 0.03). No such difference was       this could not, therefore,
    omnivorous women                           noted in the postmenopausal patients.                 directly explain the results
    aged 33 ± 6 and 57 ± 5 years,              Postmenopausal patients showed significantly lower

    Table 15.  (continued)

    Study protocol                             Results                                               Comments                        Reference
    respectively and with those in             ceruloplasmin levels (mean = 0.309 ± 0.011 g/litre)   No control for smoking
    a group of 12 pre- and 11                  than the corresponding control groups (means =
    postmenopausal vegetarian                  0.387 ± 0.013 and 0.355 ± 0.11 g/litre,               The investigators concluded
    women aged 34 ± 7 and                      respectively, P < 0.01), this being more pronounced   that the high serum
    59 ± 5 years, respectively who             when the control groups were pooled (P < 0.001).      copper/ceruloplasmin ratio
    were all free of breast cancer.            Again, there was no overall significant               in the breast cancer patients
    Fasting serum samples were                 change with time during the study year.               may reflect disordered
    collected on three consecutive             The copper/ceruloplasmin ratios were higher in        copper metabolism in this
    days, typically four times in a year       both groups of patients, these increases being        disease (serum levels
                                               significant in the premenopausal group when           determined after diagnosis)
                                               compared with the corresponding omnivorous
                                               controls (P <0.05) and in the postmenopausal
                                               patients when compared with both the omnivorous
                                               (P < 0.001) and vegetarian (P < 0.01) control
                                               groups. The ratio in the postmenopausal patients
                                               (mean = 3.94 ± 0.096 µg/g) was significantly higher
                                               than in the premenopausal patients (mean = 3.44 ±
                                               0.061 µg/g; P < 0.001)

    dose-response trend in this regard (Kok et al., 1988; Coates et al.,
    1989; Overvad et al., 1993). Moreover, there has been no association
    between intake of copper and cancer, in those few analytical
    epidemiological studies in which it has been investigated (Cavallo et
    al., 1991; Dabek et al., 1992; Prasad et al., 1992).

         There is therefore little convincing evidence that copper plays
    an aetiological role in the development of cancer in humans.

    8.3.3  Dermal exposure

         Sources of topical exposure to copper have come from its use in
    pigments, ornaments, jewellery, dental amalgams, and IUDs, and as an
    antifungal agent and an algicide. Though copper algicides are used in
    the treatment of water in swimming pools and reservoirs, there are no
    reports of toxicity from these applications.

         Copper or copper salts may induce allergic contact dermatitis in
    susceptible individuals. Signs and symptoms include itching, redness,
    swelling, vesicle formation and pustulation.  Patch-testing to
    identify the sensitized state generally involved using covered 24-48 h
    contact with 0.5-5.0% copper sulfate in water or petrolatum.  Numerous
    reports have been published on the allergic response to unintentional
    and defined dermal exposure to copper or preparations containing
    copper (Hackel et al., 1991; Nordlend & Linden, 1991; Klapheck et al.,
    1994; Krolczyk et al., 1995), however, the exposure concentrations
    leading to any effect are poorly characterized in most cases.

         Routine patch testing of 1190 eczema patients found that only 13
    (1.1%) cross-reacted to 2% copper sulfate in petrolatum.  The
    investigators warned of the possibility that contamination of copper
    with nickel (a well-established contact allergen) might have been the
    cause of the apparent reaction to copper (Karlberg et al., 1983). In
    an investigation of copper and zinc status in 22 asthmatic, 21
    eczematous and 19 healthy Italian children (age-matched), the
    asthmatic group had higher mean values for serum and hair copper
    concentrations, and the eczematous group had higher mean hair copper
    concentrations, than did healthy controls.  Estimated dietary copper
    intakes were said to be similar for the three groups and ranged from
    90 to 111% of the "safe and adequate" intakes (Di Toro et al., 1987).

    8.4  Disorders of copper homoeostasis: populations at risk

         Because copper is an essential metal, there are homoeostatic
    mechanisms to maintain copper levels within defined limits. However,
    there are a number of disorders in homoeostatic mechanisms which can
    result in deficiency or toxicity from exposure to copper at levels
    which are tolerated by the general population. In addition to this,
    gross overexposure to copper can overwhelm the homoeostasis mechanisms
    in the normal individual.  The hereditary copper metabolic disorders
    are Menkes disease and Wilson disease.

    8.4.1  Menkes disease

         Menkes disease is an X-linked recessive disorder of copper
    metabolism that occurs in approximately 1 in 200 000 live births.
    Clinically the condition resembles a copper deficiency state and is
    characterized by skeletal abnormalities, severe mental retardation,
    neurological degeneration and death in early childhood. The symptoms
    of Menkes disease result from a deficiency of copper and its effects
    on the function of copper-dependent enzymes.

         The gene for the condition has been isolated (Chelly et al.,
    1993; Mercer et al., 1993; Vulpe et al., 1993) and designated  MNK. 
    The gene codes for a 1500-amino-acid P-type cation transporting
    ATPase, with strong homology to the bacterial and yeast cation
    transporting ATPases. The  MNK gene also has strong homology to the
    gene that is defective in Wilson disease (see section 8.4.2) (Bull et
    al., 1993; Thomas et al., 1995).

         Although the gene involved is widely expressed (except in liver),
    and copper actually accumulates in some cells (such as fibroblasts,
    kidney and placenta), the primary defect is a marked reduction in the
    first phase of copper transport. Most of the copper entering mucosal
    cells from the diet does not enter the portal circulation and travel
    to the liver and elsewhere. As a result, in most tissues, enzymes that
    depend upon copper for their functions will be inactive or have
    reduced activity. This may be the reason for the diverse clinical
    symptoms observed in Menkes patients. The MNK protein has structural
    similarities to Mg(II), Na(I), K(I), and Ca(II) transporters from
    various organisms. P-type ATPases have a conserved aspartate residue
    which is phosphorylated in the course of cation transport and have
    specific metal-binding sequences. The metal binding sequences are
    similar to those of P-type ATPases of bacteria, characterized by a
    G-M-T-C-XX-C motif. The Menkes disease and Wilson disease genes both
    encode proteins with six of these metal-binding sequences in the
    N-terminal half of the molecules, and multiple hydrophobic (probably
    membrane spanning) sequences nearer the C-terminal. They share a 59%
    amino-acid sequence identity with each other, and, respectively, share
    43% and 33% identities with the bacterial transporter CopA (Solioz et
    al., 1994). In Menkes disease the liver is not overtly affected,
    whereas in Wilson disease the liver is the primary site of damage. The
    gene for Menkes disease (also called  Mc1) has been mapped to band
    q13 on chromosome X (Mercer et al., 1993), and cloned, again by three
    independent research groups (Mercer et al., 1993; Vulpe et al., 1993;
    Chelly et al., 1993).

         The primary defect appears to involve defective expression of a
    transporter that transfers copper across the basolateral membrane of
    intestinal mucosal cells.  The transporter also may play a role in
    other cells, because it is widely expressed. It seems possible that
    its function might be to aid in copper efflux from cells, since Menkes
    fibroblasts accumulate the metal and fail to express the  MNK gene.
    This may not be the case in other tissues where accumulation has not
    been observed. In Menkes disease, intestinal absorption of copper, or

    its transfer across the placenta to the fetus, does not totally
    exclude copper from the body, since this is incompatible with life.
    Some cell types in tissues such as the intestine accumulate copper
    (Waldrop & Ettinger, 1990) which is subsequently lost as the
    intestinal cells are sloughed. The bulk of the metal is believed to
    accumulate in the Menkes-affected cell in metallothionein complexes.
    The lack of copper transport across the gut is one factor in
    production of a copper deficiency in most tissues.  Most likely,
    transporters for other metal ions can be used, at least to some
    extent, for copper transfer. Nevertheless, there is a still a serious
    copper deprivation in most tissues of the body, with the consequence
    that copper-dependent enzymes in all areas are affected and have a
    diminished function.

         Lysyl oxidase has been shown to be important in cross-linking
    collagen and elastin and its lack of activity may explain the
    connective tissue lesions.  Low levels of cytochrome c oxidase may
    contribute to poor thermal regulation.  Tyrosinase deficiency would be
    expected to lead to hypopigmentation of the skin and hair.  The pili
    torti (twisted or "kinky" hair) observed in Menkes patients is related
    to the cross-linking failure of keratin which is dependent on copper.
    Deficiency of cytochrome c oxidase, SOD and dopamine betahydroxylase
    may result in neurological degeneration, mainly by oxygen free
    radicals (Bankier, 1995).

         The clinical features observed in Menkes patients are a direct
    result of the failure of copper to be incorporated into specific
    copper-dependent enzymes (Kodama, 1993). Hence, Menkes disease mimics
    a deficiency in copper.  Babies with Menkes disease are often born
    prematurely; and although they appear to have fine, normal-looking
    hair they often have problems associated with temperature instability,
    jaundice and feeding (Bankier, 1995).  Many pass the developmental
    milestones of head control and responsive smile, but by the age of 3
    months they develop loss of head control and begin to have seizures.
    They have truncal hypotonia (a condition of diminished tone of the
    skeletal muscles and diminished resistance of muscles to passive
    stretching) and progressive spasticity of the limbs.  The hair becomes
    fragile, lustreless and hypopigmented.  The hair feels to the touch
    like steel wool, owing to pili torti.  The skin becomes hypopigmented
    and hyperextensible (cutis laxa) and the joints become hypermobile
    (Martin et al., 1994).

         The bones are osteoporotic with flared metaphyses of the long
    bones, rib fractures and possible wormian bones (small irregular bones
    in the sutures between the bones of the skull) visible by cranial
    radiography.  In the case of severe occipital horn syndrome the main
    effect is bone spurs, perhaps because of disordered connective tissue
    function and neurological problems (Kaler et al., 1994). The
    vasculature is tangled and elongated owing to numerous splits and
    fragmentations in arterial elastic fibres and thickened intima.
    Alterations in the central nervous system include severe mental
    retardation, seizures and ataxia which are due to intense degenerative
    changes of the brain and the cerebellum with a pronounced alterations

    of the Purkinje cells (Iwata et al., 1979).  Subdural and cerebral
    haematoma may occur.  There is progressive deterioration until death
    occurs, usually by the age of 5. Urinary tract diverticulum (a pouch
    or sac produced by herniation of the mucous membrane through a defect
    of the lining of the urinary tract) is common.

         The majority of patients with Menkes disease present with severe,
    classical symptoms although individuals with milder symptoms and/or
    longer survival have been observed (Haas et al., 1981; Gerdes et al.,
    1988).  A spectrum of mutations adversely affecting protein expression
    has been observed in severely affected Menkes patients.  The diseases
    X-linked cutis laxa (Levinson et al., 1993; Yeowell et al., 1994),
    occipital horn syndrome (Kaler et al., 1994) and milder Menkes
    phenotypes result from mutations that only diminish or alter  MNK 

    8.4.2  Wilson disease

         Samuel A.K. Wilson described a disorder of the nervous system
    associated with liver cirrhosis. Wilson wrote that the disease, "...is
    familial, invariably fatal (and caused by) a toxin generated in
    connection (with) the hepatic cirrhosis that is always found after
    death" (Wilson, 1912). Following this lead in 1920, Hall concluded
    that Wilson disease occurred only in individuals who inherited a
    defective gene (Hall, 1921) which Bearn later showed to be recessive
    (Bearn, 1960). It was not until 1948 that Cumings identified that
    copper was indeed the toxin in Wilson disease, finding that the liver
    and brain of patients had an extremely high content of the metal
    (Cumings, 1948).

         Wilson disease is the most extensively described inherited
    disorder of copper metabolism. The gene is distributed worldwide,
    having been demonstrated in virtually all races.  Current global
    estimates indicate that the incidence rate of the disease is
    approximately 1 in 30 000 live births, with prevalency ranging from 15
    to 30 per million.  The gene frequency varies between 0.3 and 0.7%,
    corresponding to a heterozygote carrier rate of slightly greater than
    1 in 100.

         Genetic studies from a large Israeli-Arab kindred identified a
    linkage between the Wilson disease locus and the erythrocyte enzyme
    esterase D, thereby establishing that the gene mutation responsible
    for Wilson disease was located on chromosome 13 (Frydman et al.,
    1985).  Using multipoint linkage techniques, the abnormal gene for
    Wilson disease was localized more specifically to 13q14-q21.  In 1993,
    a candidate gene for Wilson disease  (WND) was reported independently
    by several different groups of investigators, using slightly different
    strategies for positional cloning (Bull et al., 1993; Petrukhin et
    al., 1993; Tanzi et al., 1993).  The  WND gene consists of a
    transcript of approximately 7.5 kilobases, which is expressed
    primarily in liver, kidney and placenta; it has also been detected in
    heart, brain, lung, muscle and pancreas, albeit at much lower levels.
    The full-length cDNA sequence of the  WND gene (Bull et al., 1993;

    Tanzi et al., 1993) predicts a protein of 1411 amino acids which is a
    member of the cation-transporting P-type ATPase subfamily, highly
    homologous to the Menkes disease gene product and the
    copper-transporting ATPase (CopA) found in copper-resistant strains of
     Enterococcus hirae.

         From sequence analysis of the cDNA, the WND protein is predicted
    to possess a metal-binding domain (containing five specific binding
    sites), an ATP-binding domain, a cation channel and phosphorylation
    region, and a transduction domain responsible for the conversion of
    the energy of ATP hydrolysis to cation transport.  To date, more than
    30 disease-specific mutations in the Wilson disease gene have been
    identified, and it has been postulated that different mutations at
    that locus may explain the clinical variability.  Moreover, the
    variety of mutations identified in the Wilson disease gene potentially
    may affect copper transport to varying degrees, and at different
    cellular sites (Schilsky, 1994).  However, detailed genetic and
    epidemiological studies suggest that the variability in clinical
    expression observed in Wilson disease patients may not be solely a
    consequence of allelic heterogeneity, since marked differences in
    presentation, age of onset and disease course have been observed in
    family members who have inherited two identical mutant alleles
    (Walshe, 1995).

         Developments in the molecular genetics of Wilson disease have
    provided a means for carrier detection and early diagnosis (Sternlieb,
    1993).  In fact, several studies using haplotype analysis of relatives
    with closely linked markers have permitted precise carrier detection
    with less than 1-2% error.  There also is a report of prenatal
    exclusion of Wilson disease by analysis of DNA polymorphism in a
    chorionic villus biopsy performed at 9 weeks gestation (Cossu et al.,
    1992).  Unfortunately, the use of genetic techniques in the diagnosis
    of Wilson disease has significant limitations.  Currently, DNA marker
    studies can be performed only within families, and under circumstances
    where the diagnosis has already been established definitely in at
    least one family member by standard biochemical methods.  The index
    patient's DNA is then used as a reference to recognize the
    disease-carrying chromosomes in other members of the family.  However,
    spontaneous chromosomal rearrangements can cause such markers to be
    uninformative, thereby limiting the diagnostic reliability. These
    findings indicate considerable potential difficulties for DNA-based
    genetic screening, since most patients will possess alleles with two
    different mutations of the Wilson disease gene (Schilsky, 1994).
    Given the rapidity and accuracy of biochemical analyses in
    establishing the diagnosis of Wilson disease, as well as the
    aforementioned limitations of genetic testing, standard biochemical
    methods should continue to be utilized in the evaluation of most
    suspected cases.  In addition, genetic screening of young family
    members of patients afflicted with the disorder would facilitate early
    diagnosis and permit initiation of therapy in the presymptomatic

         It is postulated that the harmful effects of excess copper are
    mediated by the generation of free radicals, which deplete cellular
    stores of glutathione and oxidize lipids, enzymes and cytoskeletal
    proteins.  Indeed, it has been shown that a number of intracellular
    systems are disrupted by elevated copper concentrations, including
    organellar membranes, DNA, microtubules, and various enzymes and
    proteins, although the principal cellular target of copper toxicity is
    unknown.  In the earliest stages of hepatocellular injury,
    ultrastructural abnormalities involving the endoplasmic reticulum,
    mitochondria, peroxisomes and nuclei have all been identified
    (Sternlieb, 1990).  These changes, in conjunction with diminished
    mitochondrial enzyme activities, may be important steps in the
    pathophysiological events leading to lipid peroxidation and
    triglyceride accumulation in the hepatocyte.

         Wilson disease patients exhibit impaired biliary excretion of
    copper, which is believed to be the fundamental cause of copper
    overload.  The prompt reversal of abnormal copper metabolism in Wilson
    disease patients following orthoptic liver transplantation confirms
    that the primary defect resides in the liver.  It has been proposed
    that the Wilson disease gene product is responsible for copper
    secretion from the liver cell, either across the canalicular (apical)
    membrane of the hepatocyte or into a subcellular compartment that
    communicates with the bile canaliculus (Tanzi et al., 1993).  The
    latter is consistent with a putative lysosomal defect underlying the
    diminished biliary excretion and systemic accumulation of copper
    observed in patients with Wilson disease.  In addition, in an animal
    model of Wilson disease, the Long-Evans Cinnamon (LEC) rat, excessive
    hepatic copper accumulation occurs in the setting of diminished
    biliary excretion.  These rodents exhibit impaired entry of copper
    into the lysosomes, with normal delivery of lysosomal copper to the
    bile (Schilsky et al., 1994).  The LEC rat is a mutant strain of the
    Long-Evans rat which spontaneously develops fulminant hepatitis at 3-4
    months of age, resulting in a 40% mortality rate.  Surviving animals
    manifest chronic hepatic disease, low serum ceruloplasmin levels and
    increased copper concentrations in the liver.  Thus, the LEC rat
    shares many important clinical, biochemical and histological features
    with Wilson disease, and the recent availability of this animal model
    will probably provide new insight into the pathogenesis of the human

         The biochemical defect which leads to the accumulation of copper
    in Wilson disease is present at birth; however, clinical symptoms
    rarely are observed before the age of 5 years.  The initial signs of
    Wilson disease are generally detected in older children, adolescents
    and young adults, although case reports have documented the clinical
    onset as early as 4 years.  Wilson disease patients typically present
    with hepatic and/or neurologic dysfunction.  Less commonly, patients
    present with skeletal, cardiac, ophthalmologic, endocrinologic or
    dermatologic symptoms.  Approximately 25% of patients have involvement
    of two or more organ systems at initial evaluation, although, with the

    advent of aggressive screening, there has been a significant increase
    in the number of asymptomatic patients diagnosed. The clinical
    manifestations of Wilson disease are summarized in Table 16.

        Table 16.  Clinical manifestations of Wilson disease
    (hepatolenticular degeneration)

    Organ system           Symptoms

    Hepatic                cirrhosis, chronic active hepatitis, fulminant failure
    Neurologic             bradykinesia, rigidity, tremor, ataxia, dyskinesia,
                           dysarthria, seizures

    Psychiatric            behavioural disturbances, cognitive impairment, affective
                           disorders, psychosis

    Ophthalmologic         Kayser-Fleischer rings, sunflower cataracts

    Haematologic           haemolysis, coagulopathy

    Renal                  renal tubular defects, diminished glomerular filtration,

    Cardiovascular         cardiomyopathy, arrhythmias, conduction disturbances,
                           autonomic dysfunction

    Musculoskeletal        osteomalacia, osteoporosis, degenerative joint disease

    Gastrointestinal       cholelithiasis, pancreatitis, spontaneous bacterial

    Endocrine              amenorrhoea, spontaneous abortion, delayed puberty,

    Dermatologic           azure lunulae, hyperpigmentation, acanthosis nigricans

         Hepatic involvement in Wilson disease tends to manifest at a
    younger age (mean 8-12 years) than does neurological dysfunction, and
    is nonspecific, mimicking the features of a variety of acute and
    chronic liver diseases.  Three major clinical patterns of liver
    disease are observed: cirrhosis, chronic active hepatitis and
    fulminant hepatic failure. In the early asymptomatic phase of Wilson
    disease, or in the presence of inactive cirrhosis, liver tests may be
    normal or only minimally elevated. In the majority of cases, hepatic
    injury develops insidiously and, if untreated, pursues a chronic and
    relentless course to cirrhosis. Hepatocellular carcinoma is uncommonly
    associated with Wilson disease, in contrast to haemochromatosis.

         An estimated 5-30% of patients with Wilson disease exhibit
    clinical, biochemical and histological features similar to those
    observed in chronic active hepatitis (Scott et al., 1978; Schilsky et
    al., 1991).  The diagnosis may be overlooked in these patients, since
    a significant number, almost 50% in one series (Scott et al., 1978),
    have no evidence of neurologic dysfunction or Kayser-Fleischer rings
    on ophthalmologic examination.  Serum ceruloplasmin levels also may be
    normal in the setting of severe hepatic inflammation.  It has been
    estimated that Wilson disease represents the underlying aetiology in
    5% of patients with idiopathic chronic active hepatitis who are under
    35 years of age (Schilsky et al., 1991).  A distinctive feature of
    wilsonian chronic active hepatitis is the relatively modest elevations
    of serum aminotransferase levels in the presence of severe
    hepatocellular necrosis and inflammation.

         More dramatically, Wilson disease occasionally manifests as
    fulminant hepatic failure.  These patients may be indistinguishable
    from individuals with viral-induced hepatic necrosis, and many of the
    biochemical tests used to establish the diagnosis of Wilson disease
    are abnormal in patients with other forms of fulminant hepatic failure
    (McCullough et al., 1983).  The clinical features most suggestive of
    fulminant wilsonian hepatitis include the presence of intravascular
    haemolysis, splenomegaly, and Kayser-Fleischer rings.  Biochemical
    markers indicative of Wilson disease include relatively mild
    elevations in serum transaminases despite massive hepatic necrosis,
    hyperbilirubinaemia with normal or low alkaline phosphatase levels,
    and a markedly elevated serum copper concentration.  The serum level
    of aspartate aminotransferase (ASAT) typically is higher than that of
    alanine aminotransferase (ALAT), as a result of the associated
    haemolysis.  Although uncommonly observed in wilsonian fulminant
    hepatic failure, Kayser-Fleischer rings are not pathognomonic, since
    they are occasionally seen in patients with other cholestatic hepatic
    disease.  Liver biopsy with measurement of quantitative copper may be
    helpful, although deranged clotting function may preclude this
    procedure, or necessitate the transjugular approach.  If a biopsy
    specimen is obtained, histological evidence of cirrhosis
    (predominantly micronodular) in a young patient with fulminant
    hepatitis is suggestive of Wilson disease, as is an elevated hepatic
    copper content.  Wilson disease patients with acute hepatic failure
    tend to be young and to have a fulminant clinical course, with
    survival generally no longer than days to weeks unless liver
    transplantation is performed.  Even when transplantation is
    unavailable for patients, it remains imperative to make the diagnosis
    of Wilson disease for the purpose of aggressive medical therapy and
    family screening.

         The simplest screening procedure includes a slit-lamp examination
    of the eyes, and measurement of serum ceruloplasmin and transaminase
    (ALAT, ASAT) levels. If Kayser-Fleischer rings are present on
    ophthalmologic examination and ceruloplasmin levels are below 200
    mg/litre in a patient with neurologic signs or symptoms, the diagnosis
    of Wilson disease is established. If a patient is asymptomatic,
    exhibits isolated liver disease, or lacks corneal rings, the

    coexistence of a hepatic copper concentration above 250 µg/g (dry
    weight) and a low serum ceruloplasmin level also is sufficient to make
    the diagnosis.

         The normal serum concentration of ceruloplasmin is 200-400
    mg/litre.  Although a decreased ceruloplasmin level  per se is not
    diagnostic of Wilson disease, approximately 90% of all patients, and
    85% of individuals presenting with hepatic manifestations of the
    disease, have levels that are below the normal range.

         The 10% of heterozygous carriers of the gene for Wilson disease
    who manifest diminished serum levels of ceruloplasmin, yet never
    develop clinical symptoms or signs of the disease, may cause
    diagnostic confusion.  These individuals, who represent approximately
    1 in 2000 of the general population, may present a difficult
    diagnostic dilemma if they fortuitously develop chronic active
    hepatitis or cirrhosis (of another aetiology), thereby mimicking the
    clinical, biochemical and histological features of Wilson disease.
    Normal ceruloplasmin concentrations are found in up to 15% of patients
    with Wilson disease and active liver involvement (Scott et al., 1978).

         The urinary excretion of copper is greater than 100 µg/24 h
    (normal < 40 µg/24 h) in most patients with symptomatic Wilson
    disease, reflecting increased serum levels of the readily filterable
    fraction of nonceruloplasmin copper.

         If Kayser-Fleischer rings or neurological abnormalities are
    absent, a liver biopsy for quantitative copper determination is
    essential to establish the diagnosis of Wilson disease.  Care must be
    taken to ensure that the biopsy needle and specimen container are free
    from copper contamination.  The normal hepatic copper concentration
    varies from 15 to 55 µg/g (0.24-0.87 µmol/g) dry liver.  Virtually all
    untreated patients with Wilson disease have elevated hepatic copper
    levels, ranging from 250 to as high as 3000 µg/g dry liver.  Values
    below 250 µg/g are usually attributable to the irregular distribution
    of copper in the liver, particularly in the presence of cirrhosis,
    when small fragmented biopsy samples are obtained.  The finding of a
    normal hepatic copper concentration effectively excludes the diagnosis
    of untreated Wilson disease.  However, an elevated liver copper level
    alone is insufficient to establish the diagnosis of Wilson disease,
    since concentrations above 250 µg/g may be found in other chronic
    hepatic disorders (most cholestatic conditions).  In the great
    majority of individuals with prolonged cholestasis, serum
    ceruloplasmin concentrations are either normal or increased. The
    histochemical staining of liver biopsy specimens for copper is of
    little diagnostic value in patients with Wilson disease.

    8.4.3  Hereditary aceruloplasminaemia

         Although no defect in copper metabolism has been identified in
    cases of aceruloplasminaemia, this condition is included here because
    ceruloplasmin is a genetically regulated, copper-binding protein with
    a role in iron metabolism (Harris & Gitlin, 1996) (see chapter 6).

         Recent evidence indicates that genetic abnormalities of
    ceruloplasmin synthesis occur as an autosomal recessive condition
    (Logan et al., 1994). Clinical signs and symptoms in these patients
    include mental confusion, memory loss, dementia, cerebellar ataxia,
    altered motor function, retinal degeneration and diabetes (Miyajima et
    al., 1987; Logan et al., 1994; Harris, 1995; Morita et al., 1995).
    Biochemical signs are decreased serum copper levels and absent or
    nonfunctional ceruloplasmin in plasma and impaired copper absorption
    (Harris, 1995). Isotopic tracer studies demonstrate enhanced copper
    incorporation into liver with limited release into plasma since
    ceruloplasmin synthesis is absent, yet copper delivery to tissues is
    preserved (Miyajima et al., 1987; Harris, 1995). In fact, copper
    homoeostasis appears to be minimally affected while striking
    abnormalities in iron metabolism are found.

         There is a significant decrease in serum iron, normal
    iron-binding capacity, markedly elevated serum ferritin and low
    urinary iron excretion. Iron deposition in liver, brain, pancreas and
    other tissues is markedly increased. The alterations in iron
    homoeostasis are correctable by the intravenous administration of
    ceruloplasmin (Ragan et al., 1969). On the basis of this evidence the
    clinical symptoms are most like the result of iron overload in brain,
    pancreas and other critical organs, rather than induced by a copper

    8.4.4  Indian childhood cirrhosis

         Indian childhood cirrhosis (ICC) was once a major cause of infant
    mortality on the Indian subcontinent (Kumar, 1984). The peculiar
    epidemiological, clinical and histopathological features, the
    enigmatic aetiology and the uniformly fatal outcome have baffled many
    for over a century now (Achar et al., 1960; Chawla et al., 1973;
    Bhagwat & Walia, 1980; Sethi et al., 1993).

         Epidemiologically, the illness normally strikes between the ages
    of 6 months and 3 years (Bhave et al., 1992) although it can occur up
    to 5 years of age (Nayak & Ramalingaswamy, 1975).  There is a male
    predominance and high rates of parental consanguinity, and up to 22%
    of siblings are affected.

         Clinically, the onset is generally insidious (86%). In the early
    stage of the disease the complaints are nonspecific such as abdominal
    distention, irregular fever, excessive crying and altered appetite. In
    a few children, the disease begins with jaundice, but commonly
    jaundice is a late feature. In the second clinical stage of the
    disease, the liver is characteristically firm with a "leafy" edge.
    The progress is relentless and within a few months, the patient
    progresses on to the terminal stages with jaundice,
    hepatosplenomegaly, oedema and ascites.  Death is usually due to
    intercurrent infections or terminal hepatocellular failure leading to
    haemorrhagic complications or hepatic coma.

         The standard liver function tests are usually deranged but not
    specific for the differentiation of early ICC from other childhood
    liver disorders. Serum copper is raised significantly in ICC. The mean
    serum copper values increase with the clinical progression of the
    disease (Tanner et al., 1979; Sharda & Bhandari, 1984; Sethi et al.,
    1993).  Serum ceruloplasmin levels, however, are normal or elevated,
    in contrast to Wilson disease.  Hepatic copper is increased.  A
    hepatic copper level > 800 µg/g dry weight helps distinguish ICC from
    other liver disorders occurring at this age.

         Histopathology remains the cornerstone of definitive diagnosis.
    (Parekh & Patel, 1972; Bhave et al., 1982, 1983).  The two most
    discriminatory features of ICC now recognized are typical widespread
    coarse dark brown orcein staining and intralobular pericellular
    fibrosis (Pradhan et al., 1983). Hepatocytic necrosis (seen in 97%)
    and hyaline (66%) are also diagnostic though late features. Portal
    fibrosis, inflammation and disruption of the limiting plate are seen
    in most cases, but also are seen in other liver disorders and hence
    are not of discriminatory value. Parenchymal fat is usually absent and
    cholestasis is a late feature (Pandit & Bhave, 1983).  Raised hepatic
    copper, indicated by orcein staining, is seen consistently in ICC.
    Intensity of orcein staining correlates significantly with the
    histopathological grade of the disease (Sethi et al., 1993).

         Various aetiological agents have been implicated in ICC, but none
    has so far been confirmed. Tanner et al. (1983) stated that "early
    introduction of copper-contaminated animal milk is of aetiological
    importance", based on the observation that ICC was predominantly seen
    in children who were bottle-fed rather than breast-fed, and that milk
    stored in brass vessels prior to feeding became contaminated with high
    levels of copper.  Experimentally, boiling and storing of milk in
    untinned brass vessels raises its copper concentration more than 60
    times, and copper and brass vessels have been used traditionally in
    some parts of India to boil and store milk and water.  Although
    ingestion of large amounts of copper in early infancy may be a factor
    in the aetiology, it cannot fully explain the disease. Approximately
    half of the patients presenting with ICC had received milk which had
    been previously stored in brass vessels (Sharda & Bhandari, 1984).

         In a study in India, a group of 32 children who developed
    cirrhosis had a significantly higher mean value of serum copper
    measured after diagnosis than a control group of 10 healthy
    age-matched children. The use of brass utensils to carry, boil and
    store milk occurred in only 14 (44%) of the cases, and increased serum
    copper levels were not limited to these.  In another 82 children
    suffering from cirrhosis, liver biopsies revealed raised liver
    concentrations of copper in all cases, and levels increased with the
    severity of the disease (Sethi et al., 1993).

         In some cases, other family members and siblings had received
    milk from the same source as the ICC cases but were found to have
    normal serum and urinary copper levels (Sharda & Bhandari, 1984).
    Furthermore, that ICC has been seen in children who have been

    breast-fed suggests that copper is unlikely to be the sole cause of
    the illness (Sethi et al., 1993).

         Because of the familial occurrence and high consanguinity, a
    genetic aetiology of ICC has been suspected (Agrawal et al., 1979;
    Sethi et al., 1993). Chandra (1976) reported a pedigree analysis
    compatible with autosomal recessive inheritance.  Although both serum
    and hepatic concentrations increased with the severity of the disease,
    the copper content is variable at the same stage of the disease.
    Thus, genetic heterogeneity in ICC has been postulated (Sethi et al.,

         The copper chelator  d-penicillamine has been given to early ICC
    patients, and histological improvement and remission in up to 65% of
    patients has been claimed (Tanner et al., 1987).  This is a single
    study on only 29 patients; therefore, more work needs to be done to
    definitely determine the role of  d-penicillamine in the treatment of

         There has been a reduction of ICC in India (Bhave et al., 1992).
    Whether this reduction is due to the reduction of the use of brass
    vessels, or due to increasing intercaste marriages leading to genetic
    dilution, or both, is yet unclear.

         A similar reduction in fatal infantile liver cirrhosis in a
    region of Austria has been reported (Müller et al., 1996).  An
    ecogenetic aetiology proposed in these conditions requiring a
    convergence of a genetic predisposition with a high copper intake
    could also be a prerequisite for the development of ICC.  However,
    whether ICC represents a specific form of infantile copper toxicosis
    (ICT) or is an unrelated infantile cirrhosis is yet to be determined.
    The relative importance of the role of environmental exposure to
    copper and the genetic predisposition to copper accumulation have not
    yet been determined.

    8.4.5  Idiopathic copper toxicosis, or non-lndian childhood

         Scattered reports of early childhood cirrhosis similar to ICC,
    referred to as copper-associated idiopathic copper toxicosis (ICT)
    have appeared from some Western countries (Walker-Smith & Blomfield,
    1973; Müller-Höcker et al., 1987; Adamson et al., 1992; Gormally et
    al., 1994).  It is unclear whether the aetiology of this disease is
    the same as that of ICC as seen in India (section 8.4.4).
    Müller-Höcker et al. (1987, 1988) described the first three cases in
    Germany with histological and clinical features of ICC, including very
    high liver copper levels.  Eife et al. (1991) reported a total of 22
    such cases (13 fatal) in Germany up to 1990 and attributed them to
    ICT.  All the families involved from Germany and elsewhere, lived in
    rural areas and were supplied with soft and acidic water from private
    wells using copper pipes.  The exposed children were breast-fed only
    briefly or not at all and their formula had been made up with well
    water, presumably contaminated with copper.  Details on three of the

    aforementioned German cases were given by Müller-Höcker et al. (1987,
    1988), Schramel et al. (1988) and Weiss et al. (1989).  The water
    copper levels (non-representative single values) varied from 0.4 to
    15.5 mg Cu/litre.  These values were not measured during the time of
    exposure, but several months later.  The authors attributed the
    illness to copper toxicosis, possibly in connection with an unproven
    genetic predisposition and/or unusual high copper exposure of the
    babies via the formulas.

         Müller et al. (1996) reported on the largest non-Indian series of
    cases of a disease they regarded as identical to ICC or ICT.
    Unfortunately they were unable to obtain liver samples to confirm high
    copper values, and relied on photographs for histology to demonstrate
    the similarity with ICC.  In the Tyrol region of Austria between 1900
    and 1974, 138 fatal cases of this cirrhosis were found.  Detailed
    family pedigree analysis suggested that susceptibility to the disease
    was inherited in an autosomal recessive fashion and that the
    copper-rich diet of the region induced the symptoms (experiments
    duplicating methods of milk preparation using copper vessels suggested
    copper levels of up to 60 µg/litre).  Many similarly fed infants did
    not develop cirrhosis.  There have been no cases since 1974.  The
    authors speculated that this could be due to the replacement of copper
    and brass vessels, although increased mobility of the population and
    fewer consanguineous marriages may have diluted the gene pool reducing
    the number of homozygous children.  This report provides a likely
    explanation for the causation and natural history of copper-associated
    ICT in Austria and possibly elsewhere.

         A number of case reports on childhood cirrhosis associated in
    most cases with only intermediate hepatic copper levels (< 400 µg/g
    dry weight) have been described worldwide, but no environmental copper
    exposure was evident (Lim & Choo, 1979; Maggiore et al., 1987; Aljajeh
    et al., 1994; Baker et al., 1995).

         In order to test the hypothesis that ICT is an entirely
    environmental condition, Scheinberg & Sternlieb (1994) reported on
    three Massachusetts, USA, towns where drinking-water was known to
    contain high levels of copper (8.5-8.8 mg Cu/litre on first-draw
    samples after 6 h of stagnation).  Between 1969 and 1991, mortality of
    3000 children under the age of 6 years with liver and other diseases
    were studied.  During that period there were 135 deaths among the
    study population but none from cirrhosis or any form of liver disease.
    The sample size of this study was insufficient to fully test the
    proposed hypothesis.

         Fewtrell et al. (1996) reported 220 patients aged up to 7 years
    with liver disease in the United Kingdom in 1991-1993.  Copper
    exposure in tap water was mostly below 3 mg/litre, but in 15 cases
    higher levels may have occurred.  In this series of patients too no
    cases of ICT were detected.

         A retrospective, multicentre study (Schimmelpfennig et al., 1996)
    detected a total of 103 cases of early childhood cirrhosis of
    different causes for the years 1982-1994 in Germany.  The three cases
    described in detail by Müller-Höcker et al. (1987, 1988) were not
    included in this study.  In only two cases were the exact conditions
    of increased copper exposure reliably reconstructed and other
    aetiologies of cirrhosis excluded.  The concentrations of copper in
    the tap water in these two cases were 9-26 mg/litre owing to specific
    conditions of the individual water supplies.  These concentrations may
    have been the cause of one fatal case and may have led to severe liver
    disease in the other.  Recently a case of adult liver cirrhosis
    associated with a daily copper intake of 0.5-1.0 mg Cu/kg body weight
    was described (see section 8.3.2) (O'Donohue et al., 1993).  Based on
    these collective data, a purely environmental basis for ICT cannot be
    confirmed or excluded; thus, the cause of liver injury remains

    8.4.6  Chronic liver diseases

         Copper retention occurs as a result of impaired biliary
    excretion. As reviewed recently by Zucker & Gollan (1996), conditions
    such as primary biliary cirrhosis, primary sclerosing cholangitis,
    extrahepatic biliary obstruction or atresia, intrahepatic cholestasis
    of childhood and chronic active hepatitis can lead to liver copper
    levels above 250 µg Cu/g dry weight.  These patients can be
    distinguished from those with Wilson disease on the basis of history,
    physical findings and elevated or normal serum ceruloplasmin levels.
    The presence of hepatic disease requires caution in the provision of
    dietary copper.  Correction of biliary output in the cholestatic
    condition may lead to decrease in liver copper levels (Ohi & Lilly,

    8.4.7  Copper in infancy

         Fetal copper metabolism is different from that in children or
    adults.  Neonates have high levels of copper in the liver and low
    levels of serum copper and ceruloplasmin (Epstein, 1983) and elevated
    levels of metallothionein that decrease after birth.  After the age of
    about 6 months both liver copper and serum copper levels come within
    the adult range.  The ratio of hepatic concentration of copper in
    newborns to that of an adult human is 15 : 4 (Goyer, 1991).

         Acquired copper deficiency is a clinical syndrome that occurs
    mainly in infants (Shaw, 1992), although it has also been described in
    children and in adults. Copper deficiency is usually the consequence
    of decreased copper stores at birth (see chapter 6), inadequate
    dietary copper intake, poor absorption, elevated requirements induced
    by rapid growth or increased copper losses. Excretion of copper is
    usually via the bile, but if renal tubular reabsorption is impaired
    urinary losses may be quite high. The multiple factors that may lead
    to deficiency commonly coexist in copper-deficient subjects. Copper
    deficiency is more frequent in preterm infants, especially of very low
    birth weight, owing to their reduced copper stores at birth given the

    smaller relative size of the liver and higher requirements determined
    by their high growth rate (Widdowson & Dikerson, 1964; Widdowson et
    al., 1974; Dauncey et al., 1977; Sutton et al., 1985; Hurley & Keen,

         Infants fed exclusively diets based on cow's milk are more prone
    to develop copper deficiency because of the low copper content of milk
    and limited absorption of this mineral in cow's milk. In contrast,
    breast-fed infants absorb more copper; this may be due to the lower
    casein content of human milk or to factors present in human milk which
    enhance copper absorption (Naveh et al., 1981; Lönnerdal et al.,
    1985). In developing countries, where infant feeding is often based on
    cow's milk enriched with a high concentration of refined
    carbohydrates, copper deficit may be more prevalent because fructose
    and other refined sugars lower copper absorption.

         On the basis of published information, the most common cause of
    copper deficiency is insufficient copper supply during the nutritional
    recovery of malnourished children (Shaw, 1992).  These infants present
    several factors which are frequently associated to copper deficiency:
    history of low birth weight, short duration of breast-feeding, a diet
    based on cow's milk and a highly refined carbohydrate, or increased
    losses of nutrients due to diarrhoeal disease and frequent infections.
    During nutritional recovery they grow 5-10 times as fast as normal for
    their age group, thus increasing the nutrient requirement.

    8.4.8  Malabsorption syndromes

         Copper deficiency has been reported in subjects with
    malabsorption syndromes such as coeliac disease, tropical sprue,
    cystic fibrosis, partial gastrectomy or short bowel syndrome due to
    intestinal resection (Williams, 1983; Rodriguez et al., 1985; Hayton
    et al., 1995).  Copper deficit should be suspected in infants with
    prolonged or recurrent diarrhoeal episodes, abnormal bile loss,
    intestinal resections, or loss of intestinal contents from intestinal
    fistula (Williams, 1983; Castillo-Duran et al., 1988). Castillo-Duran
    et al. (1988) evaluated the magnitude of copper loss in 14 infants
    during acute diarrhoeal episodes requiring hospitalization. The
    results were compared with those obtained in 15 matched control
    infants. Faecal losses of copper were twice as high in the diarrhoea
    group as in the control subjects. This group presented a negative
    copper balance up to 7 days after hospital admission. Copper losses
    were directly related to faecal weight. Furthermore, Rodriguez et al.
    (1985) compared the copper status of 19 children exhibiting chronic
    diarrhoea with two control groups (19 healthy and 11 malnourished
    children). Plasma copper levels were 30% lower and hair copper content
    decreased 3-4-fold in the group with chronic diarrhoea relative to the
    control groups.

         High oral intakes of zinc and iron decrease copper absorption and
    may lead to copper deficiency (Prasad et al., 1978; Williams, 1983).
    This phenomenon is used as a therapeutic strategy in Wilson disease
    where high zinc intake (40-50 mg/day) has been demonstrated to lower

    copper absorption. Copper deficiency has been also documented in
    subjects receiving penicillamine or other cation chelating agents, or
    high doses of oral alkali therapy which enhance copper losses
    (Williams, 1983).

    8.4.9  Parenteral nutrition

         Patients fed with intravenous nutrient mixtures lacking
    sufficient copper will develop symptomatic deficiencies after 3-12
    months (Shike et al., 1981).  In adults, this presents as an
    iron-resistant anaemia, with a mark fall in neutrophils. In children,
    as well as the haematological abnormality, there are marked effects in
    bone: characteristic radiological changes, greater ease of fracture
    and reduced bone age (Shaw, 1992).

         It has been shown that infusion of 0.3 mg Cu/day will maintain a
    70 kg adult in copper balance (Shike et al., 1981).  However, in
    patients with high volume fistula or diarrhoeal losses additional
    copper may be needed.  The adult normative requirements of 1.3 mg
    Cu/day will maintain plasma copper within the reference interval and
    prevent the development of deficiency disease (Shenkin et al., 1987).
    An increased amount of copper may be required in patients who have
    high volume fistula fluid or diarrhoeal losses.

         The neonatal requirements for copper will vary according to such
    factors as premature delivery and low birth weight.  It has been
    suggested that approximately twice as much copper is required by the
    pre-term infant compared to the term infant (Shaw, 1992; WHO, 1996).

         Where there is evidence of choleostasis, copper supplements in
    both adults and children should be reduced or withheld and the patient
    monitored for any signs of developing copper toxicity.

    8.4.10  Haemodialysis patients

         Copper homoeostasis mechanisms available for regulating
    gastrointestinal absorption of copper are bypassed by parenteral
    administration.  Copper toxicity in patients on haemodialysis is not
    common.  In two studies of four patients exposed to poorly defined
    concentrations of copper in the dialysis fluid (0.056 to > 0.11
    mg/litre) headache, sweating, nausea, hypotension, stupor and coma
    were reported (Klein et al., 1972; Lyle et al., 1976).  Similar signs
    and symptoms were reported in three patients exposed to copper
    concentrations between 5.1 and 8.8 mg/litre of dialysate (Manzler &
    Schreiner, 1970).

    8.4.11  Cardiovascular diseases

         Changes in copper concentrations have been associated with
    ischaemia (Kinsman et al., 1990), as well as various cardiovascular
    and cerebrovascular related problems (Peterson et al., 1990).
    Reviewing the relationship between ischaemic heart disease and copper
    deficiency, Sorenson (1989) found evidence that copper deficiency can

    elevate blood pressure. Impaired tissue formation has been associated
    with copper deficiency, particularly with the cardiovascular system
    (Farquharson et al., 1989; McCormick et al., 1989; Saari & Johnson,
    1990; Tinker et al., 1990).  Variation in copper intake may cause
    significant changes in the SOD level in certain cardiac tissue (Askari
    et al., 1990).

         There are some reports concluding that elevated serum copper
    levels (nondietary copper exposure) are implicated in the onset of
    cardiovascular disease.  In two double-blind studies, groups of 7 or 8
    males took a supplement of copper gluconate providing 2 or 3 mg
    Cu/day, respectively, for 6 weeks.  Groups of 6 males formed control
    groups in each case.  The data suggested that 2 and 3 mg Cu/day could
    increase LDL cholesterol and total serum cholesterol, respectively.
    However, the control groups showed a variability in levels that made
    these findings questionable.  At 3 mg Cu/day, there was an increase in
    the haemoglobin level after 6 weeks (Medeiros et al., 1991).  An
    earlier study found no significant changes in the serum levels of
    copper, zinc, magnesium, triglyceride, serum glutamic-oxaloacetic
    transaminase (SGOT), gamma-glutamyl transpeptidase (GGT), lactate
    dehydrogenase (LDH) or alkaline phosphatase, in a group of 7 subjects
    ingesting 10 mg Cu/day for 12 weeks as copper gluconate.  Both treated
    and placebo groups reported nausea, diarrhoea, heartburn and back
    pain.  The small group sizes should be noted (Pratt et al., 1985).

         In England a correlation study, with measurements made after
    diagnosis of coronary heart disease, has shown higher serum copper
    levels in cardiovascular disease patients (Punsar et al., 1975).  A
    follow-up study in the Netherlands compared the copper and zinc intake
    in cardiovascular mortality; the adjusted risk of death from
    cardiovascular disease showed a U-shaped pattern which was four times
    higher in subjects in the highest quartile for serum copper (> 1.43
    mg/litre), but a twofold excess mortality was also observed in
    subjects with low serum copper (< 1.05 mg/litre) (Kok et al., 1988).
    It is noteworthy that causal interpretation of these data is difficult
    because the disease might have affected serum copper levels.
    Furthermore, the possibility that elevated serum copper levels are the
    result of preclinical disease could not be ruled out.  Also,
    information on vitamin C, iron status and other nutrients that are
    associated with copper is not available.  In another prospective
    study, baseline serum copper levels were measured in 1666 randomly
    selected Finnish males aged 42-60 years in 1984-1988, and the cohort
    followed until December 1989.  When divided into tertiles of initial
    serum copper, the highest tertile experienced acute myocardial
    infarction in 4.6% of the subjects, compared with 3.6% in the medium
    tertile and only 0.9% in the lowest tertile.  After adjustments, the
    relative risks for the three groups were 4.0, 3.5 and 1.0,
    respectively (Salonen et al., 1991).  It should be stressed that
    elevated serum copper could be a consequence rather than a causal
    factor for acute myocardial infarction.

         The same group of authors reported that the mean increase in the
    maximal common carotid intima media thickness after 2 years was
    greater in men with high serum copper concentrations, those with low
    serum selenium concentrations and those with raised serum LDL
    cholesterol concentrations.  They concluded that there was a
    synergistic effect of copper, a low serum concentration of selenium,
    and LDL cholesterol concentration in atherogenesis (Salonen et al.,

         The association between serum ceruloplasmin level and the
    subsequent incidence of myocardial infarction and stroke were studied
    in a nested case-control study in Finland.  High serum ceruloplasmin
    levels were significantly associated with higher future odds of
    myocardial infarction but not of stroke, which support the hypothesis
    that a high serum ceruloplasmin level is a risk factor for myocardial
    infarction (Reunanen et al., 1992).  This was consistent with the
    described positive relationship between high serum copper and the
    aggregation of classical risk factors (McMaster et al., 1992). Several
    investigators (Taggart et al., 1986; Fraser et al., 1989) reported
    that ceruloplasmin is a positive acute-phase reactant and increases in
    response to injury and infection in parallel with other plasma protein
    markers such as C-reactive protein.

         All these observations may seem incongruous when juxtaposed with
    the copper-deficiency theory (Klevay, 1975), but they are not in
    conflict with the theory because high serum copper does not prove high
    copper absorption.  Experiments with animals reveal that the opposite
    may be true (Klevay, 1988, 1992).  Thus, the role of elevated serum
    copper (unrelated to dietary copper exposure) in the aetiology of
    cardiovascular disease remains a matter of controversy and

    8.5  Occupational exposure

         It has been reported that occupational exposure to copper fume
    results in metal fume fever (Armstrong et al., 1983) and a similar
    condition has been reported from inhalation of finely ground
    copper-oxide dust (Schiatz, 1949).  Air concentrations capable of
    producing these effects are not well defined.  Schiatz (1949) reported
    on conditions in a postwar factory in which ventilation systems were
    inoperative.  In this case, exposures were likely to be unusually high
    compared to plants with adequate industrial hygiene.

         Most industrial exposures are to a mixture of copper and other
    contaminants, and assessing the effects of copper alone from such
    studies is extremely difficult.  This restricts the usefulness of much
    of the data on Bordeaux mixture sprayers (Pimentel & Menezes, 1977;
    Plamenac et al., 1985), from the mining and smelting of copper
    (Ruoling & Mengxuan, 1990; Chen et al., 1993) and from the maintenance
    of moulds in a paper mill (Srivastava et al., 1992). Copper refinery
    studies are less likely to be confounded by mixed exposures.  Studies
    where effects could reasonably be attributed to copper are discussed

         A large historical prospective study of 3550 men working for at
    least 1 year in the tank house of nine copper refineries in the USA
    (Logue et al., 1982) provided no statistically significant evidence of
    an increased risk of cancer.

         Suciu et al. (1981) reported on a clinical study of workers
    exposed to copper dust during the sieving and electrolysis processes.
    Exposures at the time of the clinical examinations were very high,
    ranging from 464 mg Cu/m3 in 1971 to 111 mg Cu/m3 in 1973 [present
    widely recognized exposure limits are typically 1 mg Cu/m3 (ILO,
    1991)].  Signs and symptoms studied and their occurrence included
    hepatomegaly in 55.6%, digestive disorders in 10-15%, and a range of
    respiratory signs and symptoms.  Normal serum copper values in
    unexposed workers were reported as 0.76-1.17 mg/litre.  In 1970-1973,
    the proportion of workers with serum copper above the normal range
    increased from 40% to 92%. Using a number of assumptions, absorption
    of copper can be estimated as being in the range of 200 mg/day.  The
    absence of control data and information on methods used for measuring
    exposure severely limit the usefulness of this study (Suciu et al.,

         In another study, Gleason (1968) reported symptoms similar to the
    common cold with sensations of warmth and stuffiness of the head in
    workers polishing copper plates using an aluminium oxide abrasive on
    buffing wheels.  Air samples in front of the buffing wheel were
    reported at 0.12 mg Cu/m3 but at times estimated to be a factor of
    2-3 times higher.  Microscopic examination indicated the particulates
    to be metallic copper rather than copper-oxide dust.

         No adequate studies were found on the effects of occupational
    exposures to copper on fertility or fetal development.


    9.1  Bioavailability

         Copper usually has limited bioavailability in environmental
    media, and this needs to be carefully considered in all assessments of
    its environmental impacts.  Bioavailability refers to the degree to
    which total chemical in the environment (e.g. water, sediment, food
    items) can actually be taken up by organisms (Rand & Petrocelli,
    1985).  The more bioavailable a chemical is, the greater the potential
    for toxicity or bioaccumulation.  Bioavailability can be affected by
    the speciation of a chemical (i.e. certain species will be more or
    less able to interact with and pass through the absorptive surfaces of
    organisms), but can also be affected by other physicochemical
    properties of the media which regulate uptake of chemicals.

    9.1.1  Bioavailability in water

         A large body of environmental literature demonstrates that
    bioavailability is generally poorly related to the concentration of
    total metal in water.  Major factors reported to limit copper
    bioavailability are adsorption to suspended particles, complexation by
    dissolved organic matter and complexation by some inorganic ligands
    such as carbonate (Sunda & Guillard, 1976; Brungs et al., 1976; Allen
    & Brisben, 1980; Giesy et al., 1983; Borgmann & Ralph, 1983, 1984;
    Borgmann & Charlton, 1984; Meador, 1991; Verweij, 1992; Erickson et
    al., 1996).  Copper toxicity is usually found to decrease with
    increasing water hardness, possibly because calcium and copper compete
    for adsorption sites on biological surfaces, so that greater calcium
    concentrations will limit copper adsorption (Zitko & Carson, 1976;
    Howarth & Sprague, 1978; Chakoumakos et al., 1979; Miller & Mackay,
    1980; Pagenkopf, 1983).  Copper toxicity has also been reported to be
    affected by pH, which may be due either to hydrogen ion affecting
    copper speciation or to the interactions of copper with biological
    surfaces (Howarth & Sprague, 1978; Miller & Mackay, 1980; Borgmann,
    1983; Meador, 1991; Erickson et al., 1996).

         Particular attention has been paid to the possibility that the
    principal bioavailable species is the free copper (cupric) ion.
    Several studies have shown a close correlation of copper toxicity to
    cupric ion activity as the concentrations of organic ligands vary
    (Sunda & Guillard, 1976; Allen & Brisbin, 1980; Meador, 1991; Verweij
    et al., 1992).  However, other studies have shown that this
    correlation is not always good for some organic ligands and organisms
    (Giesy et al., 1983; Borgmann & Charlton, 1984; Borgmann & Ralph,
    1983, 1984;  Erickson et al., 1996).  In fact, certain hydrophobic
    copper complexes appear to have high bioavailability (Ahsanullah &
    Florence, 1984).  Studies which evaluated the effect of pH on copper
    toxicity also do not show a close correlation of toxicity with cupric
    ion activity (Borgmann 1983; Meador, 1991).  Toxicity on the basis of
    cupric ion will also vary with varying water hardness, although if
    this is due to competitive interactions it does not contradict the
    notion that cupric ion is the principal bioavailable species.  More

    information and analysis regarding the "free ion activity model" for
    metal toxicity and metal bioavailability is provided in a review by
    Campbell (1995).

         The bioavailability of Cu(I) has been largely ignored since
    soluble or complexed forms of Cu(I) have not been thought to occur in
    significant amounts in aerobic environments.  However, studies by
    Moffett & Zika (1987) speculate that Cu(II) can be directly or
    indirectly reduced to Cu(I) by photochemical processes.  If this
    should occur in seawater, chloride ions might stabilize the Cu(I)
    through complex formation.

         Whatever the mechanisms, bioavailability can vary widely and must
    be considered in any interpretation and application of toxicity data
    such as those presented later in this chapter.  Additional
    consideration must be given to the condition of organisms and any
    physicochemical exposure conditions which affect organism
    susceptibility without affecting bioavailability, such as temperature
    and sodium concentrations (Erickson et al., 1987, 1996).  Some
    empirical strategies exist for doing this.  The US EPA water quality
    criteria for copper (US EPA, 1984) are adjusted for hardness, based on
    regression analysis of studies in which toxicity was evaluated at
    various hardness levels.  This addresses only some aspects of
    bioavailability, and EPA procedures allow for criteria to be modified
    based on toxicity tests in site water which evaluate bioavailability.
    Welsh et al. (1993) provide empirical equations for the effects of pH
    and organic carbon on the acute toxicity of copper to fathead minnows.
    Erickson et al. (1987) proposed similar equations for several
    physicochemical factors affecting acute copper toxicity.  Such
    empirical approaches have considerable utility, but can be expensive
    to develop.  Some recent research has introduced predictive models
    which are more mechanistically based and have a potential for
    providing better extrapolations.  Predicting effects of copper on fish gill function

         Gills of freshwater fish have two important physiological
    functions; transport of gas (oxygen, carbon dioxide, ammonia) and
    uptake of active ions (sodium, calcium) (Wood, 1992; Playle, 1997). At
    environmentally realistic levels for anthropogenically contaminated
    waters, metals exert their toxic effects by binding to these sodium
    and calcium pump-associated ligands in a highly specific fashion,
    thereby inhibiting the inward transport of the essential nutritive
    ions.  These ligands are, therefore, the proximate receptors for the
    metals; the free cationic forms of the metals are the most potent in
    binding to these receptors.  For cupric and cadmium ions, strong
    relationships between the gill metal burden and mortality have been
    determined experimentally (MacRae et al., in press).  Thus, it may be
    possible to predict toxicity from gill metal burden for these two
    metals and potentially other cationic metals.

         Viewed in the above context, the specific receptor ligands on the
    gill are entirely analogous to other anionic ligands in the water
    column which may also bind the cationic metal - for example chloride,
    hydrogen carbonate and dissolved organic carbon (DOC) - and indeed the
    gill ligands will compete with these natural ligands for the metal
    (Playle et al., 1993a,b).  The final metal partitioning will depend in
    part on the affinities and numbers of natural ligands relative to gill
    ligands.  Naturally occurring cations in the water column (e.g.
    sodium, calcium, hydrogen) will compete with the metal for both the
    natural anionic ligands and gill receptor ligands.  Aquatic
    geochemical speciation programs such as MINEQL+ and MINTEQA2
    (Allison et al., 1991; Schecher & McAvoy, 1992) are specifically
    designed to deal with these competitive interactions and can be used
    to produce accurate equilibrium models of the metal partitioning among
    the various ligands in the water, provided the water chemistry is
    known.  At present, these programs do not contain binding constants
    for the gill receptor ligands and therefore deal only with
    partitioning within the water column.  However, they allow the user to
    add constants for other ligands at will.  A problem with these
    modelling approaches is that the biomembrane-water interaction is
    treated as an equilibrium situation, whereas it is, in fact, a dynamic
    reaction and kinetic factors (rate constants) should also be taken
    into account.

         Recently, methods have been developed to determine conditional
    equilibrium binding constants of copper and other metals to the gill
    receptor ligands (Janes & Playle, 1995).  In brief, these involve
    experimental determination of equilibrium gill metal burden after
    exposure of the fish (3 h) to environmentally relevant levels of the
    metal in the presence of various concentrations of natural and/or
    synthetic ligands with known metal-binding constants.  Analogous
    competition experiments can be run in the presence of various
    concentrations of natural cations to determine the conditional binding
    constants of the gill receptors for such cations.  These constants can
    then be added into chemical speciation calculation programs to make a
    prediction of gill receptor loading with metal, and therefore
    toxicity, in any water with known chemistry.

         The advantages of this predictive modelling approach include the

    *    it is mechanistically based
    *    for the first time in aquatic toxicology it allows estimation of
         metal dose at the receptor surface directly associated with
    *    it takes all important water chemistry factors into account (not
         just hardness, for example)
    *    it can deal with multiple metals simultaneously.

         This approach to modelling toxicity allows for flexible,
    site-specific criteria based on the known chemistry of the receiving
    water and the known chemistry of the gill surface.  This approach is
    also currently being investigated for freshwater invertebrates.

    9.1.2  Bioavailability of metals in sediments

         Determining the bioavailability of metals sorbed to sediments is
    a key to understanding their potential to accumulate in aquatic
    organisms and to induce toxic effects.  Considerable published data
    indicate that total metal concentrations on sediments are not a good
    estimator of the bioavailable fraction of the total chemical present
    (Ruiz et al., 1991; DeVevey et al., 1993; Allen & Hansen, 1996).
    Total metal concentrations in sediments which produce toxic effects
    can differ by a factor of 10-100 for different sediments.  In order to
    assess the potential for toxicity based on chemical measurements, the
    bioavailable fraction of the total metal present needs to be
    estimated.  A number of approaches to determining metal
    bioavailability associated with sediments have been evaluated,
    including carbon normalization and sorption of metals in oxic
    freshwater sediments to particulate carbon and the oxides of iron and
    manganese (Jenne, 1987).

         Recently, the dominant role of the sediment sulfides in
    controlling metal bioavailability has been demonstrated (DiToro et
    al., 1990, 1991; Ankley et al., 1991).  Sulfides are common in many
    freshwater and marine sediments and are the predominant form of sulfur
    in anaerobic sediments (usually found as iron sulfide).  The ability
    of sulfide and metal ions to form insoluble precipitates with water
    solubilities well below the toxic threshold of dissolved metal is well
    known (DiToro et al., 1990).  This accounts for the lack of toxicity
    from sediments and sediment pore waters even when high metal
    concentrations are present (Ankley et al., 1991).  The same authors
    have shown that the solid-phase sediment sulfides that are soluble in
    weak cold acid, termed acid volatile sulfides (AVS), are a key factor
    in controlling the toxicity of heavy metals (copper, cadmium, nickel,
    lead, zinc).  Toxicity due to these metals is not observed when they
    are bound to sediment and when, on a molar basis, the concentration of
    AVS is greater than the sum of the molar concentrations of metals.
    When the ratio of the sum of the simultaneously extracted metals to
    AVS concentration exceeds 1.0 on a molar basis, toxic effects due to
    metals may be expressed, if the metal(s) are not complexed by other
    ligands.  The key concept here is that the metal : AVS ratio can be
    used to predict the fraction of the total copper concentration present
    in sediment that is bioavailable.

         Limitations to the AVS : metal ratio approach occur when the AVS
    concentration is low.  This could occur in fully oxidized sediments.
    Most sediments have at least a small zone where the sediments are oxic
    near the sediment-water interface.  The importance of this zone has
    been demonstrated for copper relative to AVS and accumulation of
    copper in midge  (Chironomus tentans) (Besser et al., 1996).  In
    these situations, other phases (i.e. iron and manganese oxides,
    dissolved organic carbon and particulate organic carbon) can play an
    important and more dominant role in determining the bioavailability of
    copper.  The available data suggest that AVS concentrations may be
    sufficient in both freshwater and marine ecosystems to be the dominant

    sorbing phase for copper and other metals, except in fully aerobic

    9.2  Essentiality

         Copper is an essential element for all biota.  Copper was
    identified in plant (Bucholtz, 1816; Meissner, 1817) and animal
    (Sarzeau, 1830; Harless, 1847) systems in the nineteenth century and
    postulated to be a biological catalyst in the early twentieth century
    (Fleurent & Levi, 1920; Guerihault, 1920).  Subsequent nutritional
    studies demonstrated that copper and other metals were necessary for
    optimal growth of plants and animals (McHargue, 1925, 1926, 1927a,b;
    Arnon & Stout, 1939; Woolhouse, 1983).  Copper was shown to be an
    essential element for animals by Hart et al. (1928) who demonstrated
    that copper, as well as iron, is necessary to prevent anaemia in rats.
    Copper is also essential for the utilization of iron in the formation
    of haemoglobin (Friberg et al., 1979); hence its involvement in

    9.2.1  Animals

         To satisfy their internal metabolic demands, all species in a
    given habitat are adapted to the natural concentration range of
    essential elements. Therefore, laboratory-generated no-observed-effect
    concentrations (NOECs) substantially below the natural background
    concentration of copper require further attention as they appear to
    violate evolutionary principles. This may be explained by the concept
    of the optimal concentration band of essential elements (OCEE). This
    concept is well known in the field of ecotoxicology of essential
    elements, but has not so far been accommodated in the regulatory
    context. Thus although ecotoxic at high concentrations, copper may
    also be limiting or cause symptoms of deficiency at low ambient
    bioavailable concentrations.

         Most crustaceans and molluscs possess the copper-containing
    haemocyanin as their main oxygen-carrying blood protein.  Haemocyanin
    doubles their requirement for copper compared to other invertebrates
    (Hopkin, 1993).

         White & Rainbow (1985) calculated theoretical estimates for the
    minimum metabolic requirements of copper in molluscs and crustaceans.
    Enzymatic requirements for both groups were estimated to be 26.3 mg
    Cu/kg (dry weight).  The possession of haemocyanin as a respiratory
    pigment adds a further nonenzymatic metabolic requirement of 125 mg
    Cu/kg for certain gastropod molluscs and 57.4 mg Cu/kg for some
    crustaceans such as decapods.  However, Depledge (1989) recalculated
    the amount of copper required by decapod crustaceans to be 82.8 mg/kg
    (dry weight).  Hopkin (1993) estimated that terrestrial isopods
    require a minimum whole-body concentration of 50 mg Cu/kg. Evidence on
    copper concentrations of certain decapod crustaceans in the deep sea
    suggests that circumstances exist where there is insufficient
    bioavailable copper for the decapods to meet all their metabolic
    copper requirements (Rainbow, 1988). Small specimens of the

    mesopelagic caridean  Systellaspis debilis, for example, have low
    copper concentrations (30 mg/kg dry weight), body concentrations
    reaching only 100 mg/kg in large adults. According to the theoretical
    calculations of Depledge (1989) the smaller  S. debilis would only
    have sufficient absorbed copper to match enzymatic needs, whereas
    larger adults have sufficient copper for haemocyanin requirements as
    well.  This is indeed the case; Rainbow & Abdennour (1989) found that
    small  S. debilis contained little, if any haemocyanin, large animals
    containing a more typical haemocyanin complement. Moreover, juvenile
     S. debilis undertake limited vertical migrations. This may be
    related to the shortage of haemocyanin in juveniles, indicating that
    insufficient bioavailable copper in the mesopelagic environment may
    limit activity levels until sufficient copper has been accumulated to
    allow the synthesis of increased haemocyanin concentrations.  Ambient
    copper availability in the deep ocean is so low that levels of copper
    in juvenile crustaceans are a reflection of copper deficiency. Any
    such deficiency is only overcome in adults which have had sufficient
    time to accumulate body copper concentrations meeting all metabolic

         Analysis of concentrations of copper in invertebrates from
    uncontaminated sites suggests that some terrestrial invertebrate
    species may be copper deficient (Hopkin, 1993).  In mammals,
    molybdenum has been shown to influence the tissue and blood levels of
    copper.  Copper deficiency may occur in mammals when the intake of
    molybdenum is excessive (Friberg et al., 1979).  This is thought to be
    due to the formation of copper molybdate.

         Problems related to copper and molybdenum metabolism have been
    widely reported in grazing domestic livestock, and there are some
    reports of concern for wildlife (Ward & Nagy, 1976; Flynn et al.,
    1977; Robbins, 1983).  The metabolism of copper, molybdenum and
    inorganic sulfate is extremely complex and interrelated (Underwood,
    1977).  The interactions of copper and molybdenum can result in two
    toxic scenarios; excess copper-deficient molybdenum, or deficient
    copper-excess molybdenum.  In the presence of inorganic sulfur it is
    impossible to delineate between the toxicity of one and deficiency of
    the other (Buck et al., 1976).  Deficiency or excess of copper and
    molybdenum are most prominent among ruminants and directly related to
    copper-molybdenum balance in soil and forage.

         King et al. (1984) examined copper and molybdenum levels in
    white-tailed deer from a uranium-mining district of Texas, USA, where
    molybdenosis was reported in cattle.  Liver copper levels ranged from
    0.47 to 0.94 µg/g in all samples, and there was no difference between
    mined and unmined areas.  Only 1 deer of 36 examined contained
    detectable levels of molybdenum.  The authors suggest that 6 deer with
    liver copper levels < 1.0 µg/g were probably suffering from copper
    deficiency that was not molybdenum-induced.  Keinholz (1977) reported
    that mean copper and molybdenum levels in liver of deer from a
    molybdenum mining area were 40 and 1 µg/g, respectively, above control

         Ward & Nagy (1977) demonstrated that mule deer were able to
    withstand much higher dietary levels of molybdenum (1000 µg/g) than
    domestic livestock.  The authors point out, however, that the diet
    used was a pelleted concentrate which may have affected availability
    of molybdenum to the deer.  They did observe that mule deer rejected
    feed with excess molybdenum.  The ability of wildlife to select feeds
    low in molybdenum would reduce the chances of toxicity.

         A copper deficiency in moose on the Alaskan Kenai peninsula
    impaired hair and hoof keratinization, and reduced reproduction (Flynn
    et al., 1977). Adult females in the Kenai moose population had a 53.5%
    pregnancy rate compared with 91.6% for moose in another area of
    Alaska. Copper levels in the moose browse (5.7 µg/g) are considered
    marginal for domestic livestock.  Examination of tissue molybdenum and
    sulfur levels led the authors to believe that the copper deficiency
    was not molybdenum induced (Flynn et al., 1976).

         Aulerich & Ringer (1976) showed that addition of 25 or 50 µg Cu/g
    to the diet stimulated growth of young mink (dark ranch phase).  Up to
    200 µg Cu/g in the diet had no effect on adult mink reproduction but
    there was increased kit mortality at this level (Aulerich et al.,
    1982). Liver copper levels increased in proportion to dietary levels,
    but supplemental copper had no effect on the concentration of zinc or
    iron in mink liver.  The acute (21-day) LC50 (intraperitoneal
    injection) of copper sulfate and copper acetate in adult mink was 7.5
    and 5.0 mg/kg, respectively (Aulerich et al., 1982).

         There is a marked difference between species in their ability to
    tolerate high levels of copper.  Levels that are toxic to ruminants
    (30-50 µg Cu/g) are well tolerated by nonruminants.  A difference in
    the rate of copper absorption from the diet between ruminants and
    nonruminants may partially explain the difference in sensitivity (Buck
    et al., 1976).  Rats, swine and mink can tolerate up to 200-250 µg
    Cu/g in the diet (Aulerich et al., 1982).

         There is also some indication that the source or quality of
    dietary protein may be a factor in copper toxicity.  Suttle & Mills
    (1966) observed severe copper toxicosis in swine receiving whitefish
    meal but not in those receiving soybean-oil meal, with both diets
    containing up to 425 µg Cu/g.  It is also possible that the effects of
    dietary protein source on copper toxicity are related to their
    concentrations of elements such as zinc and iron, both of which have
    been shown to protect swine from the adverse effects of high (250-750
    µg/g) levels of dietary copper (Ritchie et al., 1963).

    9.2.2  Plants  Aquatic plants

         Copper must be provided as a micronutrient (as copper chloride or
    copper sulfate) in the culture media for growing algae (McLachlan,
    1973).  Copper participates, as part of the plastocyanin molecules, in
    the electron transport during photosynthesis, and as co-factor in a

    number of enzymatic reactions and metabolic pathways (Bidwell, 1979;
    De Boer, 1981; Lobban et al., 1985).  Terrestrial plants

         Copper is an essential micronutrient for normal plant nutrition
    (Woolhouse, 1983; Marschner, 1986; Fernandes & Henriques, 1991;
    Larcher, 1995), because this element is constituent of a number of
    plant enzymes (Adriano, 1986; Fernandes & Henriques, 1991), some of
    which are listed in Table 7.  Copper is required in small amounts:
    5-20 mg/kg in plant tissue is adequate for normal growth (Nriagu,
    1979; Clarkson & Hanson, 1980; Howeler, 1983; Stevenson, 1986), less
    than 4 mg/kg is considered deficient (Robson & Reuter, 1981; Howeler,
    1983; Marschner, 1986) and more than 20 mg/kg is considered toxic
    (Stevenson, 1986).  However, depending on the plant species, plant
    organ, developmental stage, and nitrogen supply, these ranges can be
    larger (Thiel & Finck, 1973; Robson & Reuter, 1981).  Adriano (1986)
    reports a variety of soil types which are deficient in copper for
    normal plant growth including peat and muck soils, alkaline and
    calcareous soils, highly leached sandy and acid soils, and soils
    heavily fertilized with nitrogen, phosphorus or zinc.  Zinc is
    expected to serve as an uptake competitor.  Typical visible symptoms
    of copper deficiency are stunted growth, distortion of young leaves,
    necrosis of the apical meristem, and wilting and bleaching of young
    leaves (Rahimi & Bussler, 1973).  Copper deficiency results in
    insufficient lignification of the cell walls of the xylem vessels
    (Rahimi & Bussler, 1974; Pissarek, 1974) indicating that the degree of
    lignification is a good indicator of nutritional copper status in

    9.3  Toxic effects: laboratory experiments

         Since copper is an essential metal for aquatic and terrestrial
    organisms, care must be taken when interpreting toxicity test results.
    For all organisms there will be an optimum concentration range, with
    copper being toxic or deficient above or below this optimum range.  A
    wide variety of factors will influence this optimum range including
    previous exposure, test conditions and species sensitivity.

    9.3.1  Microorganisms  Water

         Dutka & Kwan (1981) reported a 15-min Microtox EC50 at 3800 µg
    Cu/litre.  Microtox EC50 (15 min) values were reported at 1200
    µg/litre for a copper chloride solution and at 600 µg/litre in sewage
    (Codina et al., 1993).  Blaise et al. (1994) calculated 5-, 15-,
    30- and 60-min EC50s in Microtox tests to be 1100, 150, 70 and 60 µg
    Cu/litre, respectively.  Carlson-Ekvall & Morrison (1995) report that
    the 30-min EC50 for  Photobacterium phosphoreum was 136 µg Cu/litre.
    The toxicity of copper in the presence of various organic substrates
    identified in sewage sludge was found to vary from < 20 µg/litre for
    ethyl xanthogenate to > 500 µg/litre for tannic acid.

         Codina et al. (1993) calculated copper EC50 values for two
     Pseudomonas fluorescens growth inhibition tests, a baker's yeast
     (Saccharomyces cerevisiae) test, a respiratory inhibition test with
    baker's yeast and a respiratory inhibition test with  P. fluorescens.
    The EC50 values were 51.7, 48.7, 73.2, 78.8 and 150.9 mg Cu/litre,

         Berk et al. (1985) calculated a 15-min EC50, based on inhibition
    of ciliate chemotactic response, to be 150-160 µg Cu/litre for the
    freshwater ciliate  Tetrahymena sp.  Copper concentrations of 5 and
    50 µg/litre were found to be significantly inhibitory to chemotactic
    responses of the marine ciliates  Miamiensis avidus and  Paranophrys
    sp., respectively.

         In a static test system Schafer et al. (1994) exposed the
    freshwater ciliate  Tetrahymena pyriformis to copper.  They
    calculated 48-h and 96-h EC50s, based on growth inhibition to be
    8.017 and 10.18 mg Cu/litre, respectively; NOECs were 3.563 and 3.818
    mg Cu/litre, respectively.

         Madoni et al. (1992) isolated seven ciliate species from the
    activated sludge of a sewage treatment works.  The 24-h LC50s ranged
    from 1.45 µg Cu/litre for  Blepharisma americanum (free-swimming
    form) to 64 µg Cu/litre for  Euplotes affinis (a crawling form).
    Madoni et al. (1994) isolated a further two ciliates
     (Spirostomum teres and  Drepanomonas revoluta) and found 24-h
    LC50s to be 3.51 and 1.75 µg Cu/litre, respectively.

         Tijero et al. (1991) studied the effect of copper on an anaerobic
    digester system.  A concentration threshold of 20 mg Cu/litre was
    reported, and a 50% reduction in digester yields was found at a copper
    concentration of 40 mg/litre.

         Isolda & Hayasaka (1991) studied the effect of copper (20 and
    1000 mg/litre) on the microbial processes in pond sediment for 4
    weeks.  Copper had no significant effect on glucose mineralization,
    nitrogen fixation or dehydrogenase activity.  Methanogenesis was
    significantly reduced at both copper concentrations and the highest
    exposure significantly reduced phosphatase activity.

         Flemming & Trevors (1988) studied the effect of copper on nitrous
    oxide (N2O) reduction in anaerobically incubated freshwater sediment
    at 15°C.  A concentration-dependent decrease in sediment pH and a
    significant decrease in nitrous oxide reduction were observed at
    copper concentrations ranging from 500 to 5000 mg/kg.  However, when
    copper-amended microcosms were pre-incubated to allow the sediment pH
    to return naturally to pH 7.1, an inhibitory effect on nitrous oxide
    reduction was only observed at 5000 mg Cu/kg.

         Martínez et al. (1991) calculated the 60-min EC50, based on
    3H-thymidine incorporation (a measure of bacterial heterotrophic
    activity), to be 32 µg Cu2+/litre for naturally occurring bacteria
    from the river Rhone (Mediterranean Sea) plume.  Tubbing et al. (1995)

    found EC50s, based on 3H-thymidine and 3H-leucine incorporation and
    proteolytic activity, to be 28-100, 28-90 and 585-1997 µg Cu/litre,

         Schreiber et al. (1985) exposed the marine bacterium
     Vibrio alginolyticus to copper under aerobic and anaerobic
    conditions.  The copper concentration at which there was a 50%
    reduction in heat production (TC50) was used to compare the toxicity
    under aerobic and anaerobic conditions. Copper was more toxic to the
    bacterium in anaerobic culture (TC50 = 133 µg/litre (2.1 µmol/litre))
    than in aerobic culture (TC50 = 406 µg/litre (6.4 µmol/litre)).  The
    addition of organic chelators (EDTA and nitrilotriacetic acid)
    protected the anaerobic cultures from the toxic effects of copper,
    indicating that copper-organic complexes are not toxic to the
    bacterium.  Soil

         Toxicity of copper to soil microorganisms is summarized in Table

         Chang & Broadbent (1981) calculated the threshold (EC10) and
    EC50 concentrations, based on the inhibition of carbon dioxide
    production in a silt loam soil amended with alfalfa and sewage sludge,
    to be 4.2 and 22 mg/kg (65.6 and 347 nmol/g) for DTPA-extractable
    copper (bioavailable copper).

         Rogers & Li (1985) incubated soil for 6 days in the presence of
    copper.  EC50s, based on inhibition of soil dehydrogenase activity,
    were 29 mg Cu/kg for soil enriched with 1% alfalfa and 53 mg Cu/kg for
    soil that was not enriched.

         Lighthart et al. (1983) measured soil microbial respiration in
    five soil types after treatment with copper.  After a 45-day
    incubation at 20°C the lower level treatments (3.2 and 32 mg Cu/kg,
    0.05 and 0.5 mmol/kg) had little effect, with mean inhibitions of less
    than 20%. Higher levels of 320 and 3200 mg Cu/kg (5 and 50 mmol/kg)
    inhibited respiration by up to 35% and 60%, respectively.  Bremner &
    Douglas (1971) report that copper concentrations of 50 mg/kg inhibited
    soil urease activity by 13-16% following a 5-h incubation period.

         Doelman & Haanstra (1984) found that short-term (2 weeks)
    exposures to copper (150-8000 mg/kg) caused decreases in the rate of
    soil respiration.  Long-term (up to 18 months) exposure was less clear
    cut.  In sand there was a significant decrease at copper
    concentrations of 400 mg/kg and in sandy peat there was a significant
    decrease at 1000 mg/kg.  The effect of copper in silty loam and clay
    was less apparent with a significant decrease and increase at 8000
    mg/kg for the two soil types, respectively.  Doelman & Haanstra (1986)
    calculated EC50s, based on inhibition of soil urease activity.  After
    6 weeks EC50s were 260, 570, 1370 and 4200 mg Cu/kg for sand, sandy
    loam, clay and sandy peat, respectively, and after 18 months they were
    680, 1990, 1080 and 1970 mg Cu/kg, respectively.

        Table 17.  Toxicity of copper to soil microorganisms

    Organisms        Parameter        End-point                        Concentration                              Reference

    Soil             EC10 and EC50    inhibition of CO2 production     4.2 and 22 mg/kg for silt loam soil        Chang &
    microorganisms                                                     amended with alfalfa and sludge            Broadbent (1981)

                     EC50             inhibition of soil               29 mg/kg for soil enriched with 1%         Rogers & Li
                                      dehydrogenase activity           alfalfa; 53 mg/kg for soil not enriched    (1985)

                     45-day EC50      soil respiration                 320 and 3200 mg/kg resulted in             Lighthart et
                                                                       35 and 60% inhibition                      al. (1983)

                     5-h EC50         inhibition of soil urease        inhibition between 13% and 16%             Bremner &
                                      activity                                                                    Douglas (1971)

                     6-week EC50      inhibition of urease activity    260 mg/kg in sand to 4200 mg/kg            Doelman &
                                                                       in sandy peat                              Haanstra (1986)

                     18-month EC50    glutamic acid reduction          55 mg/kg in sand to 1000 mg/kg             Haanstra &
                     (significant     time                             in sandy peat                              Doelman (1984)

                     18-month ED50    reduction of arylsulfatase       287 mg/kg in sand to 6991 mg/kg            Haanstra &
                                      activity                         in sandy peat                              Doelman (1991)

                     6-month EC50     microbial biomass                890 mg/kg in sandy loam;                   Frostegard et
                                                                       4321 mg/kg in humus                        al. (1993)

                     15-week EC50     population growth                up to 5000 mg/kg when exposed in           El-Sharouny et
                                                                       soil; 10 mg/kg when exposed in agar        al. (1988)

    Soil ciliate     7-day EC10 and   population growth                331.5 and 971.6 µg/litre                   Janssen et al.
    (Colpoda         EC50                                                                                         (1995)

         Haanstra & Doelman (1984) report that copper significantly
    reduced glutamic acid decomposition time, in an 18-month incubation,
    at 55 mg/kg in sand, at 400 mg/kg in silty loam and clay and at 1000
    mg/kg in sandy peat.  Haanstra & Doelman (1991) calculated 18-month
    ED50s, based on reduction of arylsulfatase activity, ranging from 287
    mg Cu/kg (4.51 mmol/kg) in sand to 6991 mg Cu/kg (110 mmol/kg) in
    sandy peat.

         Frostegĺrd et al. (1993) incubated forest humus and arable soil
    (sandy loam) with copper for 6 months at 22°C. EC50s, based on a
    decrease in the ATP content, were 4321 and 890 mg Cu/kg (68 and 14
    mmol/kg) for the two soils, respectively.  An EC50, based on a
    reduction in respiration, was > 8134 mg Cu/kg (> 128 mmol/kg) for
    forest humus.  In both soil types, copper exposure caused gradual
    changes in the phospholipid fatty acid composition.

         El-Sharouny et al. (1988) studied the effects of copper (500,
    2000 or 5000 mg/kg) on soil mycoflora.  The application of copper
    sulfate to the soil resulted in a significant increase in the count of
    total fungi after 1 week.  There was little further increase after 5
    weeks but at the end of the 15-week exposure there were significant
    increases.  The increases were mainly due to  Aspergillus niger,
     A. sydowii,  A. versicolor,  Penicillium chrysogenum and
     Rhizopus stolonifer.  When similar species were exposed via agar
    medium there were significant decreases at all copper exposures (10,
    50 and 100 mg/kg), the highest exposure eliminating all but
     Aspergillus niger which survived at very low levels.

         Janssen et al. (1995) found the 7-day EC10 and EC50 for the soil
    ciliate  Colpoda cucculus, based on population growth, to be 331.5
    and 971.6 µg Cu/litre (5.22 and 15.3 µmol/litre), respectively.

    9.3.2  Aquatic organisms  Plants

         Care should be taken in interpreting published algal assay
    results for copper.  Most of the algal assay EC50 results reported in
    the literature refer to studies of cell division rate carried out in
    full culture media.  Culture media contain chemicals such as iron,
    manganese, citrate, silicate and EDTA which bind copper and reduce its
    toxicity.  When the algal cells are removed from the culture medium,
    washed, and the assay carried out in a natural water (seawater or
    river water) the cell division rate is usually much more sensitive to
    copper (Stauber & Florence, 1987; Stauber, 1995).  Acute toxicity of
    copper to freshwater and marine algae is summarized in Table 18.

         Wurtsbaugh & Horne (1982) exposed a natural phytoplankton
    association from Clear Lake, California, USA, to copper for a period
    of 6 days.  Chlorophyll  a and nitrogen fixation were significantly
    reduced at copper concentrations of > 20 µg/litre and carbon fixation
    was significantly reduced at > 10 µg/litre.  Biomass estimates

    indicated that the blue-green alga  Aphanizomenon flos-aquae was more
    sensitive to copper than were diatoms.

         Wong & Chang (1991) reported that copper concentrations of 250
    µg/litre significantly reduced the growth rate of
     Chlorella pyrenoidosa: the alga did not grow at copper
    concentrations of 500 and 750 µg/litre.  Photosynthetic rate and
    chlorophyll  a during the log phase were significantly reduced at 100
    µg Cu/litre.

         Metaxas & Lewis (1991) found that the marine diatoms
     Skeletonema costatum and  Nitzschia thermalis did not grow at total
    copper concentrations above 32 µg/litre (0.5 µmol/litre) and 38
    µg/litre (0.6 µmol/litre), respectively.  At lower concentrations
     Skeletonema showed increasing growth rate and lag phase with
    increasing copper concentrations whereas  Nitzschia showed decreasing
    growth with increasing copper exposure.

         Visviki & Rachlin (1994b) studied the effects of copper on the
    algae  Dunaliella salina and  Chlamydomonas bullosa in acute (96 h)
    and chronic (8 month) exposures.  Acute exposures of 378 and 49.6 µg
    Cu/litre (5.94 and 0.78 µmol/litre) for the two species, respectively,
    had no significant effect on the ultrastructure of cells.  However,
    chronic exposure (0.03 µg Cu/litre (4.9 × 10-4 µmol/litre)) caused
    significant increases in lipid number and relative volume of
     Dunaliella and significant increases in cell volume, and decreases
    in periplasmalemmal space and cell wall relative volumes in

         A 50% reduction in the total algal cell volume of
     Selenastrum capricornutum in standard algal assay medium (SAAM)
    occurred at 85 µg Cu/litre after 14 days.  For
     Chlorella stigmatophora grown in 28% artificial seawater plus SAAM
    for 21 days a value of 70 µg Cu/litre was found for the same parameter
    (Christensen et al., 1979).

         Winner & Owen (1991a) found that copper (20 and 40 µg/litre)
    caused significant reductions in community richness of phytoplankton
    exposed for 5 week periods during different seasons of the year.
    Copper significantly changed the algal divisions (percentage
    composition of total phytoplankton) during the spring and autumn but
    not during the summer.

         Winner & Owen (1991b) exposed the green alga
     Chlamydomonas reinhardii to copper in 72-h tests. The NOECs based on
    deflagellation and changes in cell density varied from 12.2 to 49.1 µg
    Cu/litre and from 12.2 to 43.0 µg Cu/litre for the two parameters,

         Schäfer et al. (1993) found 7-day and 10-day EC50s, based on
    growth inhibition, to be 31.5 µg Cu/litre for the green alga
     Chlamydomonas reinhardii in flow-through tests with copper sulfate.

    Table 18. Toxicity of copper to algae

    Organism                     Conditionsa   Temperature   Copper salt  Parameter  End-point          Concentration    NOEC      Reference
                                               (°C)                                                    (µg/litre)       (µg/litre)

    Green alga                   stat          20            sulfate      72-h EC50  growth inhibition  79               5         Schafer et al.
    (Chlamydomonas                                                                                                                (1994)
    reinhardii)                  flow          24            sulfate      96-h EC50  growth inhibition  47               ND        Schafer et al.
    Green alga                   stat          24-26         sulfate      72-h EC50  growth inhibition  47               ND        Nyholm (1990)
    (Selenastrum capricornutum)  stat          24-26         sulfate      72-h EC50  biomass            35               ND        Nyholm (1990)

    Marine alga                                15            chloride     96-h EC50  growth inhibition  50               ND        Visviki &
    (Chlamydomonas bullosa)                                                                                                        Rachlin (1994a)

    Green alga                   stat          20            sulfate      72-h EC50  growth inhibition  120              5.6       Schafer et al.
    (Scenedesmus subspicata)                                                                                                       (1994)

    Marine alga                  ND            15            chloride     96-h EC50  growth inhibition  481              ND        Visviki &
    (Dunaliella minuta)                                                                                                            Rachlin (1991)

    Marine alga                  ND            15            chloride     96-h EC50  growth inhibition  377              ND        Visviki &
    (Dunaliella salina)                                                                                                            Rachlin (1994a)

    a Stat = static conditions (water unchanged for duration of test); flow = flow-through conditions (copper concentration in water continuously
      maintained); ND = no data available.

         Shanmukhappa & Neelakantan (1990) exposed the unicellular algae
     Synechosystis aquatilis to copper. They found 6-h EC50s, based on
    chlorophyll reduction, were 650 µg Cu/litre.  A slightly reduced EC50
    (720 µg Cu/litre) was found when algae were exposed to copper in the
    presence of humic acid (10 µg/litre).

         There are several studies which have assessed the effects of
    copper on various marine algae.  Hall et al. (1979) found that the
    growth rate (as measured by an increase in wet weight) of
     Ectocarpus siliculosus (a tolerant strain) decreased from a mean
    value of 756% in controls to 86% in algae exposed to 500 µg Cu/litre.
    The nontolerant strain was unable to grow under the two experimental
    copper exposures (250 and 500 µg/litre).

         Reed & Moffat (1983) studied the responses of tolerant and
    nontolerant isolates of the green alga  Enteromorpha compressa to
    copper concentrations of up to 610 µg/litre (9.6 µmol/litre).  They
    found that none of the physiological processes that were tested (cell
    viability, net photosynthesis, intracellular potassium and
    dimethylsulfoniopropionate content) were affected by the highest
    exposure concentration with the tolerant isolate.  However, the
    nontolerant isolate showed symptoms of copper toxicity at all copper
    exposures ranging from 114 to 610 µg/litre (1.8 to 9.6 µmol/litre).
    The authors concluded that this copper tolerance was genetically
    determined as the progeny retained the same pattern of response to
    copper enrichments.  On the other hand, Correa et al. (1996) found
    that the progeny of copper-tolerant isolates of
     Enteromorpha compressa from northern Chile responded in the same
    manner as the progeny of non-tolerant isolates of the same species.
    Chilean  Enteromorpha compressa grew well at 100 µg Cu/litre (from a
    copper-polluted site) and at 10 µg Cu/litre (from a nonpolluted site)
    and it was concluded that physiological plasticity rather than
    genotype was involved in tolerance to copper.

         Stauber & Florence (1987) found that copper ions depressed both
    cell division and photosynthesis in the marine diatom
     Asterionella glacialis (101.6 µg Cu/litre; 1.6 × 10-6 mol/litre)
    and the freshwater green alga  Chlorella pyrenoidosa (63.5 µg
    Cu/litre; 10 × 10-7 mol per litre). Ionic copper concentrations
    (176.5 µg Cu/litre; 27.8 × 10-7 mol per litre) which were inhibitory
    to cell division in the marine diatom  Nitzchia closterium had no
    effect on photosynthesis, respiration, ATP production, electron
    transport or membrane ultrastructure.  The authors suggest that the
    main toxic effect of copper on  N. closterium is to act within the
    cytosol; a different toxic mechanism was apparently operating with
     A. glacialis and  C. pyrenoidosa because both cell division and
    photosynthesis were affected by copper.  Lipid-soluble organocopper
    complexes were found to be much more toxic than ionic copper.  Stauber
    & Florence (1985a,b) showed that the toxicity of ionic copper to
     Nitzchia closterium was reduced by the addition of manganese(III)
    and iron(III) hydroxides to the culture medium.  Stauber & Florence
    (1987) demonstrated that the addition to the algal growth medium of
    trivalent metal ions such as aluminium, iron, chromium, or divalent

    metals such as manganese and cobalt (which can be oxidized by algae to
    trivalent species) reduced the toxicity of copper ions.  The trivalent
    species form a layer of hydrated metal oxide around the cell which
    adsorbs copper ions.

         Chung & Brinkhuis (1986) assessed the effects of copper at 5, 10,
    50, 100 and 500 µg/litre on the early stages of development in the
    kelp  Laminaria saccharina.  It was found that the release of
    meiospores from copper-tested sorus materials was reduced by
    concentrations of 50 µg Cu/litre.  Settlement and germination of
    meiospores were not affected by concentrations of up to 500 µg
    Cu/litre.  Development of gametophytes and gametogenesis were delayed
    at concentrations of > 50 µg Cu/litre.  Growth of the sporophytes was
    inhibited at concentrations > 10 µg Cu/litre.  Hopkin & Kain (1978)
    found that sporophyte growth and gametophyte germination of
     Laminaria hyperborea were inhibited at 10 and 100 µg Cu/litre,

         Brown & Rattigan (1979) exposed the pondweeds  Elodea canadensis
    and  Lemna minor to copper.  A 24-h IC50 (photosynthetic oxygen
    evolution) was calculated to be 150 µg Cu/litre for  Elodea.  In
    28-day tests copper concentrations of 3100 and 130 µg/litre caused 50%
    plant damage in  Elodea and  L. minor, respectively.  Dirilgen &
    Inel (1994) calculated a 7-day IC50, based on frond growth, to be
    1540 µg Cu/litre for the duckweed  Lemna minor.  Invertebrates

     Acute and short-term toxicity

         The acute toxicity of copper to freshwater and marine
    invertebrates are summarized in Tables 19 and 20, respectively.  For
    freshwater invertebrates 48-h L(E)C50s range from 5 µg Cu/litre for a
    daphnid species to 5300 µg Cu/litre for an ostracod; 96-h LC50s for
    marine invertebrates range from 29 µg Cu/litre for the bay scallop to
    9400 µg Cu/litre for the fiddler crab.

         Kaitala (1988) reported an 8-day LC50 for mussels
     (Mytilus edulis) at 127 µg Cu/litre and a 10-day LC50 for clams
     (Macoma baltica) at 54 µg Cu/litre.  Beaumont et al. (1987) exposed
    veliger larvae of common mussel  (M. edulis) and scallop
     (Pecten maximus) to copper for 15 days.  LC50s were calculated to
    be 400 and 85 µg Cu/litre for the two species, respectively.

         Centeno et al. (1993) studied the effect of temperature (10-30°C)
    on the 24-h LC50 of copper on the third instar of the crustacean
     Streptocephalus proboscideus. A significant increase in toxicity was
    observed at the highest temperature tested.  Zou & Bu (1994) observed
    an increase in the acute toxicity of copper to the water flea
     Moina irrasa with increasing temperature (20-30°C).  Snell et al.
    (1991) found that the acute toxicity of copper to the rotifer
     Brachionus calyciflorus was significantly increased at 10, 25 and
    30°C when compared with tests at both 15 and 20°C.

        Table 19.  Acute toxicity of copper to freshwater invertebrates (24-h to 96-h L(E)C50s)a

    Organism         Size/ age   Conditionsb   Temperature  Hardness (mg   pH       Copper     Parameter     Concentration   Reference
                                               (°C)         CaCO3/litre)            salt                     (µg/litre)

    Amnicola sp.     eggs        stat          17           50             7.6      ND         24-h LC50     4500            Rehwoldt et al.
                     eggs        stat          17           50             7.6      ND         96-h LC50     9300            (1973)
                     adult       stat          17           50             7.6      ND         24-h LC50     1500            Rehwoldt et al.
                     adult       stat          17           50             7.6      ND         96-h LC50     900             (1973)

    Goniobasis       ND          stat          15           154            8.5      sulfate    96-h LC50     390             Paulson et al.
    livescens                                                                                                                (1983)

    Lithoglyphus     ND          flow          15           22             7.2      chloride   96-h LC50     8               Nebeker et
    virens                                                                                                                   al. (1986)

    Juga plecifera   ND          flow          15           22             7.2      chloride   96-h LC50     15              Nebeker et
                                                                                                                             al. (1986)

    Physa integra    ND          flow          15           45             7.7      sulfate    96-h LC50     39              Arthur &
                                                                                                                             Leonard (1970)

    Campeloma        ND          flow          15           45             7.7      sulfate    96-h LC50     1700            Arthur &
    decisum                                                                                                                  Leonard (1970)

    Potamopyrgus     juvenile    flow          15           298            8.0      sulfate    48-h LC50     58              Watton &
    jenkinsi         juvenile    flow          15           298            8.0      sulfate    96-h LC50     54              Hawkes (1984)
                     prime adult flow          15           298            8.0      sulfate    48-h LC50     112             Watton &
                     prime adult flow          15           298            8.0      sulfate    96-h LC50     77              Hawkes (1984)
                     senescent   flow          15           298            8.0      sulfate    48-h LC50     87              Watton &
                     adult                                                                                                   Hawkes (1984)
                     senescent   flow          15           298            8.0      sulfate    96-h LC50     79

    Table 19.  (continued)

    Organism         Size/ age   Conditionsb   Temperature  Hardness (mg   pH       Copper     Parameter     Concentration   Reference
                                               (°C)         CaCO3/litre)            salt                     (µg/litre)

    Bristle worm
    Nais sp.         ND          stat          17           50             7.6      ND         24-h LC50     2300            Rehwoldt et al.
                     ND          stat          17           50             7.6      ND         96-h LC50     90              (1973)

    Lumbriculus      ND          stat          25           290            6.6      nitrate    96-h LC50     130             Shubauer-Berigan
    variegatus       ND          stat          25           290            7.3      nitrate    96-h LC50     270             et al.
                     ND          stat          25           290            8.3      nitrate    96-h LC50     500             (1993)

    Water fleas
    Daphnia magna    6-24 h      stat          25           80-100         7.4-7.8  sulfate    24-h LC50     380             Ferrando et
                                                                                                                             al. (1992)
                     ND          stat          17-19        44-53          7.4-8.2  chloride   48-h EC50     9.8             Biesinger &
                     ND          stat          17-19        44-53          7.4-8.2  chloride   48-h EC50     60 with food    Christensen
                     < 24 h      stat          21.6         143            7.1-7.9  oxide      48-h EC50     26              Lewis (1983)
                     ND          stat          ND           175            8.1      sulfate    48-h EC50     23-27c          LeBlanc (1982)
                     < 24 h      stat          20           ND             6.5      sulfate    48-h LC50     7               Oikari et al.
                     < 24 h      stat          20           ND             6.5      sulfate    48-h LC50     45 humic        (1992)
                     < 24 h      stat          ND           45             7.2-7.4             48-h LC50     54              Mount &
                     < 24 h      stat          ND           45             7.2-7.4             48-h LC50     53              Norberg (1984)
                     < 24 h      stat          20           130-160        8.2-9.5  sulfate    72-h LC50     86.5            Winner &
    Daphnia pulex    < 24 h      stat          20           130-160        8.2-9.5  sulfate    72-h LC50     86              Farrell (1976)

    Daphnia parvula  < 24 h      stat          20           130-160        8.2-9.5  sulfate    72-h LC50     72              Winner &
    Daphnia ambigua  < 24 h      stat          20           130-160        8.2-9.5  sulfate    72-h LC50     67.7            Farrell (1976)
                     ND          stat$         28.5         200            7.9                 48-h LC50     54.6            Vardia et al.
    Daphnia          ND          ND            ND           ND                      ND         96-h LC50     9.4             (1988)
    Daphnia hyalina  1.27 mm     stat          10           ND             7.2      chloride   48-h LC50     5               Baudouin &
                                                                                                                             Scoppa (1974)

    Table 19.  (continued)

    Organism         Size/ age   Conditionsb   Temperature  Hardness (mg   pH       Copper     Parameter     Concentration   Reference
                                               (°C)         CaCO3/litre)            salt                     (µg/litre)
    Moina irrasa     < 24 h      stat          20           < 5            8.0      chloride   48-h LC50     5.9             Zou & Bu (1994)

    Moina macrocopa  ND          stat$         24-27        ND             6.5      sulfate    48-h LC50     80              Wong (1992)

    Ceriodaphnia     < 12 h      stat          25           57             8.2      sulfate    48-h LC50     13.4            Oris et al. (1991)
    dubia            < 12 h      stat          25           45             7.7      ND         48-h LC50     17-32d          Belanger et al.
                     < 12 h      stat          25           179            8.3      ND         48-h LC50     67e             (1989)
                     < 12 h      stat          25           94             8.15     ND         48-h LC50     34-37d          Belanger et al.
                     < 12 h      stat          25           179            8.3      ND         48-h LC50     78-81e          (1989)

                     ND          stat          25           290            6.2      nitrate    48-h LC50     9.5             Schubauer-Berigan
                     ND          stat          25           290            7.1      nitrate    48-h LC50     28              et al.
                     ND          stat          25           290            8.6      nitrate    48-h LC50     200             (1993)

    Ceriodaphnia     < 4 h       stat          ND           45             7.2-7.4  ND         48-h LC50     17              Mount &
    reticulata                                                                                                               Norberg (1984)

    Simocephalus     < 24 h      stat          ND           45             7.2-7.4  ND         48-h LC50     57              Mount &
    vetulus                                                                                                                  Norberg (1984)

    Cyclops          1.27 mm     stat          10           ND             7.2      chloride   48-h LC50     2500            Baudouin &
    abyssorum                                                                                                                Scoppa (1974)

    Eudiaptomus      1.27 mm     stat          10           ND             7.2      chloride   48-h LC50     500             Baudouin &
    padanus                                                                                                                  Scoppa (1974)

    Amphipods        ND          stat          17           50             7.6      ND         24-h LC50     1200            Rehwoldt et al.
    Gammarus sp.     ND          stat          17           50             7.6      ND         96-h LC50     910             (1973)

    Gammarus         3-5 mm      stat$         12           151            6.8-7.2  ND         48-h LC50     47              Taylor et al.
    pulex            3-5 mm      stat$         12           151            6.8-7.2  ND         96-h LC50     37              (1991)
                     ND          stat$         ND           104            8.33     ND         48-h LC50     41              Stephenson
                     ND          stat$         ND           104            8.33     ND         96-h LC50     21              (1983)

    Table 19.  (continued)

    Organism         Size/ age   Conditionsb   Temperature  Hardness (mg   pH       Copper     Parameter     Concentration   Reference
                                               (°C)         CaCO3/litre)            salt                     (µg/litre)
    G. pulex         ND          stat$         ND           249            8.33     ND         48-h LC50     183             Stephenson
    (contd).         ND          stat$         ND           249            8.33     ND         96-h LC50     109             (1983)

    Gammarus         ND          stat          8            240            8.1      chloride   24-h LC50     179             Pantani et al.
    italicus                                                                                                                 (1990)

    Gammarus         ND          flow          15           45             7.7      sulfate    96-h LC50     20              Arthur &
    pseudolimnaeus                                                                                                           Leonard (1970)

    Hyallela azteca  ND          stat          25           290            6.2      nitrate    96-h LC50     17              Scubauer-
                     ND          stat          25           290            7.1      nitrate    96-h LC50     24              Berigan et al.
                     ND          stat          25           290            8.4      nitrate    96-h LC50     87              (1993)

    Echinogammarus   ND          stat          7.5-8.5      240            7.9      chloride   96-h LC50     720             Pantani et al.
    tibaldii                                                                                                                 (1995)

    Crangonyx        4 mm        stat          13           50             6.75     sulfate    48-h LC50     2440            Martin &
    pseudogracilis   4 mm        stat          13           50             6.75     sulfate    96-h LC50     1290            Holdich (1986)

    Asellus          7 mm        stat          13           50             6.75     sulfate    96-h LC50     9210            Martin &
    aquaticus                                                                                                                Holdich (1986)

    Cypris           ND          stat$         28.5         200            7.9      ND         48-h LC50     5363            Vardia et al.
    subglobosa                                                                                 96-h LC50     277.3           (1988)

    Brachionus       juvenile    stat          20           36.2           7.3      sulfate    24-h LC50     200             Couillard et al.
    calyciflorus                                                                                                             (1989)
                     ND          stat          25                                              24-h LC50     26              Snell et al.
                     ND          stat          25           80-100         7.4-7.8  sulfate    24-h LC50     76              Ferrando et al.

    Table 19.  (continued)

    Organism         Size/ age   Conditionsb   Temperature  Hardness (mg   pH       Copper     Parameter     Concentration   Reference
                                               (°C)         CaCO3/litre)            salt                     (µg/litre)
    Brachionus       neonate     stat          25           80-100         7.4-7.8  sulfate    24-h LC50     19              Snell &
    rubens                                                                                                                   Persoone

    Keratella        ND          stat          20           ND             8.3      chloride   24-h LC50     101             Borgmann &
    cochlearis                                                                                                               Ralph (1984)

    Streptocephalus  2nd/3rd     ND            20           8-10           6.4-6.6  sulfate    24-h LC50     120             Centeno et al.
    proboscideus     instar                                                                                                  (1993)

                     2nd/3rd     ND            20           71-110         7.6-7.8  sulfate    24-h LC50     210             Centeno et al.
                     instar                                                                                                  (1993)

                     2nd/3rd     ND            20           250-327        7.9-8.2  sulfate    24-h LC50     520             Centeno et al.
                     instar                                                                                                  (1993)

    Freshwater       adult       flow          15           17             6.96     sulfate    96-h LC50     34 (total Cu)   Daly et al.
    shrimp                                                                                                                   (1990a)
    Paratya          adult       flow          15           17             6.96     sulfate    96-h LC50     16 (Cu ion)     Daly et al.
    australiensis                                                                                                            (1990a)

    Chironomus sp.   ND          stat          17           50             7.6      ND         24-h LC50     650             Rehwoldt et al.
                     ND          stat          17           50             7.6      ND         96-h LC50     30              (1973)

    Chironomus       3rd instar  stat          13           25             6.3      sulfate    48-h EC50     327             Khangarot &
    tentans                                                                                                                  Ray (1989)
                     1st instar  stat          19-22        42.7           7.6      sulfate    96-h EC50     16.7            Gauss et al.
                     1st instar  stat          19-22        109.6          7.8      sulfate    96-h EC50     36.5            (1985)
                     1st instar  stat          19-22        172.3          8.1      sulfate    96-h EC50     98.2            Gauss et al.
                     4th instar  stat          19-22        42.7           7.6      sulfate    96-h EC50     211             (1985)
                     4th instar  stat          19-22        109.6          7.8      sulfate    96-h EC50     977             Gauss et al.
                     4th instar  stat          19-22        172.3          8.1      sulfate    96-h EC50     1184            (1985)

    Table 19.  (continued)

    Organism         Size/ age   Conditionsb   Temperature  Hardness (mg   pH       Copper     Parameter     Concentration   Reference
                                               (°C)         CaCO3/litre)            salt                     (µg/litre)
    C. tentans       1st instar  stat$         20           71             ND       chloride   96-h LC50     298             Nebeker et al.
    (contd).         2nd instar  flow          20           84             ND       chloride   96-h LC50     773             (1984a)
                     3rd instar  flow          20           84             ND       chloride   96-h LC50     1446            Nebeker et al.
                     4th instar  flow          20           84             ND       chloride   96-h LC50     1690            (1984a)

    Chironomus       4th instar  stat          20           40-48          7.2-7.6  sulfate    48-h LC50     739             Kosalwat &
    decorus                                                                                                                  Knight (1987a)

    Chironomus       2nd instar  stat$         12           151            6.8-7.2             48-h LC50     1200            Taylor et al.
    riparius         2nd instar  stat$         12           151            6.8-7.2             96-h LC50     700             (1991)

    a    EC50s based on immobilization; ND = no data available.
    b    Stat = static conditions (water unchanged for duration of test); stat$ = static renewal conditions
         (water changed at regular intervals); flow = flow-through conditions (copper concentration in water
         continuously maintained).
    c    Range of means for three duplicate tests.
    d    Range of tests from different culture sources; parental diet was synthetic.
    e    Range of tests from different culture sources; parental diet was algal.

    Table 20.  Acute toxicity of copper to marine invertebrates (24-h to 96-h L(E)C50S)a

    Organism            Size/age    Conditionsb   Temperature  Salinity   pH     Salt       Parameter     Concentration    Reference
                                                  (°C)         (%)                                        (µg/litre)

    Bay scallop         juvenile    stat$         20           25         ND     chloride   96-h LC50     29               Nelson et al.
    Argopecten                                                                                                             (1988)

    Surf clam           juvenile    stat$         20           25         ND     chloride   96-h LC50     51               Nelson et al.
    Spisula                                                                                                                (1988)

    Squid               larvae      stat          8.6          30         8.1    chloride   96-h LC50     309              Dinnell et al.
    Loligo opalescens                                                                                                      (1989)

    Cabezon             larvae      stat          8.3          27         7.9    chloride   96-h LC50     95               Dinnell et al.
    Scorpaenichthys                                                                                                        (1989)

    Amphipod            24 h        stat          20           32         ND     sulfate    96-h LC50     110              Ahsanullah &
    Allorchestes        adult       stat          20           32         ND     sulfate    96-h LC50     500              Florence (1984)

    American lobster    larvae      stat          20           30.5       ND     nitrate    96-h LC50     48               Johnson &
    Homarus                                                                                                                Gentile (1979)

    Dungeness crab      larvae      stat          8.5          30         8.1    chloride   96-h LC50     96               Dinnell et al.
    Cancer magister                                                                                                        (1989)

    Fiddler crab        24-29 mm    stat          29           25         ND     sulfate    96-h LC50     9420             Devi (1987)
    Uca annulipes       24-29 mm    stat          29           25         ND     sulfate    96-h LC50     12 820c          Devi (1987)

    Fiddler crab        24-29 mm    stat          29           25         ND     sulfate    96-h LC50     8380             Devi (1987)
    Uca triangularis    24-29 mm    stat          29           25         ND     sulfate    96-h LC50     14 810c          Devi (1987)

    Table 20.  (continued)
    Organism            Size/age    Conditionsb   Temperature  Salinity   pH     Salt       Parameter     Concentration    Reference
                                                  (°C)         (%)                                        (µg/litre)
    Ragworm             20 mm       stat          12           7.3        ND     nitrate    96-h LC50     357              Ozoh (1992a)
    Hediste             20 mm       stat          12           29.2       ND     nitrate    96-h LC50     512              Ozoh (1992a)
    diversicolor        20 mm       stat          22           7.3        ND     nitrate    96-h LC50     247              Ozoh (1992a)
                        20 mm       stat          22           29.2       ND     nitrate    96-h LC50     500              Ozoh (1992a)

    Tisbe battagliai    nauplius    stat$         20           34-35    6.2-8.2             nitrate       96-h LC50        64Hutchinson et
                        adult       stat$         20           34-35    6.2-8.2             nitrate       96-h LC50        88al. (1994)

    Tisbe holothuriae   ND          stat          22           38         ND     sulfate    48-h LC50     370              Verriopoulos
                                                                                                                           & Dimas (1988)

    Brachionus          neonate     stat          25           15         7.7    ND         24-h LC50     120              Snell &
    plicatilis          neonate     stat          25           30         7.7    ND         24-h LC50     130              Persoone

    Sand shrimp         adult       flow          13.7         30.1       7.9    chloride   96-h LC50     898              Dinnell et al.
    Crangon sp.                                                                                                            (1989)

    Penaeid shrimp      protozoeal  stat          27           30-34      8.7    sulfate    48-h LC50     160              Wong
    Metapenaeus         mysid       stat          27           30-34      8.7    sulfate    48-h LC50     1580             et al.
    ensis               postlarva   stat          27           30-34      8.7    sulfate    48-h LC50     4760             (1993)

    Mysid shrimp        3 days      stat                       ND         ND     chloride   96-h LC50     17               Martin et al.
    Holmesimysis                                                                                                           (1989)

    Grass shrimp
    Palaemonetes        juvenile    stat$         20           10         ND     ND         48-h LC50     2100             Burton &
    pugio                                                                                                                  Fisher (1990)
                        ND          stat          22           25       8.3-8.7             acetate       96-h LC50        3700Curtis et al.

    Table 20.  (continued)

    a  EC50s based on immobilization; ND = no data available.
    b  Stat = static conditions (water unchanged for duration of test); stat$ = static renewal conditions (water changed at regular intervals);
       flow = flow-through conditions (copper concentration in water continuously maintained).
    c  Animals collected from a polluted site (6.8-30.6 µg Cu/litre).

         Ozoh (1992c) found that sediment affected the acute response of
    both juvenile and adult ragworms  (Hediste diversicolor) to copper.
    Without sediment, increasing salinity (7.3 to 30.5%) and increasing
    temperature (12 to 22°C) reduced the acute toxicity of copper, whereas
    in the presence of sediment increasing temperature and increasing
    salinity increased the acute toxicity of copper.

         Snell & Persoone (1989a,b) exposed neonate rotifers to copper for
    24 h; NOEC was 20 µg Cu/litre at a salinity of 15%, 50 µg Cu/litre at
    30% for the marine rotifer  Brachionus plicatilis and 9.4 µg/litre
    for the freshwater rotifer  Brachionus rubens.  Ozoh & Jones (1990)
    studied the effects of salinity and temperature on the toxicity of
    copper to ragworm  (Hediste diversicolor) in 96-h tests.  Larvae at 1
    day old were more susceptible to copper than at 7 days old.
    Increasing salinity from 7.6 to 30.5% reduced copper toxicity.

         Stephenson (1983) reported that copper was 4-6 times more toxic
    to  Gammarus pulex in soft water (100 mg CaCO3/litre) than in hard
    water (250 mg CaCO3/litre). Similar findings were reported by Gauss
    et al. (1985) for first and fourth instar midge  Chironomus tentans 
    exposed to copper in acute toxicity tests.  First instar larvae were
    the most sensitive with 96-h EC50s, based on immobilization, at 16.7
    µg Cu/litre in soft water (40 mg CaCO3/litre) and 98.2 µg Cu/litre in
    hard water (170 mg CaCO3/litre).  The 48-h LC50s of copper to
     Ceriodaphnia dubia increased from 35 to 79 µg/litre when the water
    hardness was increased from 94 to 170 mg CaCO3/litre (Belanger et
    al., 1989).  Increasing the hardness from 8-10 to 250-327 mg
    CaCO3/litre decreased the 24-h LC50 of copper to the third instar of
    the crustacean  Streptocephalus proboscideus irrespective of the
    temperature (Centeno et al., 1993).  In similar tests a significant
    increase in toxicity was noted at pH 6.0 when compared with tests
    carried out at pHs ranging from 7.6 to 10.0.

         Shaner & Knight (1985) found that alkalinity had a significant
    effect on the acute toxicity of copper-bearing sediments to
     Daphnia magna.  The 24-h LC50s were 1332 and 1578 mg/kg at
    alkalinities of 600 and 1000 mg bicarbonate/litre, respectively.
    LC50s predicted by multiple regression models calculated levels
    ranging from 1146 to 5966 mg/kg at alkalinities ranging from 571 to
    2286 mg bicarbonate/litre.

         The addition of humic acid reduced the acute toxicity of copper
    to daphnids.  The mean 72-h LC50 increased from 28.3 µg Cu/litre in
    water containing no added humic acid to 53.2 µg Cu/litre in water to
    which 1.5 mg humic acid/litre had been added (Winner, 1984).  Giesy et
    al. (1983) showed a positive correlation between LC50 values for
    exposure of  Simocephalus serrulatus to copper and total dissolved
    carbon concentration.  Pantani et al. (1995) reported similar results
    for the amphipod  Echinogammarus tibaldii with the acute toxicity of
    copper being reduced by the addition of humic acid, fluvial sediment
    or bentonite.  Humic acid significantly reduced the acute toxicity of
    copper to  Daphnia pulex at all levels of water hardness tested (58,
    115, and 230 mg CaCO3/litre) (Winner, 1985).  Winner (1984) exposed

     Daphnia pulex to 30 µg Cu/litre in the presence of varying amounts
    of humic acid for up to 30 days.  Daphnids showed significantly better
    survival in water containing 0.38, 0.75 or 1.50 mg humic acid/litre
    than in water to which no humic acid had been added. In the absence of
    humic acid only one brood of young was produced because of premature
    deaths of females.  The addition of 0.38 mg humic acid/litre produced
    49 broods but the mean brood size was significantly smaller than humic
    acid controls.  Daphnids maintained on 30 µg Cu/litre and 1.50 mg
    humic acid/litre produced 101 broods with brood sizes significantly
    larger than humic acid controls.  Meador (1991) determined that humic
    acid decreased the toxicity of  Daphnia magna on the basis of total
    copper, but toxicity was constant on the basis of cupric ion activity.
    In contrast, Borgmann & Ralph (1983) and Borgmann & Charlton (1984)
    found that free metal concentrations did not provide a constant
    measure of copper toxicity to  Daphnia magna as organic matter
    concentrations changed.

         McLeese & Ray (1986) found the toxicity of copper to the marine
    shrimps  Crangon septemspinosa and  Pandalus montagui to be reduced
    when complexed with EDTA.  LC50s (144-h) were 1400 and 50 µg
    CuCl2/litre for the two species, respectively, whereas LC50s for
    copper-EDTA complexes were > 30 000 µg/litre.  However, 144-h LC50s
    for the clam  Macoma balthica were 6000 µg/litre regardless of the
    form of copper exposure.  In solutions containing nitrilotriacetic
    acid (NTA) or glycine, uncomplexed copper(II) ions were found to be
    the most acutely toxic form of copper to the freshwater shrimp
     Paratya australiensis.  However, although the copper-NTA complex did
    not contribute to the toxic effect, the copper-glycine complex appears
    to be mildly toxic (Daly et al., 1990a).  The acute toxicity of copper
    to  P. australiensis was shown to decrease in solutions of increasing
    alkalinity.  Additional experiments revealed that the tolerance to
    copper at higher alkalinities was caused by a combination of
    physiological effects associated with increased ionic strength of the
    test waters and changes in metal speciation (Daly et al., 1990b).  The
    presence of natural organic matter significantly reduced the toxicity
    of copper to  P. australiensis (Daly et al., 1990c).  Daly et al.
    (1992) found that post-moult shrimps were more sensitive to acute
    copper toxicity than individuals at other stages of the moult cycle.

         Baird et al. (1991) found that the 48-h EC50 for different
    clones of  Daphnia magna ranged from 10.5 to 70.7 µg Cu/litre.
    Pre-exposure of daphnids  (Daphnia magna) to 10 µg Cu/litre resulted
    in a significant reduction in the subsequent acute toxicity: 48-h
    LC50s in pre-exposed animals ranged from 58 to 80 µg Cu/litre whereas
    those of unexposed daphnids ranged from 23 to 27 µg Cu/litre (LeBlanc,

         Collyard et al. (1994) found little effect of age class (0.2-22
    days) on the acute (96-h) toxicity of copper to the amphipod
     Hyallela azteca. With the exception of the 6-8-day age class, which
    appeared to be the most sensitive to copper, the 95% confidence limits
    overlapped for the different age groups.  The 48-h LC50s for the
    penaeid shrimp  Metapenaeus ensis at different developmental stages

    were 160 µg Cu/litre (protozoaeal), 1580 µg Cu/litre (mysid) and 4760
    µg Cu/litre (post-larval), showing that tolerance to acute copper
    toxicity increased with age (Wong et al., 1993).

         Sosnowski et al. (1979) found that the sensitivity (72-h LC50)
    of the copepod  Acartia tonsa was strongly correlated with field
    population density and food ration.  The 72-h LC50s ranged from 9.0
    to 78.0 µg Cu/litre.  There was an inverse correlation between the log
    LC50 and adult  A. tonsa density at the time of collection.  The log
    LC50 increased with increasing food ration.  Lewis (1983) exposed
     Daphnia magna to copper in 48-h static toxicity tests at six
    different loading densities.  There was a trend of increasing toxicity
    at the lower density levels.  However, the differences in LC50 values
    were not biologically significant, with the maximum difference being
    approximately threefold.

         Dave (1984) found the 48-h EC50, based on immobilization, for
    unfed and fed daphnids  (Daphnia magna) to be 6.5 and 18.5 µg
    Cu/litre, respectively.  Neonates of  Ceriodaphnia dubia from mothers
    reared on an algal diet were 1.4-1.5 times more resistant to copper in
    acute toxicity tests than those reared on a synthetic diet (Belanger
    et al., 1989).

         Nell & Chvojka (1992) reported that copper concentrations of 8
    µg/litre significantly reduced the growth of Sydney rock oysters
     (Saccostrea commercialis) in 4-week studies.  Exposure of oysters to
    copper concentrations ranging from 8 to 64 µg/litre and 20 ng tributyl
    tin oxide/litre showed an additive effect on growth.

         Bodar et al. (1989) exposed parthenogenetic eggs of
     Daphnia magna to copper concentrations of 1.0, 10 and 25 mg/litre.
    Copper exposure at concentrations exceeding 1.0 mg/litre significantly
    reduced the total development of daphnid eggs.  However, stages 1 and
    2 (which take about half of the development time from egg to juvenile)
    showed only a slight decrease in the mean lifetime of individuals in
    these stages at copper concentrations of 10 and 25 mg/litre.
    Therefore the toxicity of copper, apparent in the total developmental
    effect, is exerted at stages 3-6.

         Hutchinson et al. (1994) exposed copepods  (Tisbe battagliai) to
    copper for 7 days.  NOECs for nauplius survival, adult survival and
    reproduction were 10, 18 and 6 µg Cu/litre, respectively; LOECs were
    18, 32 and 10 µg Cu/litre, respectively.  A subchronic value was
    calculated as the geometric mean of the highest NOEC and the lowest
    LOEC; these were 13 µg Cu/litre for nauplius survival, 24 µg Cu/litre
    for adult survival and 8 µg Cu/litre for reproduction.

     Long-term and reproductive toxicity

         Ringwood (1992) exposed gametes and early life stages of sea
    urchins  (Echinometra mathaei) and the bivalve  Isognomon 
     californicum to copper.  EC50s, based on fertilization,
    for sea urchins (1 h) and bivalves (2 h) were 14 and 55 µg Cu/litre,

    respectively; NOECs were 5.0 and 20 µg Cu/litre, respectively.  A 48-h
    EC50 (embryo survival) was 7.0 µg Cu/litre with a NOEC of 1.0 µg
    Cu/litre.  A NOEC for bivalve growth (96 h) was 1.0 µg Cu/litre.

         Stromgren & Nielsen (1991) studied the effect of copper on
    spawning, growth and mortality in larval, juvenile and mature common
    mussel  (Mytilus edulis).  EC50s, based on larval growth (10 days)
    and adult spawning frequency (30 days), were 5-6 and 2 µg Cu/litre,
    respectively. The larval 10-day LC50 was estimated to be
    approximately 10 µg Cu/litre.

         Macdonald et al. (1988) exposed yellow crab  (Cancer anthonyi) 
    embryos to copper in 7-day tests.  Copper concentrations > 1000
    µg/litre significantly reduced survival.  Hatching of embryos and
    larval survival were significantly reduced at 10 µg Cu/litre; no
    embryos hatched at copper concentrations of 100 µg/litre or more.

         Biesinger & Christensen (1972) exposed  Daphnia magna to copper
    for 3 weeks.  A 3-week LC50 of 44 µg Cu/litre was found; the 3-week
    EC50, based on reproductive impairment, was 35 µg Cu/litre.  Dave
    (1984) reports a 21-day EC50 (immobilization) of 1.4 µg Cu/litre.

         Cowgill & Milazzo (1991) carried out a three-brood toxicity test
    on  Ceriodaphnia dubia.  EC50s based on total progeny, mean brood
    number and mean brood size were found to be 357, 348 and 326 µg
    Cu/litre for metallic copper, and 305, 341 and 304 µg Cu/litre for
    copper nitrate.

         Oris et al. (1991) studied the effects of copper on the
    reproduction of the cladoceran  Ceriodaphnia dubia.  Chronic survival
    values, which were calculated as the geometric mean between NOEC and
    LOEC, were 34.6 and 24.5-34.6 µg Cu/litre for 4- and 7-day tests,
    respectively.  EC50s, based on mean total young per female, were
    38.2-40.4 and 30.7-30.8 µg Cu/litre for the two tests, respectively.

         In 21-day tests  Daphnia magna showed 100% mortality at 110 µg
    Cu/litre.  No significant effect on the intrinsic rate of natural
    increase was observed up to and including 36.8 µg Cu/litre. The
    carapace length was significantly reduced at 36.8 µg Cu/litre (Van
    Leeuwen et al., 1988).

         LeBlanc (1985) studied the competitive interactions between
     Daphnia magna and  Daphnia pulex in 28 day exposures to copper (10
    and 30 µg Cu/litre) .  D. pulex populations consistently exceeded
     D. magna populations when cocultured in the absence of copper or
    temporary exposures to 10 µg Cu/litre.  Exposure to 30 µg Cu/litre
    severely reduced initial population growth of  D. pulex without
    affecting  D. magna.  By day 14  D. magna populations were dominant;
    however,  D. pulex population growth was not completely suppressed
    and by the end of the experiment (28 days)  D. pulex had gained
    population dominance.  The reduced size of  D. magna suggested that
     D. pulex was out-competing  D. magna for available food.

         Ingersoll & Winner (1982) carried out 70-day toxicity tests with
     Daphnia pulex.  There was no significant effect on reproduction but
    survival was significantly reduced at 10 µg Cu/litre giving an NOEC of
    5 µg Cu/litre.  However, in pulse toxicity tests daphnids were exposed
    to 20 µg Cu/litre for 360 min/day (an average water concentration of 5
    µg Cu/litre).  Pulse exposures resulting in significant decreases in
    survival, brood size and body length, and delays in the age at which
    young were first produced.

         Winner & Farrell (1976) exposed four species of daphnid to copper
    at concentrations ranging from 20 to 100 µg Cu/litre for up to 130
    days.  All four species exhibited reductions in survival at
    concentrations > 40 µg Cu/litre.   Daphnia magna exhibited a
    decrease in the instantaneous rate of population growth at 60 µg
    Cu/litre;  whilst the same parameter was affected at > 40 µg Cu/litre
    for  D. pulex, D. parvula and D. ambigua. Daphnia ambigua produced
    significantly smaller broods at concentrations > 40 µg Cu/litre
    whereas mean brood size did not decrease in  D. pulex and  D. parvula
    until the concentration exceeded 60 µg Cu/litre.  Mean brood size in
     D. magna was unaffected by copper exposure.

         De Nicola Giudici & Migliore (1988) studied the long-term
    toxicity of copper on the freshwater isopod  Asellus aquaticus.  In
    30-day tests copper (5 µg Cu/litre) had no significant effect on
    female survival or birth rate.  There was no significant effect on
    growth during embryonic development; however, copper treatment during
    juvenile development reduced body growth in 90-day exposures.

         The development and hatchability of midge  (Chironomus decorus) 
    eggs were unaffected by copper (as copper sulfate) concentrations
    ranging from 100 to 5000 µg/litre.  All larvae survived a 72-h
    exposure except those at 5000 µg Cu/litre which died after only
    partial emergence.  The growth of larvae was significantly reduced
    when they were reared in copper-spiked food-substrate.  An EC50 based
    on growth was 1602 mg Cu/kg (Kosalwat & Knight, 1987b).

         Hatakeyama (1988) studied the effects of copper on the
    reproduction of chironomids  (Polypedilum nubifer) through water
    (10-40 µg Cu/litre) and food (22-5180 µg Cu/g dry weight).  Emergence
    success decreased to 74%, 38%, 16% and 2% of control values at 10, 20,
    30 and 40 µg Cu/litre, respectively.  The number of egg clusters
    produced by adults also decreased in accordance with the increase in
    copper concentration from 242 in controls to 31 at 30 µg Cu/litre; at
    40 µg Cu/litre eggs were not oviposited.  A significant decrease in
    emergence success occurred with food contaminated with 1770 mg Cu/kg:
    no emergence occurred at 5200 mg Cu/kg.

         In flow-through life cycle tests with caddisfly
     (Clistoronia magnifica) concentrations of > 17 µg Cu/litre
    prevented completion of the life cycle, and a significant reduction in
    adult emergence occurred at 13 µg Cu/litre.  The NOEC was found to be
    8.3 µg Cu/litre (Nebeker et al., 1984b).

         Arthur & Leonard (1970) exposed the amphipod
     Gammarus pseudolimnaeus and the snails  Physa integra and
     Campeloma decisum for 6 weeks to concentrations of 2.9-28.0 µg
    Cu/litre.  For all three species, reduced survival and other
    significant adverse effects occurred at concentrations of 14.8 µg
    Cu/litre and above, but no effects were noted at concentrations of 8.0
    µg Cu/litre and below.  In 100-day exposure to copper,
     Gammarus pulex populations densities were not affected at
    concentrations up to 11.0 µg Cu/litre, but were reduced at
    concentrations of 14.6 µg Cu/litre and above (Maund et al., 1992).

         Phipps et al. (1995) determined 10-day LC50s for the amphipod
     Hyalella azteca, the dipteran larva  Chironomus tentans, and the
    oligochaete  Lumbriculus variegatus to be 31, 54, and 35 µg Cu/litre,
    respectively, in Lake Superior (Canada) water (21-24°C).  Nebeker et
    al. (1986) reported 30-day LC50s for the snails  Juga plicifera and
     Lithoglyphus virens to be less than 8 µg Cu/litre.  The 11-week
    LC50 for zebra mussel  (Dreissena polymorpha)  was reported to be
    130 µg Cu/litre (Kraak et al., 1992).  The marine copepod
     (Tisbe furcata)was determined to have a 96-h LC50 of 178 µg
    Cu/litre, but concentrations as low as 56 µg Cu/litre were estimated
    to significantly reduce the intrinsic rate of population increase
    (Bechmann, 1994).

     Biochemical, physiological and behavioural effects

         Lin et al. (1992) exposed Pacific oysters  (Crassostrea gigas) 
    to copper; 8-16 mg Cu/litre caused a significant increase in
    filtration rates whereas concentrations > 32 mg/litre reduced
    filtration rates.  Glycine uptake rate was inhibited and the volume
    specific glycine transport declined in the presence of copper.

         Krishnakumar et al. (1990) exposed green mussels
     (Perna viridis) to 25 µg Cu/litre for 2 weeks.  Copper decreased
    ammonia-nitrogen excretion and significantly decreased filtration
    rate, O : N ratio, the scope for growth and growth efficiency; there
    was a nonsignificant increase in oxygen uptake.  Microscopic
    examination of digestive glands revealed a significant increase in
    lysosomal lipofuscin content and percentage incidence of tubule
    dilation.  Digestive cells showed extensive vacuolation of the
    cytoplasm.  Copper exposure caused almost 100% cilia loss and tubule

         Kraak et al. (1992) studied the effect of copper on the
    filtration rate in zebra mussel  (Dreissena polymorpha) over a 9-11
    week period.  Filtration rate was unaffected at concentrations of 13
    µg Cu/litre.  Expressed as a percentage of the controls the average
    filtration rates of mussels exposed to 53, 72 and 90 µg Cu/litre were
    44%, 33% and 27%, respectively.  The EC50, based on filtration rate,
    did not differ significantly from the 48-h EC50 (41 µg Cu/litre;
    Kraak et al., 1994) during 9 weeks (43 µg Cu/litre).  The NOEC for the
    same parameter over 48 h was 16 µg Cu/litre (Kraak et al., 1994).

         Ferrando & Andreu (1993) calculated 24-h EC50s, based on
    filtration and ingestion rates, to be 43 and 53 µg Cu/litre for
     Brachionus calyciflorus and 59 and 90 µg Cu/litre for
     Daphnia magna.

         Redpath & Davenport (1988) studied the action of three metals on
    pumping rate in the common mussel  (Mytilus edulis); they found that
    pumping was stopped by shell valve adduction at copper concentrations
    in the range 20.8-25.6 µg Cu/litre.

         Mussels  (Mytilus galloprovincialis) exposed to 40 µg Cu/litre
    showed a significant increase in the levels of malondialdehyde
    (indicative of the peroxidative process) and a decrease in the
    concentration of glutathione in gills and digestive gland.  The
    lipofuscin content in lysosome of the digestive gland was
    significantly increased (Viarengo et al., 1990).

         Gill and hepatopancreas glycogen levels were significantly
    reduced in freshwater mussels  (Lamellidens corrianus) exposed to
    concen trations of 100, 200 or 400 µg Cu/litre for up to 168 h
    (Rajalekshmi & Mohandas, 1993).

         Ferrando et al. (1993) studied the feeding rates of rotifers
     (Brachionus calyciflorus) fed on the microalgae.  A 5-h EC50, based
    on feeding rate, was calculated to be 32 µg Cu/litre.

         Weeks (1993) found a significant reduction in the feeding rate of
    the talitrid amphipod  Orchestia gammarellus at dietary
    concentrations of 688 mg Cu/kg during 48-h tests.  However, no
    significant effect was found at concentrations up to and including 817
    mg Cu/kg in 20-day exposures.

         Phelps et al. (1983) studied the effects of copper-enriched
    sediment on the burrowing behaviour of littleneck clams
     (Protothaca staminea).  Above a threshold of 5.8 mg Cu/kg added to
    dry sediment, the time for 50% of the clams to burrow (ET50)
    increased logarithmically with increasing sediment copper
    concentration.  Clams exposed to sediment mixed with a strong
    chelating agent and copper showed no significant change in burrowing

     Interactions with other chemicals

         Konar & Mullick (1993) studied the toxicity of different metal
    mixtures on zooplankton  (Diaptomus forbesi) in 48-h acute tests.
    Zinc and iron individually were found to behave antagonistically in
    combination with copper but synergistically when all three metals were
    in combination.  Copper in combination with lead alone, zinc and lead,
    iron and lead, and a combination of all four metals showed a
    synergistic interaction.

         Vranken et al. (1988) exposed free-living marine nematodes
     (Monhystera disjuncta) to copper in metal mixtures (mercury, zinc
    and nickel).  In 96-h tests, based on survival, all paired metal
    mixtures acted in a less than additive manner.  However, in EC50
    tests, based on developmental inhibition, the response was not as
    clear cut: the joint effect of copper with zinc, and copper with
    nickel was synergistic.  Copper-mercury combinations did not reveal a
    clear mode of interaction.

         Kaitala (1988) found that the presence of copper ions stimulated
    the accumulation of zinc and magnesium in mussels  (Mytilus edulis). 
    Zinc concentrations were 25% higher and zinc 100% higher than in the
    absence of copper.  Copper did not influence the uptake of magnesium
    in burrowing clams  (Macoma baltica) and zinc was not accumulated at
    all.  Vertebrates

     Lethality and growth effects

         The acute toxicity of copper to freshwater and marine fish is
    summarized in Tables 21 and 22, respectively.  The 96-h LC50s for
    freshwater fish range from 2.58 µg Cu/litre (Arctic grayling) to 7340
    µg Cu/litre (bluegill).  For marine fish, 96-h LC50 values range from
    60 µg Cu/litre for chinook salmon to 1690 µg Cu/litre for killifish.
    However, a 48-h LC50 for killifish was calculated to be 19 000 µg
    Cu/litre.  The toxicity of copper to amphibia is summarized in Table
    23.  For larvae of  Bufo melanostictus and  Xenopus laevis,
    respectively, 48-h LC50s of 446 and 1700 µg Cu/litre were found.

         Erickson et al. (1996) found the acute toxicity of copper to
    fathead minnow  (Pimephales promelas) to vary widely depending on the
    chemical characteristics of the water.  Increased pH, hardness,
    sodium, dissolved organic matter and suspended solids each caused
    toxicity to decrease on the basis of total copper concentrations, and
    96-h LC50s, based on total copper, ranged from 7 to 305 µg Cu/litre
    (0.11 to 4.8 µmol/litre) over the whole range of conditions tested in
    flow-through tests.  The results did not show a particularly good
    correlation of toxicity to cupric-ion-specific electrode measurements.
    The authors concluded that the effects of the different test
    conditions on copper speciation have an important role in determining
    toxicity; however, factors unrelated to chemical speciation also
    influenced toxicity.

         Smith & Heath (1979) studied the effect of temperature on the
    acute (24-h) toxicity of copper to five species of freshwater fish.
    There was considerable variation between species. There was a tendency
    for a higher sensitivity at higher temperatures in goldfish, channel
    catfish and rainbow trout; the converse was found for bluegill.
    However, the differences caused by temperature were a factor of 2 or
    less whereas the interspecies differences were as much as sixfold.

        Table 21.  Acute toxicity of copper to freshwater fish (48-h and 96-h LC50S)
    Organism         Size/age    Conditionsa    Temperature   Hardness (mg   pH           Copper            Parameter   Concentration    Reference
                                                (°C)          CaCO3/litre)b               salt                          (µg/litre)
    Chinook salmon   0.66 g      stat           11-13         211            7.4-8.3      sulfate (25.3%)   96-h LC50   58               Hamilton
    Oncorhynchus     0.87 g      stat           11-13         211            7.4-8.3      sulfate (25.3%)   96-h LC50   54               Buhl
    tshawytscha      alevin      flow           12            24             7.1          ND                96-h LC50   26               Chapman
                     swim-up     flow           12            24             7.1          ND                96-h LC50   19               (1978)
                     parr        flow           12            24             7.1          ND                96-h LC50   38               Chapman
                     smolt       flow           12            24             7.1          ND                96-h LC50   26               (1978)

    Coho salmon      0.41 g      stat           12            41.3           7.1-8.0      sulfate           96-h LC50   15               Buhl &
    Oncorhynchus                                                                                                                         Hamilton
    kisutch                                                                                                                              (1990)
                     6 g         stat$          13.5          33             7.0-7.5      ND                96-h LC50   17 (Cu2+)        Buckley
                     6 g         stat$          13.5          33             7.0-7.5      ND                96-h LC50   164              (1983)
                                                                                                                        (total Cu)
                     2.7 kg      flow           9.4           20             7.29         chloride          96-h LC50   46               Chapman

    Rainbow trout    0.60 g      stat           12            41.3           7.1-8.0      sulfate           96-h LC50   13.8             Buhl &
    Oncorhynchus                                                                                                                         Hamilton
    mykiss                                                                                                                                (1990)
                     1 g         stat           12            44             7.1          Count-N*          96-h LC50   20.4             Mayer &
                     1 g         stat           12            44             7.1          Count-NS*         96-h LC50   121              Ellersieck
                     1.6 g       stat           13            44             7.1          sulfate (98%)     96-h LC50   135              (1986)
                     alevin      flow           12            24             7.1          ND                96-h LC50   28               Chapman
                     swim-up     flow           12            24             7.1          ND                96-h LC50   17               (1978)
                     parr        flow           12            24             7.1          ND                96-h LC50   18               Chapman
                     smolt       flow           12            24             7.1          ND                96-h LC50   29               (1978)
                     adult       flow           9.2           42             7.57         chloride          96-h LC50   57               Chapman

    Table 21.  (continued)
    Organism         Size/age    Conditionsa    Temperature   Hardness (mg   pH           Copper            Parameter   Concentration    Reference
                                                (°C)          CaCO3/litre)b               salt                          (µg/litre)
    Brook trout      juvenile    flow           12            ND             ND           sulfate           96-h LC50   110              McKim &
    Salvelinus                                                                                                                           Benoit
    fontinalis                                                                                                                           (1971)

    Cutthroat trout  2.1 g       flow           13            194            7.8          chloride          96-h LC50   83.3             Chakoumakos
    Salmo clarki     9.4 g       flow           13            194            7.8          chloride          96-h LC50   221              et al.
                     25.6 g      flow           13            194            7.8          chloride          96-h LC50   243              (1979)
    Arctic grayling  alevin      stat           12            41.3           7.1-8.0      sulfate           96-h LC50   23.9-131c        Buhl &
    Thymallus        fry         stat           12            41.3           7.1-8.0      sulfate           96-h LC50   9.6              Hamilton
    arcticus         0.34 g      stat           12            41.3           7.1-8.0      sulfate           96-h LC50   2.58             (1990)

    Fathead minnow   1 g         stat           17            44             7.1          Count-N*          96-h LC50   35.9             Mayer &
    Pimephales       1.1 g       stat           17            44             7.1          Count-NS*         96-h LC50   154              Ellersieck
    promelas         1.2 g       stat           18            272            7.4          sulfate (98%)     96-h LC50   838              (1986)
                     22 mm       flow           20-26         202            7.5-8.2      sulfate           96-h LC50   490              Pickering
                     55 mm       flow           20-26         202            7.5-8.2      sulfate           96-h LC50   460              et al.
                     3.2-4.2 cm  stat           22            40-48          7.2-7.9      acetate           96-h LC50   390              Curtis et
                     47 mm       flow           24            200 (154)      8.0          ND                96-h LC50   490              Geckler
                                                                                                                                         et al.
                     56 mm       flow           24            200 (154)      8.0          ND                96-h LC50   440              (1976)

    Bluntnose minnow 15-16 mm    flow           25            200            7.9-8.3      sulfate           96-h LC50   230              Horning
    Pimephales                                                                                                                           Neiheisel
    notatus                                                                                                                              (1979)
                     84 mm       flow           24            200 (154)      8.0                            96-h LC50   340              Geckler
                                                                                                                                         et al.
    Bluegill         1.2 g       stat           17            44             7.1          Count-N*          96-h LC50   3280             Mayer &
    Lepomis          1.2 g       stat           17            44             7.1          Count-N*          96-h LC50   13 700           Ellersieck
    macrochirus      1 g         stat           24            44             7.4          oxychloride (99%) 96-h LC50   980              (1986)

    Table 21.  (continued)
    Organism         Size/age    Conditionsa    Temperature   Hardness (mg   pH           Copper            Parameter   Concentration    Reference
                                                (°C)          CaCO3/litre)b               salt                          (µg/litre)
    L. machrochirus  1.5 g       stat           18            44             7.1          sulfate (98%)     96-h LC50   884              Mayer &
    (contd).         1.5 g       stat           18            272            7.4          sulfate (98%)     96-h LC50   7340             Ellersieck
                     18.6 g      flow           24            200 (154)      8.0          ND                96-h LC50   8300             Geckler
                     19.2 g      flow           24            200 (154)      8.0          ND                96-h LC50   10 000           et al.

    Green sunfish    1.1 g       stat           18            44             7.1          sulfate (98%)     96-h LC50   3510             Mayer &
    Lepomis          1.1 g       stat           18            272            7.4          sulfate (98%)     96-h LC50   3400             Ellersieck
    cyanellus                                                                                                                            (1986)

    Pumpkinseed      ND          stat           28            55             8.0          ND                96-h LC50   2700             Rehwoldt
                                                                                                                                         et al.
    Lepomis gibbosus                                                                                                                     (1972)

    Goldfish         0.9 g       stat           18            272            7.4          sulfate (98%)     96-h LC50   13 800           Mayer &
    Carassius                                                                                                                            Ellersieck
     auratus                                                                                                                             (1986)

    Golden shiner    ND          flow           ND            72.2           7.5          chloride          96-h LC50   8460             Hartwell
                                                                                                                                         et al.
    Notemigonus                                                                                                                          (1989)

    Banded killifish ND          stat           28            55             8.0          ND                96-h LC50   840              Rehwoldt
                                                                                                                                         et al.
    Fundulus                                                                                                                             (1972)

    Striped bass     ND          stat           28            55             8.0          ND                96-h LC50   4000             Rehwoldt
                                                                                                                                         et al.
    Roccus saxatilis                                                                                                                     (1972)

    White perch      ND          stat           28            55             8.0          ND                96-h LC50   6400             Rehwoldt
                                                                                                                                         et al.

    Table 21.  (continued)
    Organism         Size/age    Conditionsa    Temperature   Hardness (mg   pH           Copper            Parameter   Concentration    Reference
                                                (°C)          CaCO3/litre)b               salt                          (µg/litre)
    Roccus                                                                                                                               (1972)

    American eel     ND          stat           28            55             8.0          ND                96-h LC50   6000             Rehwoldt
    Aguilla                                                                                                                              et al.
    rostrata                                                                                                                             (1972)

    Carp                         stat           28            55             8.0          ND                96-h LC50   800              Rehwoldt
                                                                                                                                         et al.
    Cyprinus carpio                                                                                                                      (1972)
                     3.5-5.5 g   stat           20            50             7.5          ND                48-h LC50   118              Peres &
                     3.5-5.5 g   stat           20            100            7.5          ND                48-h LC50   289              Pihan
                     3.5-5.5 g   stat           20            300            7.5          ND                48-h LC50   751              (1991a)
                     3.5 cm      stat$          20            ND             7.1          sulfate           96-h LC50   300              Alam &
                     6.5 cm      stat$          15            ND             7.1          sulfate           96-h LC50   1000             Maughan

    Fantail          36.8 mm     stat           19-21         ND             ND           sulfate           96-h LC50   333d             Lydy &
    Etheostoma       36.8 mm     stat           19-21         ND             ND           sulfate           96-h LC50   385e             Wissing
    flabellare                                                                                                                           (1988)

    Johnny darter    39.2 mm     stat           19-21         ND             ND           sulfate           96-h LC50   489d             Lydy &
    Etheostoma       39.2 mm     stat           19-21         ND             ND           sulfate           96-h LC50   569e             Wissing
    nigrum                                                                                                                               (1988)

    Rainbow darter   41 mm       flow           24            200 (154)      8.0          ND                96-h LC50   320              Geckler
                                                                                                                                         et al.
    Etheostoma                                                                                                                           (1976)

    Orangethroat     55 mm       flow           24            200 (154)      8.0          ND                96-h LC50   850              Geckler
    darter                                                                                                                               et al.
    Etheostoma                                                                                                                           (1976)

    Table 21.  (continued)
    Organism         Size/age    Conditionsa    Temperature   Hardness (mg   pH           Copper            Parameter   Concentration    Reference
                                                (°C)          CaCO3/litre)b               salt                          (µg/litre)

    Stoneroller      60 mm       flow           24            200 (154)      8.0          ND                96-h LC50   290              Geckler
    Campostoma                                                                                                                           et al.
    anomalum                                                                                                                             (1976)

    Creek chub       64 mm       flow           24            200 (154)      8.0          ND                96 h LC50   310              Geckler
    Semotilus                                                                                                                            et al.
    atromachulatus															 (1976)

    Blacknose dace   47 mm       flow           24            200 (154)      8.0          ND                96-h LC50   320              Geckler
    Rhinichthys                                                                                                                          et al.
    atratulus                                                                                                                            (1976)

    Brown bullhead   39 mm       flow           24            200 (154)      8.0          ND                96-h LC50   540              Geckler
    Ictalurus                                                                                                                            et al.
    nebulosus                                                                                                                            (1976)

    Striped shiner   55 mm       flow           24            200 (154)      8.0          ND                96-h LC50   790              Geckler
    Notropis         55 mm       flow           24            200 (154)      8.0          ND                96-h LC50   1900             et al.
    chrysocephalus   (1.7 g)                                                                                                             (1976)

    Mudfish          27.1 g      stat           ND            ND             ND           sulfate           96-h LC50   4301             Ebele et
    Clarias                                                                                                                              al. (1990)

    Cichlid          82 g        stat           22            ND             7.24         ND                96-h LC50   1059             Al-Akel
    Oreochromis                                                                                                                          (1987)

    a    Stat = static conditions (water unchanged for duration of test); stat$ = static renewal
         conditions (water changed at regular intervals); flow = flow-through conditions
         (copper concentration in water continuously maintained); ND = no data available.

    Table 21.  (continued)

    b    Alkalinity in parentheses (mg/litre).
    c    Range of LC50s for fish from different sources.
    d    Fish collected in winter
    e    Fish collected in summer.

    Table 22.  Acute toxicity of copper to marine fisha

    Organism            Size/age    Conditions   Temperature  Salinity    pH         Copper       Parameter    Concentration    Reference
                                                 (°C)         (%0)                    salt                      (µg/litre)

    Chinook salmon      1.6 g       stat         11-13        brackish    7.6-8.1    sulfate      96-h LC50    60               Hamilton &
    Oncorhynchus                                                                     (25.3%)                                    Buhl (1990)

    Coho salmon         smolt       flow         13           28.6        8.1        chloride     96-h LC50    601              Dinnell et
    Oncorhynchus                                                                                                                al. (1989)

    Topsmelt            larvae      stat         21           33          ND         chloride     96-h LC50    238              Anderson et
    Atherinops affinis                                                                                                          al. (1991)

    Tidewater           19 days     stat         25           20          ND         nitrate      96-h LC50    140              Mayer (1987)
    silverside                                                                       (34%)
    Menidia peninsulae

    Spot                adult       stat         25           20          ND         nitrate      96-h LC50    280              Mayer (1987)
    Leiostomus                                                                       (34%)

    Sheepshead minnow   larvae      stat$        25           34-35       6.2-8.2    nitrate      96-h LC50    > 220 fed        Hutchinson
    Cyprinodon                                                                                                                  et al. (1994)

    Rivulus marmoratus  0.03-0.1 g  flow         26-27        14          ND         ND           96-h LC50    1250-1610        Lin & Dunson

    Killifish           0.02-0.13 g flow         26-27        14          ND         ND           96-h LC50    1690             Lin & Dunson

    Fundulus            juvenile    stat$        20           10          ND         ND           48-h LC50    19 000           Burton &
    heteroclitus                                                                                                                Fisher (1990)

    Dab                 16.9 g      flow         12           34.6        7.7        nitrate      96-h LC50    300              Taylor et al.
    Limanda limanda                                                                                                             (1985)

    Table 22.  (continued)

    Organism            Size/age    Conditions   Temperature  Salinity    pH         Copper       Parameter    Concentration    Reference
                                                 (°C)         (%0)                    salt                      (µg/litre)
    Grey mullet         0.87 g      flow         12           34.6        7.7        nitrate      96-h LC50    1400             Taylor et al.
    Chelon labrosus                                                                                                             (1985)

    Shiner perch        adult       flow         13.2         29.5        7.8        chloride     96-h LC50    418              Dinnell et
    Cymatogaster                                                                                                                al. (1989)

    a  Stat = static conditions (water unchanged for duration of test); stat$ = static renewal conditions (water changed at regular intervals);
       Flow = flow-through conditions (copper concentration in water continuously maintained); ND = no data available.

         Chakoumakos et al. (1979) found the acute toxicity of copper to
    cutthroat trout  (Salmo clarki) to be inversely correlated with both
    water hardness and alkalinity.  The 96-h LC50s ranged from 15.7 µg
    Cu/litre at low alkalinity (20.1 mg CaCO3/litre) and hardness (26.4
    mg/litre) to 367 µg Cu/litre at high alkalinity (178 mg/litre) and
    hardness (205 mg/litre).  The most important copper species causing
    toxicity within the pH range tested were Cu2+, Cu(OH)+ and
    Cu(OH)20.  The concentration of each of these species varies with pH
    and alkalinity.  Lower pHs favour Cu2+; higher pHs favour Cu(OH)+
    and Cu(OH)20.  Lower alkalinities favour all three species.

         Peres & Pihan (1991a) reported a similar relationship between
    acute copper toxicity and hardness for both carp  (Cyprinus carpio) 
    and rainbow trout  (Oncorhynchus mykiss): 48-h LC50s for trout
    ranged from 25 µg Cu/litre to 560 µg Cu/litre at a water hardness
    ranging from 10 to 300 mg/litre. For carp, LC50s ranged from 118 to
    751 µg/litre whilst the hardness ranged from 50 to 300 mg/litre.  The
    toxicity of copper to rainbow trout decreased with increasing
    hardness.  LC50s (15 days) ranged from 18 µg Cu/litre at a hardness
    of 12 mg CaCO3/litre to 96 µg Cu/litre at a hardness of 97 mg/litre.

         Miller & Mackay (1980) tested the effects of different hardness
    (12-99 mg CaCO3/litre) and alkalinity (10-51 mg CaCO3/litre) on
    acute toxicity of copper to rainbow trout.  Toxicity was inversely
    related to hardness at all alkalinities and to alkalinity at higher
    hardness.  Incipient median lethal concentrations ranged from 18 to 96
    µg Cu/litre.  Howarth & Sprague (1978) evaluated the acute toxicity of
    copper to rainbow trout in waters of various hardness, alkalinity and
    pH and reported 96-h LC50s to range from 20 to 516 µg Cu/litre.
    Cusimano et al. (1986) reported the 96-h LC50 for rainbow trout to
    vary from 66 µg Cu/litre at pH 4.7 (alkalinity -0.2 mg CaCO3/litre)
    to 2.8 µg Cu/litre at pH 7.0 (alkalinity 11 mg CaCO3/litre).

         Welsh et al. (1993) determined the acute toxicity of copper to
    larval fathead minnows in waters of varying pH and dissolved organic
    carbon (DOC).  Toxicity was inversely related to both parameters, with
    96-h LC50s ranging from 2.0 µg Cu/litre at pH 5.6 and 0.2 mg
    DOC/litre to 182 µg/litre at pH 7 and 16 mg DOC/litre.  Empirical
    regression equations were derived that could be useful for predicting
    toxicity in different waters and the slopes of these equations were
    similar to those reported by Erickson et al. (1987, 1996).

         Anderson et al. (1994) reported 7-day LC50s and NOECs from three
    different laboratories for larval topsmelt.  LC50s were 162 and 274
    µg Cu/litre and NOECs were 100 µg Cu/litre at a salinity of 34%.
    LC50s were 55.7 and 58.4 µg Cu/litre and NOECs were 32 µg Cu/litre at
    a salinity of 20%.  LC50s and NOECs were calculated for topsmelt
    which were spawned at different times of the year over a 2-year period
    (1990-1991).  NOECs for copper were 100 µg Cu/litre except two 180 µg
    Cu/litre (November 1990) and 56 µg Cu/litre (May 1991);  LC50s for
    these tests ranged from 131 to 240 µg Cu/litre.

        Table 23.  Acute toxicity of copper to amphibians

    Organism          Size/age        Conditionsa      Temperature  Hardness (mg   pH       Copper     Parameter    Concentration     Reference
                                                       (°C)         CaCO3/litre)            salt                    (µg/litre)

    Clawed toad       larvae          stat             20           ND             ND       sulfate    48-h LC50    1700              DeZwart &
    Xenopus laevis                                                                                                                    Sloof (1987)

    Toad              larvae          stat             31           185            7.4      sulfate    48-h LC50    446               Khangarot
    Bufo              larvae          stat             31           185            7.4      sulfate    96-h LC50    320               & Ray
    melanostictus                                                                                                                     (1987)

    a  Stat = static conditions (water unchanged for duration of test); ND = no data available.

         McNulty et al. (1994) carried out a series of 7-day growth and
    survival experiments with larval topsmelt  (Atherinops affinis) of
    different ages.  Fish aged 0, 3 and 5 days were less sensitive to
    copper than fish > 7 days old.  LC50s ranged from 365 µg Cu/litre
    in 0-day larvae to 137 µg Cu/litre in 20-day larvae.  NOECs were
    constant for all age groups at 180 µg Cu/litre for fish 1 and 3 days
    old and 100 µg Cu/litre for all other groups.  Pickering & Lazorchak
    (1995) exposed fathead minnow  (Pimephales promelas) to copper in a
    7-day larval survival and growth test.  The NOEC and LOEC, based on
    growth, were 25 and 50 µg Cu/litre, respectively, for larvae 1, 4 and
    7 days old.  For survival the NOEC and LOEC were 200 and 400 µg
    Cu/litre, respectively, for 1-day-old and 4-day-old larvae; however,
    for 7-day-old larvae the NOEC and LOEC were 100 and 200 µg Cu/litre.
    The subchronic value (geometric mean of NOEC and LOEC) was 35 µg
    Cu/litre.  The authors found the test to be relatively insensitive to
    changes in test conditions.

         Hutchinson et al. (1994) exposed sheepshead minnow larvae
     (Cyprinodon variegatus) to copper for 7 days.  NOECs for survival
    and growth were 120 and 220 µg Cu/litre, respectively; LOECs were 220
    and > 220 µg Cu/litre, respectively.  A subchronic value was
    calculated as the geometric mean of the highest NOEC and the lowest
    LOEC; these were 160 µg Cu/litre for survival and > 220 µg Cu/litre
    for growth.

         Seven-day survival tests with coho salmon
     (Oncorhynchus kisutch) carried out following 16 weeks exposure
    indicate that the exposed fish became significantly more tolerant of
    copper.  The 168-h LC50 for previously unexposed fish was 220 µg
    Cu/litre whereas fish exposed to 140 µg Cu/litre were 2.5 times less
    sensitive at 550 µg Cu/litre (Buckley et al., 1982).

         Collvin (1985) studied the effect of copper (1-81 µg Cu/litre) on
    the maximal growth rate of perch  (Perca fluviatilis) over a 30-day
    period.  Copper reduced maximal growth rate at concentrations > 22 µg
    Cu/litre.  Reduced growth rate was mainly an effect of reduced food
    conversion efficiency attributed to an increased metabolism caused by

         Lanno et al. (1985) fed rainbow trout  (Oncorhynchus mykiss) on
    a diet containing copper at concentrations ranging from 9 to 3088 mg
    Cu/kg for 8 weeks, and from 8.5 to 664 mg Cu/kg for up to 24 weeks.
    After 8 weeks reduced weight gain and feed intake, increased
    feed : gain ratios and mortalities were observed in trout reared on
    test diets containing > 730 mg Cu/kg.  Trout receiving 1585 or 3088
    mg Cu/kg showed pronounced food refusal.  After 16 weeks trout reared
    on a diet containing 664 mg Cu/kg had significantly lower live body
    weights, although there was no significant difference after 24 weeks.

         Mount et al. (1994) fed rainbow trout  (Oncorhynchus mykiss) on
    a brine shrimp  (Artemia sp.) diet containing 440, 830 or 1000 mg
    Cu/kg (dry weight) for up to 60 days.  Fish fed the 830 or 1000 mg
    Cu/kg diets showed 30% mortality during the experiment.  No

    significant effect of copper on growth was observed.  The authors
    conclude that waterborne copper released from  Artemia may have
    contributed to the mortality.

     Effects on reproduction and early life-stages

         Stouthart et al (1996) exposed newly fertilized common carp
     (Cyprinus carpio) eggs to copper (19.1 and 50.8 µg/litre; 0.3 and
    0.8 µmol/litre) at pH 6.3 and 7.6.  No significant effect of copper on
    egg mortality, larval heart rate and tail movement or whole-body
    potassium and magnesium content was observed at pH 7.6.  However,
    whole-body sodium and calcium were significantly decreased and larval
    mortality and larval deformation were significantly increased at the
    higher copper exposure.  At pH 6.3, exposure to 50.8 µg Cu/litre (0.8
    µmol/litre) significantly increased egg mortality and decreased heart
    rate and tail movements; premature hatching and a
    concentration-dependent increase in larval mortality and larval
    deformation were also observed.  The whole-body content of potassium,
    sodium, magnesium, and calcium were all significantly decreased by
    both copper exposures at pH 6.3.

         Anderson et al. (1991) carried out both fertilization tests and
    embryo development tests on topsmelt  (Atherinops affinis).  In
    fertilization tests percentage fertilization was measured following
    exposure of sperm to copper. The NOEC values for four fertilization
    tests ranged from 32 to > 90 µg Cu/litre; EC50s ranged from 24 to
    163 µg Cu/litre.  In embryo tests embryos were checked for up to 12
    days for viability, abnormalities, mortality and hatching success.
    The NOEC for embryo abnormalities ranged from 55 to 123 µg Cu/litre;
    EC50s ranged from 115 to 180 µg Cu/litre.  The NOEC for larval
    hatching success and for larval abnormalities ranged from 55 to 123 µg
    Cu/litre, and from 55 to 68 µg Cu/litre for the two parameters,
    respectively; EC50s ranged from 108 to 182 µg Cu/litre, and from 75
    to 190 µg Cu/litre.

         Pickering et al. (1977) exposed fathead minnow
     (Pimephales promelas) to copper (8-100 µg Cu/litre) at 6, 3 and 0
    months prior to spawning.  The prespawning exposure time had no
    significant effect on reproduction.  However, egg production was
    significantly lower at concentrations of 37 µg Cu/litre or more. The
    maximum acceptable toxicant concentration (MATC) was estimated to be
    32 µg Cu/litre.

         Dave & Xiu (1991) monitored the effects of copper chloride on
    hatching and survival of zebrafish  (Brachydanio rerio) exposed from
    post-fertilization for 16 days.  Significant mortality of embryos
    occurred at concentrations of 32 µg Cu/litre and above during the
    first day of exposure.  For exposures of 1-16 µg Cu/litre, hatch was
    significantly delayed relative to the control value of 4 days and
    embryo mortality exceeded 50%.  Delayed hatching was also reported at
    concentrations < 1 µg Cu/litre, but effect levels are very uncertain
    because exposure concentrations were unmeasured and background
    concentrations are unknown.

         Scudder et al. (1988) exposed embryos of fathead minnow
     (Pimephales promelas) to total copper concentrations ranging from
    0.6 to 621 µg Cu/litre from 5 to 10 h post-fertilization to 2 days
    post-hatch.  Significant declines in percentage survival and
    percentage total hatch were observed at 621 µg Cu/litre but not at 338
    µg Cu/litre.  A significant increase in the number of embryos with
    abnormalities was observed at > 338 µg Cu/litre.  Larval fish were
    exposed to copper at the same concentrations for 28 days post-hatch.
    Fish growth was significantly reduced and percentage abnormalities
    increased at the lowest treatment concentration (61 µg Cu/litre) and
    effects increased with increasing concentration. Percent survival was
    significantly reduced at concentrations of 113 µg Cu/litre and above
    and the 28-day LC50 was estimated to be 128 µg Cu/litre.

         Mount (1968) conducted an 11-month, full-life-cycle exposure of
    fathead minnows to copper in a hard water (hardness 200 mg
    CaCO3/litre, alkalinity 160 mg CaCO3/litre, pH 7.9).  Growth and
    survival were significantly reduced at 95 µg Cu/litre and reproduction
    was completely suppressed at 32-34 µg Cu/litre, but unaffected at
    14-15 µg Cu/litre.  In a softer water (hardness 31 mg CaCO3/litre,
    alkalinity 30 mg CaCO3/litre, pH 7.0), Mount & Stephen (1969)
    reported survival, growth, and reproduction to be significantly
    affected at 18.4 µg Cu/litre, but not at 10.6 µg Cu/litre.

         McKim et al. (1978) tested the effects of copper on the growth
    and survival of embryos and larvae of eight fish species.  The
    standing crop of fish after 30-70 days post-hatch was significantly
    reduced at exposure concentrations of 32 µg Cu/litre for rainbow
    trout, 34 µg Cu/litre for white sucker, 44 µg Cu/litre for brook
    trout, 42 µg Cu/litre for lake trout, 46 µg Cu/litre for brown trout,
    103 µg Cu/litre for lake herring, 104 µg Cu/litre for northern pike,
    and 104 µg Cu/litre for smallmouth bass.

         Seim et al. (1984) contrasted the effects of continuous and
    intermittent (4.5 h/day) exposure of steelhead trout to copper for 78
    days.  The EC50 for growth reduction on the basis of average copper
    concentrations was 46 µg Cu/litre for continuous exposure, but only 27
    µg Cu/litre for intermittent exposure.

         Sayer et al. (1989) exposed yolk-sac fry of brown trout
     (Salmo trutta) to copper concentrations of 1.2, 2.5 and 5.1 µg
    Cu/litre (20, 40 and 80 nmol/litre) at pH 4.5 and at calcium
    concentrations of 20 or 200 µmol for 30 days.  Mortalities were high
    (70-100%) at the lower calcium concentration for all three copper
    concentrations.  Only one death was observed for copper and high
    calcium exposures.  At high calcium levels, impaired sodium, potassium
    and calcium uptake were observed for all three copper concentrations.

         Horning & Neiheisel (1979) exposed bluntnose minnow
     (Pimephales notatus) to copper concentrations ranging from 4.3
    (control) to 119.4 µg Cu/litre.  Minnows exposed to 119.4 µg Cu/litre
    for 60 days were significantly smaller than the other groups.
    Survival of parental bluntnose minnows was not affected by any copper

    concentrations during the 60-day exposure.  Copper
    concentrations > 18 µg Cu/litre significantly reduced the number of
    spawnings, the total number of eggs produced and the number of eggs
    per female.  Therefore, the MATC based on reproductive impairment was
    between 4.3 and 18 µg Cu/litre.  Minnows held in "clean" water for 9
    months ceased to spawn on exposure to 119.4 µg Cu/litre.  Fish exposed
    to 119.4 µg Cu/litre for the same 9-month period began to spawn 60
    days after being transferred to "clean" water.

         McKim & Benoit (1971) exposed brook trout
     (Salvelinus fontinalis) to copper(II) concentrations ranging from
    1.9 to 32.5 µg Cu/litre for 22 months.  The highest concentration
    decreased survival and growth in adult fish, and reduced the number of
    viable eggs produced and hatchability.  No effects on adult survival,
    growth or reproduction were observed at copper concentrations of 17.4
    µg/litre or less.  Concentrations of 17.4 and 32.5 µg Cu/litre had
    marked adverse effects on survival and growth of alevins and juvenile
    fish.  Therefore, the MATC for brook trout exposed to copper (hardness
    45 mg CaCO3/litre; pH 7.5) was between 9.5 and 17.4 µg Cu/litre.

         Benoit (1975) exposed bluegills  (Lepomis macrochirus) to copper
    concentrations ranging from 12 to 162 µg Cu/litre for a period of 22
    months.  Adult bluegill survival and reproduction were significantly
    affected only at the highest copper concentration of 162 µg Cu/litre.
    A 90-day exposure of larvae transferred at hatch revealed a
    significant reduction in survival at > 40 µg Cu/litre; larval
    growth was not significantly reduced at 77 µg Cu/litre and below.

     Metabolic, biochemical and physiological effects

         Beckman & Zaugg (1988) exposed chinook salmon
     (Oncorhynchus tshawtscha) to natural springwater with an elevated
    copper concentration (48 µg/litre).  In parr, gill Na+, K+-ATPase
    activity was unaffected by an 18-h exposure, whereas in smolt there
    was significant inhibition.  In both parr and smolt there were
    significant increases in haematocrit and plasma glucose.

         Arillo et al. (1984) investigated the effect of copper at levels
    of 30-100 µg Cu/litre on a wide variety of biochemical parameters in
    the rainbow trout  (Oncorhynchus mykiss).  The exposure period was 4
    months.  Copper significantly reduced ALAD (aminolaevulinic acid
    dehydratase) activity in liver, carbonic anhydrase activity in blood,
    gill sialic acid content and the respiratory control ratio and oxygen
    consumption in liver mitochondria.

         Heath (1991) exposed bluegill  (Lepomis macrochirus) to a free
    copper concentration of 261 µg Cu/litre for 7 days.  Copper caused an
    elevation in plasma glucose of approximately 100% and a significant
    reduction in liver ATP.  Acute hypoxia stress responses such as
    hyperglycaemia, lower plasma sodium, lower liver ATP and higher plasma
    potassium were accentuated by prior exposure to copper.

         Nemcsók & Hughes (1988) exposed rainbow trout to concentrations
    of 200 or 2000 µg Cu/litre for up to 48 h.  Activities of blood
    glucose, ASAT and ALAT were significantly increased after 24 h at 2000
    µg Cu/litre and after 48 h at the lower concentration.  Significant
    decreases in acetylcholinesterase activity were observed over the same
    time periods at each of the copper concentrations.  Copper sulfate
    (200 µg/litre) had only a slight damaging effect on tissues after 24 h
    as indicated by biochemical and haematological parameters.  However,
    the addition of sulfuric acid (pH 6.5) significantly increased blood
    glucose, ASAT, ALAT and lactate dehydrogenase, and significantly
    decreased acetyl cholinesterase activity.

         Muńoz et al. (1991) exposed juvenile rainbow trout to copper (50
    µg Cu/litre) for 21 days.  Copper caused rapid and significant
    elevations of plasma cortisol levels; plasma sodium showed a
    significant decrease for 7-15 days.

         Lydy & Wissing (1988) studied the thermal resistance of fantail
    darters  (Etheostoma flabellare) and johnny darters  (E. nigrum) 
    exposed to copper at sublethal concentrations for 96 h.  Thermal
    resistance was determined using the critical thermal maxima (CTMAX)
    with loss of equilibrium as the end-point.  The mean CTMAX value for
    fantail darters exposed to 149 µg Cu/litre was 7.6°C below that of the
    control means.  In johnny darters, a concentration of 292 µg Cu/litre
    depressed the CTMAX value by 5.2°C.  The NOEC for the fantail darter
    was 42 µg Cu/litre and for the johnny darter 128 µg Cu/litre.

     Structural effects and malformations

         Benedetti et al. (1989) exposed brown bullhead
     (Ictalurus nebulosus) to concentrations of 5000 µg Cu/litre for 24 h
    and 300 µg Cu/litre for 40 days.  All fish reared for more than 1
    month in 300 µg Cu/litre showed epidermal changes such as an increase
    in mucus cell numbers and a tendency for the cells to become
    superficial.  In fish most severely impaired by copper poisoning the
    epidermis appeared thinner in patches compared with controls.  The
    gills of fish exposed to 5 mg Cu/litre were markedly damaged with
    swollen and hyperaemic lamellae, necrosis and disaggregation of the
    epithelium, whereas fish in the 300 µg Cu/litre group showed more
    variable gill damage.  Histomorphological analysis of livers from both
    groups did not reveal diffuse changes in the hepatic parenchyma.
    However, areas of patchy degeneration and isolated degenerated
    elements located within areas of normal hepatocytes were observed.
    Histochemical staining revealed that all treated fish had lower liver
    glycogen content than controls.

         Khangarot (1992) investigated ultrastructural alterations in the
    liver of snake-headed fish  (Channa punctatus) following exposure to
    50 and 100 µg Cu/litre using transmission electron microscopy.  After
    4 days at 50 µg Cu/litre there is a marked proliferation of the smooth
    endoplasmic reticulum (SER), complete degeneration of rough
    endoplasmic reticulum (RER), loss of ribosomes from the surface of
    RER, a random distribution of ribosomes throughout the cytoplasm and

    an increase in the number and size of SER cisternae.  Mitochondrial
    swelling, and the loss of internal and external mitochondrial
    membranes, were observed.  A large number of vacuoles and lysosome
    having dense bodies were observed after 7 days exposure.  The
    lysosomal matrix frequently displayed crystalline structures of
    various sizes and the nuclear size was reduced with chromatin material
    clumped within the nucleus.  Prominent changes in nuclei of fish
    hepatocytes were observed after exposure to 100 µg Cu/litre.  Rupture
    of nuclear membranes and clumping of chromatin in necrotic cell nuclei
    with the aggregation of interchromatin material at the centre of the
    nucleus were recorded.  More dilation and vesiculation were observed
    in RER after 7 days. Aggregation of SER and RER, rupturing of
    mitochondrial membrane, a decrease in the number of mitochondria and
    an increase in the number of Golgi complexes were also observed.

         Kirk & Lewis (1993) studied copper-induced changes in gills of
    rainbow trout  (Oncorhynchus mykiss) by scanning electron microscopy.
    Trout exposed to 500 µg Cu/litre for 2 h showed collapse of lamellae
    and considerable secretory activity of mucous cells.  Filament tips
    were swollen and bent, and had an increase in the number of mucous
    cells which extruded copious amounts of mucus.  Exposure of fish to
    1000 µg Cu/litre caused the gills to be covered in mucus and cellular
    debris.  There were many ruptured and exhausted mucous cells, lamellar
    fusion occurred and epithelial cells were extremely swollen throwing
    the gill surface into swellings and ridges. There was a greater
    proliferation of chloride and mucous cells, and increased mucus
    secretion compared with the lower copper exposure.

     Behavioural effects

         Pedder & Maly (1985) studied the attraction-avoidance response of
    rainbow trout at concentrations ranging from 500 to 4000 µg Cu/litre.
    There was an initial attraction response at all copper concentrations
    which led to high mortality at 3000 and 4000 µg Cu/litre.  Avoidance
    of copper was observed, following the initial period of attraction, at
    > 500 µg Cu/litre; maximum avoidance at 1.0 mg Cu/litre.  The 96-h
    EC50, based on avoidance, was between 500 and 750 µg Cu/litre.
    Hartwell et al. (1989) found the avoidance threshold for golden shiner
     (Notemigonus crysoleucas) to be 26 µg Cu/litre in flow-through

         Ellgaard & Guillot (1988) observed that exposure to copper
    elicited a hypoactive response in bluegill at all concentrations
    tested (40, 80 and 400 µg Cu/litre) and that the effect was
    concentration-dependent.  At all concentrations, locomotor activity
    appeared to fall rapidly during the first 4 days following exposure
    and then tended to plateau for the rest of the 8-day exposure.

         Steele (1989) studied the effect of sublethal exposures of copper
    (50, 100 and 200 µg Cu/litre) on the daily activity of sea catfish
     (Arius felis) after a 72-h static exposure.  Fish exposed to 0.1 or
    0.2 mg Cu/litre showed significant hyperactivity and a loss of the

    normal daily activity pattern of this species; the same two exposure
    groups showed significantly less variability in activity.  Model ecosystems and community effects

         Havens (1994a) dosed mesocosms with copper concentrations ranging
    from 2 to 200 µg Cu/litre for 5 days.  There was a significant
    negative relationship between total algal biovolume and copper dose.
    The decline in algal biovolume at higher doses (> 50 µg Cu/litre)
    reflected the loss of  Rhodomonas, Aphanizomeron, Chlamydomonas  and
     Ceratium.  The assemblage that survived was dominated by diatoms.
    There was a significant negative exponential relationship between
    zooplankton biomass and copper dose.  The most sensitive species were
    the calanoid copepods with a biomass reduction of > 50% at 20 µg
    Cu/litre; the cyclopoids were the most tolerant with more than 50% of
    the biomass of cyclopoid copepodids surviving the highest dose.

         Havens (1994b) exposed a freshwater plankton community to copper
    (140 µg Cu/litre) for 14 days.  Copper significantly reduced the dry
    weight biomass of zooplankton, ciliates, flagellates and autotrophic
    phytoplankton. Bacterial biomass was significantly increased; however,
    this resource went virtually unexploited because the most effective
    bacterial grazers (large cladocerans and protozoans) were greatly
    reduced by copper exposure.

         Hedtke (1984) exposed an aquatic microcosm to copper for 32 weeks
    under flow-through conditions.  No significant effects on material
    cycling and biological structure were observed at 4.0 µg Cu/litre. At
    9.3 µg Cu/litre primary production levels were significantly reduced
    by the end of the experiment and dissolved organic carbon production
    was substantially lower than controls. Copper concentrations > 30
    µg Cu/litre significantly lowered primary production and macroalgal
    growth, and there were substantial structural changes increasingly
    shifting from autotrophic to heterotrophic systems with increasing
    copper levels.

         Scanferlato & Cairns (1990) spiked sediment with copper
    concentrations of 10, 100 or 1000 mg Cu/kg dry sediment in an aquatic
    microcosm.  Most of the added copper remained bound to sediment
    particles during the 8-week experiment.  The lowest concentration had
    no effect on the structure or function of the microcosm.  In
    microcosms exposed to 100 mg Cu/kg (500 µg Cu/litre in overlying
    water) both chlorophyll  a content and respiration were significantly
    decreased.  Other structural and functional attributes were rather
    variable.  Significant decreases in production, respiration,
    respiration/biomass ratio, ATP and chlorophyll  a, and significant
    increases in assimilation ratio and autotrophic index were observed at
    1000 mg Cu/kg (overlying water concentration = 20 mg Cu/litre).

         Hart et al. (1992) evaluated the effects of copper on
    phytoplankton communities in 5-m diameter (40-m3 volume) enclosures
    in Island billabong (floodplain on Magela Creek, northern Australia).
    Copper (1.3 g) was added to the enclosure at a rate thought to be 10×

    the normal wet season values for Island billabong.  Concentrations of
    total copper over the 10-week experiment ranged from 2.2 to 51 µg
    Cu/litre in the treatment and 3.9 to 26 µg Cu/litre in the control.
    The addition of copper had little effect on the phytoplankton

         Winner et al. (1990) evaluated the seasonal responses of
    planktonic and benthic communities exposed to copper concentrations of
    20 or 40 µg Cu/litre in oligotrophic ponds for 5-week periods.
    Phytoplankton and zooplankton were more sensitive to copper in the
    spring than in the summer or autumn.  Zooplankton exhibited a 43% and
    an 86% reduction in density in the 20 and 40 µg Cu/litre enclosures,
    respectively.  The authors suggest that this is related to seasonal
    changes in the dissolved organic carbon content of the ponds.

         Winner & Owen (1991a) evaluated the toxicity of copper to
    daphnids in 7-day chronic tests  (Ceriodaphnia dubia) and algae in
    4-day cell reproduction tests  (Chlamydomonas reinhardii) using
    filtered pond water from Brandenburg Pond, Ohio, USA.  The studies
    were performed numerous times over a 6-month period.   C. reinhardii 
    NOECs were typically 20-40 µg Cu/litre whereas for  C. dubia they
    were 50-80 µg Cu/litre.  For both species, NOECs increased with
    increasing alkalinity and hardness.   C. reinhardii NOECs based on
    cell growth also declined with increasing dissolved organic carbon

         Winner & Owen (1991b) examined the sensitivity of freshwater
    phytoplankton communities to chronic copper exposure in 100-litre
    polyethylene enclosures in Brandenburg Pond, Ohio, USA.  The studies
    were conducted in four 5-week exposures over the course of 2 years.
    Nominal exposure levels of 0, 20 and 40 µg Cu/litre were used and
    verified analytically.  Over the course of the experiment 82 taxa of
    phototrophs were identified in the enclosures.  Seasonal variation in
    algal population density was observed with the largest depression of
    the algal populations in the two spring exposures at both the 20 and
    40 µg Cu/litre concentrations.  Summer and fall population responses
    to copper at the 20 and 40 µg Cu/litre level were minimal, although
    individual taxa were effected.  During the first spring exposure,
    effects on zooplankton and benthos were also measured (Moore & Winner,
    1989).  Small mayflies and chironomids were effected at 40 µg Cu/litre
    and no effects on benthos were observed at 20 µg Cu/litre.  Rotifers
    and copepods exhibited significant reduction in density at both 20 and
    40 µg Cu/litre.  Daphnids were not affected at either treatment level,
    which was consistent with laboratory toxicity tests with
     Ceriodaphnia dubia.

         Moore & Winner (1989) studied the effect of copper (20 and 40 µg
    Cu/litre) on zooplankton and benthos in enclosure experiments.  During
    5-week exposures copper caused significant decreases in the alga
     Uroglena, rotifers, cyclopoids and calanoid copepods. The density of
    small mayflies and chironomids was significantly decreased at the
    higher copper exposure.  Other benthic organisms such as fingernail

    clams, larger midges and mayflies were not affected by copper but
    rather by fish predation and/or adult emergence.   Daphnia achieved
    significantly higher densities at 20 µg Cu/litre than in the controls.

         Clements et al. (1989) conducted experiments in artificial
    streams to examine the influence of water quality on the
    macroinvertebrate responses to copper.  The effects of copper on the
    reduction of macroinvertebrate abundance was greater in streams of low
    hardness (53-60 mg/litre) and alkalinity (49-61 mg/litre) at a copper
    concentration of 6 µg Cu/litre compared with streams of higher
    hardness (150-157 mg/litre) and alkalinity (137-146 mg/litre), and a
    copper concentration of 15 µg/litre.  However, the responses to copper
    were highly variable among taxa.  Tanytarsini chironomids and the
    mayflies  Baetis brunneicolor and  Isonychia bicolor were
    particularly sensitive, whereas Orthocladiini chironomids and
    net-spinning caddisflies were quite tolerant of copper in experimental

         Clements et al. (1988, 1990) conducted a series of
    macroinvertebrate community toxicity tests with copper in
    laboratory- and field-constructed aquatic streams using water from the
    New River in Giles County, Virginia, USA. Rock-filled trays were
    colonized by macroinvertebrates in the New River for 30 days and then
    placed in either the laboratory- or the field-constructed streams. The
    results of the 1988 laboratory experiments indicated that 96-h
    exposures to copper (low dose = 15-32 µg Cu/litre) resulted in a
    reduction in the number of taxa present by 24-36%. In 1990, 10-day
    exposures were performed with copper and laboratory and field results
    compared. The field-constructed streams showed significantly less
    response to copper than the macroinvertebrates in laboratory-housed
    streams. The low dose (11.3 µg Cu/litre) resulted in a 10% reduction
    of taxa and 44% reduction in species abundance in field constructed
    streams as compared to 56 and 75% reduction in laboratory streams,
    respectively. Species of the order Ephemeroptera (mayflies) were the
    most sensitive in both studies.

         A field study was conducted at Shayler Run, a natural stream near
    Clermont County, Ohio, USA, to determine the effects of copper on
    stream biota (Geckler et al., 1976).  A single nominal concentration
    of 120 µg Cu/litre was chosen as the test level.  This value was
    selected because it was thought that it would be high enough to affect
    sensitive fish populations, based on laboratory chronic fish studies
    performed earlier.  This stream was also known to receive sewage from
    a small waste treatment plant 6.5 km (4 miles) upstream from the test
    area.  Measured concentrations in the treated portion of the stream
    during the study ranged from 44.1 to 96.3 µg Cu/litre. All but one
    abundant fish species in the stream and four of the five abundant
    macroinvertebrates were adversely affected by the exposure to copper.

         Leland et al. (1989) exposed oligotrophic streams to copper
    concentrations ranging from 2.5 to 15 µg total Cu/litre (12-75 ng
    Cu2+ activity/litre calculated by computer modelling). Declines in
    population density of species representing all major orders

    (Ephemeroptera, Plecoptera, Coleoptera, Trichoptera and Diptera)
    occurred at 5 and/or 10 µg total Cu/litre.  Herbivores were more
    sensitive to copper toxicity than predators.

         Saward et al. (1975) exposed a marine food chain, comprised of
    phytoplankton, bivalves  (Tellina tenuis) and fish (plaice
     Pleuronectes platessa), to copper at concentrations of 10, 30 and
    100 µg Cu/litre for 100 days.  All copper exposures reduced the
    standing crop and the rate of photosynthesis per unit of chlorophyll
     a in phytoplankton.  Copper adversely affected bivalve condition by
    means of a reduction in carbohydrate reserves and nitrogen levels.
    Fish showed reduced growth; however, no significant change in
    condition or biochemical composition was reported.

    9.3.3  Terrestrial organisms  Plants

         Generally visible symptoms of copper toxicity are small chlorotic
    leaves and early leaf fall.  Growth is stunted, and initiation of
    roots and development of root laterals are poor.  Reduced root
    development may result in a lowered water and nutrient uptake which
    leads to disturbances in the metabolism and growth retardation
    (Balsberg Pĺhlsson, 1989).

     Toxicity to plants grown hydroponically

         Beckett & Davis (1977) stated that yield alone is a poor index of
    toxicity since the height of the plateau depends on many other
    factors.  The toxic effects of a given concentration also depend on
    many factors.  However, the effect on yield of a potentially harmful
    element depends mainly on its concentration in the tissue.  Therefore,
    the tissue concentration of the element at the upper critical level
    should be relatively independent of other factors.  Davis & Beckett
    (1978) grew barley  (Hordeum vulgare), lettuce  (Lactuca sativa), 
    rape  (Brassica napus) and wheat  (Triticum aestivum) in a nutrient
    solution containing copper.  The dry matter yield of these plants was
    independent of the copper concentrations in their photosynthesizing
    tissues, up to a critical concentration (upper critical level).  The
    upper critical concentrations of copper for barley, lettuce, rape and
    wheat were, respectively, 19, 16, 21 and 21 mg Cu/litre.  Beckett &
    Davis (1978) exposed young barley plants to combinations of copper,
    zinc and nickel.  At tissue concentrations in excess of their
    respective upper critical levels the toxic effects of copper and
    nickel appear to be additive, but the combination of copper and zinc
    appears to be antagonistic.

         Taylor & Foy (1985) observed that plants of  Triticum aestivum 
    exposed to 3 mg Cu/litre (50 µmol/litre), as copper sulfate, showed
    acute signs of copper toxicity, including mild necrosis and symptoms
    of induced iron deficiency in the leaves, and inhibition of root
    growth and lateral root initiation.  Plants exposed to 50 mg Cu/litre

    (800 µmol/litre), as copper-EDTA, showed systemic toxicity symptoms
    probably reflecting iron deficiency as the primary toxic effect.
    Leaves showed mild necrosis and symptoms of iron deficiency; root
    growth, although depressed, was not as severely affected as with
    copper sulfate, and lateral root initiation was unaffected.

         Wong & Bradshaw (1982) grew perennial rye grass
     (Lolium perenne) from seed in solutions of copper sulfate for 14
    days.  Copper concentrations of 0.02 mg Cu/litre (0.3 µmol/litre)
    caused a 50% reduction in normal root growth.

         Alva & Chen (1995) examined the effects of 6.35, 317.5, 635 and
    1270 µg Cu/litre (0.1, 5, 10 or 20 µmol Cu/litre) in nutrient solution
    at pH 5.5 on growth, uptake and partitioning of copper by seedlings of
    mandarin  Cleopatra and citrumelo  Swingle rootstock.  There was a
    significant exposure-dependent decrease in both shoot and root dry
    weight.  The concentration of copper in both shoots and roots
    increased with increasing exposure concentration.  The increase in
    tissue copper concentration was more marked in roots than in shoots.
    The pronounced effect of copper on iron uptake could, in part, be
    explained by the development of iron chlorosis symptoms.

         Winter wheat plants  (Triticum aestivum L cv. Starke II) were
    grown for 7 days in split-root chambers containing nutrient solutions
    with various copper chloride concentrations (32 controls) to 635 µg
    CuCl2/litre; 0.5 to 10 µmol/litre).  Average root length and dry
    weight of the root parts exposed to 127-635 µg Cu/litre (2-10
    µmol/litre) decreased, and lateral root initiation was delayed; dry
    weight of root parts increased in control plants.  Copper was not
    exported from the roots to the other plant parts (Adalsteinsson,

         Schmidt (1988) reported IC50s for inhibition of root growth from
    germinated seeds at 1.8 mg Cu/litre for  Lupinus albus and 0.274 mg
    Cu/litre for  Cicer arietinum.

         Burton et al. (1986) studied the interactive effects of copper,
    cadmium and nickel on seedlings of Sitka spruce  (Picea sitchensis) 
    grown in nutrient solution for 42 days.  Copper concentrations of 5
    and 10 mg/litre significantly reduced seedling yield.  There were no
    significant interactive effects on yield even where copper and cadmium
    individually affected yields, but nickel and copper did interact.

         Huber et al. (1989) treated 3-year-old white, Scots and Austrian
    pine seedlings with copper (80 mg/litre, 500 µmol/litre) for up to 90
    days.  Copper significantly affected energy homoeostasis and oleoresin
    production, and induced a loss of tolerance in Scots pine and loss of
    resistance to nematodes in Austrian pine.

         Sela et al. (1988) exposed roots and shoots of
     Azolla filiculoides to copper (10 mg/litre) for 1 day.  Copper
    exposure caused losses of potassium, chloride and magnesium from
     Azolla roots.

         Root growth of seedlings of the  Agrostis capillaris cultivars
    Parys (copper tolerant), Gognian (lead/zinc tolerant) and Highland
    (non-tolerant) was measured after 14 days growth in solutions
    containing 64-762 µg Cu/litre (1-12 µmol/litre).  Highland cultivars
    showed a sharp negative exponential decline in root growth at
    concentrations > 64 µg Cu/litre (1 µmol/litre).  Gognian and Parys
    cultivars are unaffected by copper levels of 64 and 254 µg Cu/litre (1
    and 4 µmol/litre); however, at higher concentrations Parys cultivars
    are less affected by copper than Gognian (Symeonidis et al., 1985).

         Wong et al. (1994) reported that a zinc/lead tolerant cultivar of
     Festuca rubra was tolerant of copper.  The cultivar showed a high
    tolerance index (80.33%) at 50 mg Cu/litre.  A treatment of 50 mg
    Cu/litre appeared to cause little damage to root and shoot elongation.
    Fresh weights of roots were significantly reduced at copper
    concentrations of 10 mg/litre; there was no effect on fresh weight of
    shoots.  Metal transport to the shoot was minimal, indicating that the
    root may play a major role in preventing transportation of copper to
    the upper portion of the plant.

     Toxicity to plants grown in soil

         Toxicity of copper to terrestrial plants grown in soil is
    summarized in Table 24.

         Jarvis (1978) grew perennial ryegrass  (Lolium perenne) in a
    loam soil amended with copper at concentrations of 9.5, 95.3 and 953
    mg/kg (dry weight).  Significant reductions in dry weight of shoots
    and roots over 4 harvests were observed only at the highest

         Graham et al. (1986) grew carrizo citrange seedlings in sandy
    soil amended with copper at concentrations ranging from 25 to 300
    mg/kg as basic copper sulfate.  The growth of seedlings and the
    colonization by the mycorrhizal fungus  Glomus intraadices were
    reduced logarithmically with copper exposure.  Copper-induced
    reductions in seedling phosphorus uptake were more closely related to
    the inhibition of hyphal development outside the root than to the
    development of vesicles and arbuscules in the root.

         Walsh et al. (1972) applied copper sulfate and hydroxide to a
    loamy sand at rates of 15-486 kg Cu/ha for 2 years.  Rates of up to 54
    kg Cu/ha had no adverse effect on the yield of snap beans
     (Phaseolus vulgaris).  Slight decreases were noted at copper
    concentrations in excess of 130 kg/ha and marked reductions in yield
    were observed at 405 kg/ha of the hydroxide and 486 kg/ha of the
    sulfate.  Soil copper concentrations and yield were highly correlated.
    Significant reductions in the yield of snap beans were noted when more
    than 20 mg Cu/kg was extracted from soil with HCl or DTPA and when
    more than 15 mg Cu/kg was extracted with EDTA.

        Table 24.  Toxicity of copper to terrestrial plants grown in soil

    Organism            Parameter           End-point           Concentration             Reference

    Perennial           4 harvests          significant         953 mg/kg                 Jarvis
    grass                                   reduction in                                  et al.
    (Lolium                                 dry weight                                    (1978)
    perenne)                                of shoots
                                            and roots

    Snap beans          2 years             yield               significant decrease      Walsh
    (Phaseolus                                                  at concentrations         et al.
    vulgaris)                                                   > 20 mg/kg extracted      (1972)
                                                                with HCl or DTPA
                                                                or at 15 mg/kg when
                                                                extracted with EDTA

         Gettier et al. (1988) studied the response of corn  (Zea mays) 
    grown in fields amended with six annual applications of
    copper-enriched manure or copper sulfate at application rates ranging
    from 48 to 198 kg Cu/ha.  No significant effect on grain yield was
    observed in a fine sandy loam and clay loam soils.  However,
    applications of copper sulfate (60 and 198 kg/ha) caused significant
    increases in grain yield on silt loam soil.  There was no significant
    accumulation of copper by corn plants during the experiment.

          Cineraria maritima L and  Centauria moschata L were tested for
    their tolerance/sensitivity in metal-rich soils, which contained high
    levels of copper and were derived from iron ore from Lalitpur, Girar,
    India.  The two plant species growing in the mineralized soil showed
    higher accumulations of copper than those grown in non-mineralized
    soils.  The rate of photosynthesis and chlorophyll content were
    reduced in  C. maritima but not in  C. moschata, indicating that
     C. maritima is more sensitive to mineralized soil (Farooqui et al.,
    1995).  Invertebrates

         Neuhauser et al. (1985) exposed earthworms  (Eisenia fetida) to
    copper in both contact and artificial soil toxicity tests.  In 48-h
    contact tests LC50s were 6.7 µg/cm2 for copper acetate, 4.9 µg/cm2
    for copper chloride, 7.4 µg/cm2 for copper nitrate and 6.3 µg/cm2
    for copper sulfate.  There was no significant difference between the
    toxicity of the different copper salts.  In an artificial soil test (2
    weeks) the LC50 was found to be 643 mg Cu/kg.  Spurgeon et al.
    (1994), using the OECD recommended protocol, reported the 14-day LC50
    for  E. fetida to be 683 mg Cu/kg.  The 56-day LC50 and NOEC were
    555 and 210 mg Cu/kg, respectively; the EC50 and NOEC, based on

    cocoon production, were 53.3 and 32 mg Cu/kg, respectively.  Ma (1984)
    estimated that the 6-week LC50 for the earthworm  Lumbricus rubellus
    was 1000 mg Cu/kg (dry weight soil).

         Martin (1986) maintained earthworms  (Allolobophora calignosa) 
    in soil containing copper concentrations ranging from 5 to 1000 mg/kg
    for 14 days.  All worms died at 1000 mg Cu/kg; copper significantly
    reduced growth at 500 mg/kg and reduced the number of egg capsules per
    worm at 100 mg/kg.

         Van Gestel et al. (1989) exposed earthworms
     (Eisenia fetida andrei)  to copper for a 1-week preconditioning
    period followed by a further 3 weeks.  EC50s, based on cocoon
    production, were 62 mg Cu/kg for the pre-conditioning period (1 week)
    and 191 mg Cu/kg for the following 3-week exposure.  A NOEL was
    derived for the whole of the exposure period of 60-120 mg Cu/kg.
    Cocoon hatchability was not affected by copper exposure.  In 12-week
    exposures copper concentrations of 10 and 18 mg/kg stimulated growth;
    the EC50 and NOEC for growth reduction were > 100 and 56 mg Cu/kg,
    respectively (Van Gestel et al., 1991).

         Neuhauser et al. (1984) exposed earthworms  (Eisenia fetida) to
    500, 1000, 2000 and 4000 mg Cu/kg of manure (dry weight) for 6 weeks.
    Copper significantly reduced growth and cocoon production.  Similar
    results were obtained with four different copper salts (acetate,
    chloride, nitrate and sulfate).  The growth rate and reproduction had
    returned to normal after a 6-week period without copper.

         Ma (1984) studied the effects of copper on growth, reproduction
    and litter breakdown in earthworms  (Lumbricus rubellus) during
    6-week exposure periods in sandy or loam soils.  Copper concentrations
    of up to 373 mg/kg did not cause significant mortality.  In sandy soil
    the number of cocoons and litter breakdown were significantly reduced
    at 131 mg Cu/kg; body weight gain was significantly reduced at 372 mg
    Cu/kg.  In loam soil the number of cocoons were significantly reduced
    at 63 mg Cu/kg, litter breakdown at 136 mg Cu/kg and body weight gain
    was unaffected at concentrations up to 373 mg Cu/kg.  Ma (1988)
    calculated 4-week EC50s, based on cocoon production, to be 122, 68
    and 51 mg Cu/kg for the earthworms  Lumbricus rubellus, 
     Aporrectodea caliginosa and  Allolobophora chlorotica, 

         Malecki et al. (1982) exposed earthworms  (Eisenia fetida) to
    six different copper salts for 8 weeks.  Significant reductions in
    growth and reproduction (cocoon production) were observed at copper
    nitrate concentrations of 100 mg/kg (dry weight).  Copper sulfate
    caused significant reductions in reproduction at 100 mg/kg and copper
    chloride reduced growth at 500 mg/kg.  The other salts tested affected
    growth and reproduction at copper concentrations of > 1000 mg/kg.
    The least toxic salt was copper oxide which significantly affected
    growth and reproduction at > 20 000 mg/kg.  Long-term studies (20
    weeks) with copper acetate revealed significant reductions in cocoon
    production at 5000 mg Cu/kg.

         Bengtsson et al. (1986) exposed earthworms  (Dendrobaena rubida)
    to copper concentrations of 10, 100 and 500 mg/kg in soil at varying
    levels of acidity over a 3-month period.  Survival of adults, cocoon
    production and hatching success decreased with increasing acidity; the
    reduction was even greater when low pH (pH 4.5) was combined with
    copper.  Irrespective of pH, 500 mg Cu/kg in the soil rapidly caused a
    collapse of the worm population; survival and cocoon production were
    significantly lower than in controls and hatching failed entirely.

         Streit (1984) exposed orbatid mites and earthworms
     (Octolasium cyaneum) to copper in soil-filled plastic containers for
    6 weeks.  Copper concentrations of up to 200 mg/kg had no significant
    effect on the numbers of the seven predominating orbatid mite species.
    However, at copper concentrations of 40 mg/kg the earthworms either
    had been killed or had migrated to the noncontaminated half of the
    container.  The authors state that the toxicity of copper to
    earthworms depends on the particular soil type and in particular the
    organic carbon content of the soil.  For example, 4-day LC50s were
    reported at 181 mg Cu/kg in poor organic soil (3.2% carbon) and 2760
    mg Cu/kg in peat soil (42.6% carbon).

         Denneman & Van Straalen (1991) exposed the oribatid mite
     (Platynothrus peltifer) to dietary copper in 3-month reproduction
    tests.  Copper concentrations of up to 2000 mg/kg (dry weight) had no
    significant effect on survival.  The NOECs for growth and reproduction
    were found to be 9.42 and 2.65 µmol Cu/g, respectively.

         Donkin & Dusenbery (1993) exposed nematodes
     (Caernorhabditis elegans) to copper in different soil types in 24-h
    toxicity tests.  LC50s were 70, 534, 413, 1061 and 629 mg Cu/kg for
    sand, sandy loam (66% sand), sandy loam (55% sand), loam and clay
    loam, respectively.  An LC50 for nematodes exposed in water only was
    105 mg Cu/litre.

         Parmelee et al. (1993) incubated forest soil treated with copper
    (100, 200, 400 and 600 mg/kg) for 7 days.  Omnivore-predator nematodes
    and mesostigmatid and orbatid mites were the groups most sensitive to
    copper and were significantly reduced at copper levels of 100 mg/kg.
    Total nematode and microarthropod numbers declined significantly at
    copper concentrations above 200 mg/kg.  Trophic structure analysis
    revealed that the high sensitivity of nematode predators reduced
    predation and resulted in significantly increased numbers of nematodes
    at 200 mg Cu/kg.

         Marigomez et al. (1986) fed terrestrial slugs  (Arion ater) on a
    diet containing copper concentrations ranging from 10 to 1000 mg/kg
    for 27 days.  No treatment-related effect on mortality was observed.
    Copper concentrations of 100 mg Cu/kg or more showed an exponential
    change in feeding with treated slugs eating less than controls.
    Initially the feeding behaviour of slugs on a diet of 50 mg Cu/kg was
    unaffected, but by the end of the 278-day treatment they showed the
    same reduction in feeding as the higher exposures.

         Bayley et al. (1995) exposed larvae of the carabid beetle
     (Pterostichus cupreus) to copper in both the soil (500 mg Cu/kg) and
    in their food (500 mg Cu/kg fresh weight; 1357 mg Cu/kg dry weight).
    Larval mortality due to copper exposure was 69% when adjusted for
    control mortality and mainly occurred during larval metamorphosis and
    pupation.  The locomotor behaviour of male and female adult beetles
    surviving the exposure to elevated copper during larval development
    was significantly impaired.

         Gintenreiter et al. (1993) reared gypsy moth  (Lymantria dispar)
    larvae on an artificial medium contaminated with copper (10, 50, 250
    and 1250 mg/kg) from hatching or the fourth instar stage to pupation.
    All larvae died at 1250 mg Cu/kg and larval survival was significantly
    reduced at 250 mg Cu/kg.  Contamination from hatching generally
    resulted in a decrease in headcapsule width and this was significant
    at a copper concentration of 50 mg/kg.  The number of larvae hatched
    per egg cluster was significantly reduced at 50 mg Cu/kg.  NOECs were
    10 mg Cu/kg for development rate, growth and reproduction, and 50 mg
    Cu/kg for mortality in larvae exposed from the first instar.  NOECs
    were 10 mg Cu/kg for reproduction, and 50 mg Cu/kg for development
    rate, mortality and growth in larvae exposed from the fourth instar
    stage.  Ortel et al. (1993) reared moth larvae on diets containing
    copper at 10 or 50 mg/kg.  No correlation was found between the extent
    of copper contamination and parasitization success by the braconid
    wasp  (Glyptapanteles liparidis).

         Nectoux & Bounias (1988) dosed honeybees  (Apis mellifera) with
    sucrose solutions containing copper at concentrations ranging from 250
    to 2000 mg/litre.  Controls gave an LT50 of 27 days with a daily
    percentage mortality at 3%.  LT50s for dosed bees ranged from 5.1 to
    14.2 days with the daily percentage mortality ranging from 5.78% per
    day to 30.22% per day.  Vertebrates

         Dodds-Smith et al. (1992a,b) maintained shrews  (Sorex araneus) 
    on a diet containing copper at an intake of 2.13 mg/day for 12 weeks.
    There was no significant effect on growth rate during the feeding
    trial and no relationship between copper intake and mortality.

         Aulerich et al. (1982) fed young mink on a diet containing 0, 20,
    50, 100 or 200 mg Cu/kg for 153 or 357 days.  The shorter exposure did
    not significantly affect haemoglobin or haematocrit levels.
    Reproduction performance was not adversely affected, although greater
    mortality in young mink and reduced litter mass were a result of the
    higher copper exposures.  Intraperitoneal LD50s for adult mink were
    7.5 mg/kg for copper sulfate and 5.0 mg/kg for copper acetate.

         A summary of the toxicity of copper in domestic animals was
    published by the committee on animal nutrition of the National
    Research Council (1980).  The information presented indicates that

    sheep are more sensitive to copper than other domestic animals, and
    horses appear to be more tolerant than cattle, pigs, sheep or poultry.
    This is in agreement with the results presented by Smith et al. (1975)
    who fed yearling ponies a pelleted diet up to 791 mg Cu/kg with no
    visible effects after 6 months.

         The results of Hill & Williams (1965) showed that a dietary
    concentration of 266 µg Cu/g (dry weight) slightly reduced the
    live-weight gain in lamb.  At 40.7 µg Cu/g the reduction in the rate
    of live-weight gain was statistically significant.

         The use of copper as a feed additive for growth stimulation has
    attracted interest in its toxic effects.  Combs et al. (1966) studied
    the effect of the level of dietary protein in pigs fed high copper
    rations.  Their results indicated that pigs fed a diet containing 250
    ppm (mg/kg) copper and either 14 or 22% protein had daily gains of
    0.80 and 0.78 kg, respectively, while those given diets containing 500
    ppm (mg/kg) copper and either 14 or 22% protein gained 0.48 and 0.55
    kg daily, respectively.

    9.4  Field observations

    9.4.1  Microorganisms

         Mathur et al. (1979) studied a 2-ha field comprising an organic
    muck soil which had been cultivated for 45 years, having residual
    fertilizer copper applied to a distinct site for the last 15 years.
    The soil copper content ranged from 150 to 260 mg/kg (dry weight).
    The rate of carbon dioxide evolution was significantly negatively
    correlated with both the total and extractable copper contents of the
    soil.  Acid phosphatase activity significantly decreased as copper
    content increased.  Dumontet et al. (1992) reported a significant
    negative correlation between both copper and cadmium, and soil
    respiration in the 0-15-cm layer of contaminated soil from the
    vicinity of a copper-zinc smelter.  However, the authors did not find
    a significant correlation between soil copper and acid phosphatase

         Minnich & McBride (1986) studied five soils which had received
    anthropogenic copper inputs for many years: recent sludge, aged
    sludge, spillsite, vineyard and muck.  The copper concentrations in
    the soils ranged from 33 to 1445 mg/kg soil (dry weight).  No
    significant effects due to copper on carbon mineralization were
    detected.  Nitrogen metabolism was significantly increased only in the
    soil which had received recent sludge additions.

         Burton (1987) surveyed river water and sediment samples and one
    soil sample for bacterial resistance to metals.  Between 0 and 2.4% of
    bacterial populations sampled were resistant to 1 mmol Cu/litre.  The
    highest resistance was found at Crater Lake, Colorado, USA.  However,
    sediment copper concentrations were much higher at several other

    9.4.2  Aquatic organisms

         Wood (1983) found that naturally occurring marine phytoplankton
    populations show a tolerance to added cupric ions which far exceeds
    the physiological limits of phytoplankton cultures grown in chemically
    defined media.  The tolerance appears to be due to regulation of
    bioavailability of added copper by an abundance of copper-complexing
    agents.  Coastal phytoplankton were less sensitive than continental
    shelf or oceanic communities.  The toxicity of copper correlated more
    with copper-complexing capacity than with biotic species composition
    or community structure.

         Effler et al. (1980) monitored the impact of low-level copper
    applications to Cazenovia Lake, New York, USA.  The application caused
    only small increases (up to 5 µg Cu/litre) for 2-5-day periods.  The
    treatment did not achieve the desired algicidal action on the target
    phytoplankton.  There were short-term alterations in the seasonal
    succession processes within phytoplankton populations.  No significant
    effects on zooplankton or submerged macrophytes were observed.

         Hanson & Stefan (1984) summarized the effects of nearly 60 years
    of copper use as an algicide on some lakes in Minnesota, USA.  Copper
    treatments have been as high as 250 µg Cu/litre averaged over the
    entire lake.  Algal mortality has led to hypolimnetic oxygen depletion
    and affected nutrient dynamics.  Phytoplankton populations have
    shifted to greater dominance of blue-green algae.  Several major fish
    kills related to transiently high copper concentrations have been
    documented, and fish populations have shifted to less desired species.
    Sediment concentrations as high as 5600 mg Cu/kg have developed,
    affecting development of macrophytes and benthic invertebrate

         Carlson et al. (1986) investigated the effects of copper on the
    Naugatuck River, Connecticut, USA, which received multiple discharges
    from domestic and industrial sources.  Downstream from the major
    effluents, there were severe effects on fish, periphyton, and benthic
    invertebrates.  Average total copper concentrations at sampling
    stations in the affected area ranged from 50 µg Cu/litre to over 400
    µg Cu/litre, whereas stations with little or no impact had average
    concentrations < 20 µg Cu/litre.  Toxicity tests using river water
    showed reduced survival of  Ceriodaphnia dubia in samples from
    affected areas, but little or no effects from samples from unimpacted
    areas. The US EPA water quality criteria for the river varied from 5
    to 12 µg Cu/litre for 4-day average concentrations and from 7 to 18 µg
    Cu/litre for 1-h average concentrations.  These concentrations were
    exceeded even in the unaffected area.  However, toxicity tests using
    dilution water from the river showed copper to be 3-8-fold less toxic
    than did dilution water typical of laboratory tests used to establish
    the criteria.  Therefore, site-specific criteria were estimated to be
    24-43 µg Cu/litre for the 4-day average and 34-61 µg Cu/litre for the
    1-h average.  These concentrations were rarely exceeded upstream from
    the major discharges, but were routinely exceeded downstream,
    consistent with the observed biological impact.

        Grant et al. (1989) studied the tolerance of polychaete worms
     (Nereis diversicolor) to copper.  Polychaete worms collected from a
    site with surface sediment levels of 1733 mg Cu/kg were more tolerant
    when exposed to 500 µg Cu/litre in acute tests than those from a
    low-metal site (19 mg Cu/kg).  LT50 values were 70 h for worms from a
    low-metal site and 1407 h for those from the contaminated site.
    Laboratory-bred worms still retained the tolerance.  Worms showed a
    graded level of tolerance depending on field exposure.

         Han & Hung (1990) reported the case of green oysters
     (Crassostrea gigas) in the Charting mariculture area of southwestern
    Taiwan in January 1986.  The green colouration was found to be due to
    high copper content of the oyster tissue.  A survey of the area
    revealed total dissolved copper levels ranging from 5 to 23.6
    µg/litre, particulate copper levels ranging from 1 to 5.5 µg/litre and
    oyster tissue levels with a mean of 4401 mg Cu/kg (dry weight).  Green
    oysters have occasionally been observed in other areas.

         Copper-rich granules have been reported to occur in a wide
    variety of invertebrates inhabiting copper-polluted habitats.  Weeks
    (1992) observed copper-rich granules in the ventral caeca of talitrid
    amphipods  (Orchestia gammarellus) revealed by transmission electron
    microscopy.  In addition, copper deposits also appear in the physodes
    of  Fucus vesiculosis and  F. serratus (Smith et al., 1986).  The
    occurrence of intracellular deposits containing copper were reported
    in a copper-tolerant isolate of the green alga  Scenedesmus 
    (Silverberg et al., 1976).  In the latter case the metal appeared
    mainly in the nucleus, although similar structures were observed in
    the cytoplasm.  Silverberg and co-workers concluded that the
    occurrence of these inclusions could be regarded as a detoxifying
    mechanism because they were absent in the non-tolerant strains.
    Copper has been detected in polyphosphate bodies in the green alga
     Chlorella furca (Wong et al., 1994) and in the fouling diatoms
     Amphora and  Navicula (Daniel & Chamberlain, 1981).

    9.4.3  Terrestrial organisms  Tolerance

         Duvigneau & Denaeyer-De Smet (1963) studying the copper content
    in leaves of plant species growing on soils containing 500 mg Cu/kg
    (dry soil) emphasize that there is more than one tolerance mechanism
    operating.  They found that some species avoid copper toxicity by
    excluding the metal, some species accumulate the metal to very high
    concentrations and other species occupy an intermediate position.
    They conclude that there may be both exclusion and accumulation
    mechanisms evolving in different species of the same genus.

        Wu & Kruckeberg (1985) found that two legume plants
     Lupinus bicolor and  Lotus purshianus growing on copper mine waste
    in northern California, USA, with a mean soil copper content of 460
    mg/kg exhibited considerably greater copper tolerance at 0.2 mg/litre
    in nutrient solution than plants from an adjacent meadow where copper
    levels were 0.1-1.5 mg/kg.  The tolerance index of the field-collected
    plants was positively correlated with the copper concentration of the
    soil from which the plants were collected.  Wu & Lin (1990) isolated
    the nitrogen-fixing bacterium  Rhizobium loti from root nodules of
     L. purshianus growing on the copper mine and found it to have
    greater copper tolerance than rhizobium isolated from plants in a
    nearby field.  No difference was detected in uptake pattern or
    concentration of copper in tolerant and nontolerant  L. purshianus. 
    However, a copper accumulation mechanism associated with tolerance was
    found in the symbiotic rhizobium.  Effective nitrogen fixation was
    seen in copper-enriched soils.

         Kruckeberg & Wu (1992) investigated the tolerance of herbaceous
    plants colonizing copper mine waste sites in northern California, USA.
    Five of the seven species tested showed elevated copper tolerance.
    The copper-tolerant species were found at more than one copper mine.
    The mines were geographically isolated, so tolerance in these plant
    species probably evolved independently.  In  Arenaria douglasii, 
     Bromous mollis and  Vulpia microstachya the exclusion of copper
    from the shoots partly by immobilization at the roots may be a
    mechanism of tolerance.  However, in some species there were no
    differences between the uptake of copper into tissues of tolerant and
    nontolerant species.  Therefore, it would appear that different
    mechanisms of copper tolerance have evolved among the plant species
    colonizing California copper mine waste sites.

         Dickinson et al. (1991) studied the survival of sycamore
     (Acer pseudoplatanus) trees at a metal-contaminated site (copper
    refinery) in northwest England where populations of the herbaceous
    flora have evolved metal tolerance.  Cell culture growth experiments
    on explant material from shoot meristems of mature trees showed
    increased tolerance to copper.  Some of these trees predated the
    establishment of the refinery.  However, tolerance tests on tree
    seedlings showed no evidence that the trees produce tolerant
    offspring.  The tolerance of the mature trees is ascribed to
    phenotypic adaptation induced during the life of the tree as site
    contamination occurred.

         Taylor & Crowder (1984) did not find copper tolerance in the
    cattail rush  (Typha latifolia) collected from the vicinity of a
    copper smelter near Sudbury, Ontario, Canada, with soils contaminated
    with 3738 mg Cu/kg and 9372 mg Ni/kg.  Growth of both contaminated and
    non-contaminated plants was inhibited by 100 mg/kg copper-EDTA.  No
    particular  in vivo copper tolerance was found in the clones from the
    heavily contaminated site.  The metals at this site are believed not
    to be bioavailable owing to the strongly anaerobic waterlogged
    conditions or to the presence of high sulfide levels in the mud.

         Rauser & Winterhalder (1985) collected several grass species from
    the vicinity of the Sudbury copper smelter (Ontario, Canada).  They
    found clones of the grass species  Deschampsia caespitosa, 
     Agrostis gigantea and  Poa compressa to be tolerant to copper.
     Hordeum jubatum plants showed no tolerance to copper exposure.

         Frenckell-Insam & Hutchinson (1993) found copper tolerance in
    populations of the grass  Deschampsia cespitosa collected at seven
    Canadian or German mine sites, with some Sudbury area plants
    performing better in the presence of normally toxic levels of copper.
    Schultz & Hutchinson (1988) showed that this copper tolerance in
     D. cespitosa was not due to a metallothionein-like protein.

         Wainwright & Woolhouse (1977) studied three clones of
     Agrostis tenuis with respect to the effects of copper (64, 6.4 and
    0.64 mg/litre; 10-1, 10-2 and 10-3 mmol/dm3) in nutrient solution on
    growth of root segments excised from the zone of cell elongation.
    Growth (24 h) of copper-tolerant and zinc-tolerant clones was less
    inhibited by copper than was the growth of a non-tolerant clone.
    Concentrations of copper ions which inhibited root growth also caused
    leakage of potassium ions from cells.  The authors suggest that the
    loss of potassium ions from roots is due to the toxic effect of copper
    ions on the plasmalemma.  Copper fungicides and fertilizers

         During perennial tree production, amendments to soil of trace
    elements essential for growth are often necessary.  However,
    application of such amendments results in the accumulation of elements
    in topsoil.  Copper can also build up as a result of routine
    application of copper at relatively high rates with fertilizers.  In
    addition, the routine spraying of copper-based fungicides can result
    in copper accumulation.  Reuther & Smith (1953) reported an increasing
    number of Florida, USA, citrus orchards on sandy, acid well-drained
    soils affected with a chlorotic disorder of the foliage.  The authors
    linked this disorder with possible effects of high copper levels.

         Paoletti et al. (1988) reported that spring/summer fungicide
    (Bordeaux mixture) treatment of vineyards in Italy caused a decrease
    in the local population of earthworms.  In particular, a decrease in
    the number of juvenile  Allolobophora was recorded.  Other macro
    invertebrates were unaffected, both in terms of biomass and number of
    species, by fungicide applications.


    10.1  Concepts and principles to assess risk of adverse
          effects of essential elements such as copper

    10.1.1  Human health risks

         There are risks associated with low intakes as well as high
    intakes of essential elements.  The relationship between
    intake/exposure level and risk therefore has a U-shaped curve, with
    risks from deficiency at low intakes and risk of toxicity at high
    intakes (see Fig. 2).  There is a need to define an intake range that
    prevents both deficiency and toxicity for the general population.  The
    range of acceptable intakes to meet the biological requirement, as
    well as prevent risk of toxicity, may be extremely narrow.  A balanced
    and comparable scientific approach to assess risk from deficit as well
    as excess is needed when evaluating essential elements such as copper.

    10.1.2  Homoeostatic model

         The homoeostatic model describes an acceptable range of exposures
    or intake (AROI, acceptable range of oral intake) for essential trace
    elements that permits optimum health (Fig. 2).  Environmental levels
    of copper within the acceptable range of exposure do not produce
    adverse health effects among members of the general population.
    However, there are individuals or groups with disorders in
    homoeostatic mechanisms that experience health effects, either
    deficiency or toxicity, from exposures within the acceptable range.
    These disorders may be of genetic origin or from acquired disease.

    10.2  Evaluation of risks to human health

    10.2.1  Exposure of general population

         For healthy humans who are not occupationally exposed the major
    route of exposure to copper is oral.  The mean daily dietary intake of
    copper  in adults ranges between 0.9 and 2.2 mg  (see section 5.4). A
    majority of studies have found intakes to be at the lower end of that
    range.  The variation reflects different dietary habits, as well as
    different agricultural and food-processing practices used throughout
    the world.  Drinking-water may make a substantial additional
    contribution to the total daily intake of copper, particularly in
    households where corrosive waters have stood in copper pipes.  In
    homes without copper piping, or with noncorrosive water, copper intake
    from drinking-water seldom exceeds 0.1 mg/day, although intakes
    greater than a few mg per day can result from corrosive water
    distributed through copper pipes.  In general, total daily oral
    intakes are between 1 and 2 mg Cu/day, although they may occasionally
    exceed 5 mg Cu/day.  All other routes of copper exposure (inhalation
    and dermal) are insignificant in comparison to the oral route.
    Inhalation adds between 0.3 and 2.0 µg Cu/day from dusts and smoke.
    Women using copper IUDs are exposed to only 80 µg Cu/day or less from
    this source.

    FIGURE 3

    10.2.2  Occupational exposures

         Under occupational conditions where exposure to airborne copper
    is controlled at or below the widely accepted standard of 1 mg Cu/m3,
    and assuming a shift inspiratory volume of 10 m3 and a usual
    workplace distribution of particle sizes, an estimated intake of 8.5
    mg Cu/day can be calculated for occupational sources.  This may be an
    important contribution to daily intake; however, this is a worst-case
    estimate using default values and present occupational conditions
    rarely lead to this level of exposure.

    10.3  Essentiality versus toxicity in humans

    10.3.1  Risk of copper deficiency

         Clinically evident copper deficiency in adults is rarely found in
    the general population.  However, recent dietary surveys show that the
    mean population intake is suboptimal. In some regions of the world,
    such as Europe and the USA, intakes are about 20% below the
    recommended levels.  The health consequences of marginally adequate
    intakes remain to be determined.

         Infants with low birth weight (up to 15% of children worldwide)
    are particularly at risk for deficiency.  Frequent episodes of
    diarrhoea are another risk factor leading to copper deficiency. Copper
    deficiency commonly occurs during the recovery from protein energy
    malnutrition, since these infants grow rapidly and are usually fed
    diets that supply inadequate copper.

         Based on dietary interactions, individuals taking supplements of
    zinc and ascorbic acid are at risk of developing copper deficiency.

         Malabsorption states associated with copper deficiency include
    chronic diarrhoea, short bowel syndrome, partial gastrectomy, coeliac
    disease, sprue and cystic fibrosis.

         Patients receiving prolonged intravenous nutrient mixtures which
    lack sufficient copper may develop symptomatic evidence of copper

         Menkes disease (see chapter 8) is a rare (approx. 1 : 200 000)
    X-linked recessive disorder which results in a defect in the
    intestinal absorption of copper.  This disorder leads to a severe,
    symptomatic, fatal deficiency state even at copper intakes above the

         Copper deficiency has been implicated as a possible risk factor
    in the pathogenesis of cardiovascular disease.

    10.3.2   Risk from excess copper intake  General population

         When copper homoeostatic control is defective and/or copper
    intake is excessive, copper toxicity may occur.  Ingestion of excess
    copper is infrequent in humans and is usually a consequence of the
    contamination of beverages (including drinking-water) or from
    accidental or deliberate ingestion of high quantities of copper salts.
    Effects which occur at lowest levels are those on the gastrointestinal
    tract; for example, nausea, vomiting and diarrhoea.  Doses which
    induce such effects have not been well characterized and confounders
    such as microbiological quality of water supplies or other potential
    causes of the symptoms have not been adequately considered.  On the
    basis of available data, gastrointestinal illness appears to be
    associated with consumption of drinking-water containing several
    mg/litre of copper, but it is not possible to provide a precise
    number.  Symptoms disappear following a change of water supply.

         Wilson disease (hepatolenticular degeneration; see chapter 8) is
    the most common (approx. 1 : 30 000) inherited disorder of copper
    metab olism.  The mode of inheritance is an autosomal recessive trait
    which results in decreased biliary excretion of copper and in hepatic
    accumulation of the metal.

         Other extremely rare conditions such as ICC and ICT are
    characterized by copper accumulation in early childhood.  The relative
    contribution of genetic factors and/or elevated environmental exposure
    to copper remains undefined.

         A more common cause of copper accumulation in the liver is
    chronic liver disease associated with chronic cholestasis. These
    disorders include primary biliary cirrhosis, primary sclerosing
    cholangitis, extrahepatic biliary obstruction or atresia and
    intrahepatic cholestasis of childhood.  In these conditions, copper
    accumulation does not appear to be primarily responsible for hepatic

         Copper or copper salts may induce allergic contact dermatitis in
    susceptible individuals.

         There is no convincing evidence of an association between
    increased dietary copper intake and cardiovascular disease.

         Available data in humans and animals are inadequate to assess the
    reproductive/developmental effects of copper compounds.

         There is no convincing evidence that copper plays an aetiological
    role in the development of cancer in humans, on the basis of available
    epidemiological data and limited experimental data in animals.  The
    weight of evidence from  in vitro and  in vivo assays indicates that
    copper sulfate is not genotoxic.  Occupational risks

         Studies of populations of copper workers have failed to
    demonstrate systemic copper toxicity or significant excess of cancer.
    No occupational studies were found to indicate that copper exposures
    resulted in reproductive or developmental effects.

         In occupational settings, acute effects are limited to metal fume
    fever.  This condition has been produced by inhalation of fresh copper
    fume at air concentrations above 0.1 mg Cu/m3.  Similar responses to
    very finely divided copper metal and oxide dusts have been reported
    where conditions probably resulted in unusually high dust

         Chronic effects involving the liver have been reported in workers
    whose exposures were uncontrolled and likely to have been high.

    10.4  Evaluation of effects on the environment

    10.4.1  Concept of environmental risk assessment

         The science of performing environmetal risk assessments has
    evolved rapidly in recent years, with standardized techniques being
    adopted in both the USA and Europe (US EPA, 1992; OECD, 1995).  The
    key components of environmental risk assessment paradigms include
    problem formulation, analysis (which includes both exposure and
    effects analysis), and risk characterization.

         Problem formulation consists of defining the risk problem,
    assessing the population, community, or ecosystem at risk,
    establishing the model for evaluating the potential for risk and
    selecting the biological end-points and environmental media to
    analyse.  The analysis phase consists of performing detailed studies
    designed to characterize the spatial and temporal concentrations of
    the chemical of interest.  Additionally, a series of standardized
    laboratory and, in some cases field studies, are performed to evaluate
    the toxicity dose-response curve for selected end-points and species
    of interest.  The risk characterization phase integrates the exposure
    and effects data, determines the potential for co-occurrence between
    organism and contaminant and comes to a conclusion about the potential
    for risk.  The risk statement can be made in terms of a probability
    statement, frequency of time effects are expected to occur or number
    of species to be affected.  Risk is assessed by determination of the
    adequacy of the margin of safety between effects and exposure
    concentrations, and expert judgement is typically used to determine
    the acceptability of the perceived margin of safety.  There is a
    general consensus that the larger the margin of safety the lower is
    the environmental risk.  Margins of safety less than 1.0 are usually
    indicative of a higher potential for risk and may require further

    10.4.2  Components of risk assessment process for copper

         The principal components of risk assessment are exposure and
    effects characterization.  The environmental exposure has been
    assessed by reviewing the fate (transport, distribution and behaviour)
    from the point of release into and through the environmental
    compartments of air, water soil/sediment and biota. Toxicity tests
    with copper have been done on representative species of the trophic
    levels in the ecological community of interest, including algae and
    plants (primary producers), aquatic and terrestrial invertebrates
    (secondary producers) and fish and terrestrial animals (consumers).

         Since copper is an essential micronutrient, a lower limit exists
    below which deficiency will occur (see section 10.1). Thus the use of
    large safety factors in procedures to limit exposures to below toxic
    levels might result in target concentrations below essential levels.
    This potential problem has been addressed in the deficiency toxicity
    optimum concentration band for essential elements (DT-OCEE) concept
    (van Tilborg, 1996).  Because copper is a ubiquitous trace metal in
    the natural environment, it is unlikely except in some terrestrial
    regions where copper concentrations are very low, or where
    antagonistic molybdenum interactions occur, that deficiency will be a
    significant issue in the environment.  In view of these concepts, the
    environmental risk assessment paradigm for essential elements such as
    copper must (as for humans, see section 10.1.1) be expressed as a
    deficiency-toxicity model which describes an acceptable concentration
    range for copper in the environment.

         One of the key questions in ecotoxicology is to what extent
    laboratory tests in defined media under carefully controlled
    conditions are predictive of effects that will be seen in the
    environment.  Traditional toxicity testing has in the past focused on
    the acute (mortality) and chronic (e.g. growth and reproduction)
    effects of chemicals on the life stages of representative aquatic and
    terrestrial organisms.  In recent years it has been realized that the
    environmental chemistry, especially in relation to metal speciation
    and complexation, will have a signficant influence on and be a
    determinant of the outcome of laboratory toxicity tests as well as the
    effects actually seen in the environment. Several papers cited in
    chapter 9 report this circumstance (see section 9.1), which has been
    generalized in an hypothesis which describes the bioavailability of
    copper.  This has led to the now accepted view that the total copper
    in the environmental medium is not a good predictor of its
    bioavailability.  Acceptance of this concept also leads to the logical
    conclusion that the risk assessment of copper should ideally be made
    on a site-specific basis.

         Organisms may also become adapted at a local scale by
    physiological acclimation and possibly genetic changes.  Because of
    such adaptations the test-derived toxicity values will be elevated
    compared to the values for the same species from a nonadapted

    population.  On this basis it is essential that the risk assessment of
    copper should be made on a site-specific basis.

    10.5  Environmental risk assessment for copper

         For the purposes of characterizing the potential risk of copper
    to the environment there are limited data available for perfoming a
    detailed risk assessment for each environmental medium (air, water,
    soil, sediment).  The largest data set is available for the aquatic
    environment.  The intent of this section is to evaluate the available
    biological effects and exposure data for various organisms and media
    consistent with this risk paradigm, and describe ranges of
    concentrations where the potential for risk increases.

    10.5.1  Aquatic biota  Overview of exposure data

         Natural freshwater streams normally have total dissolved copper
    concentrations in the range of 1.0-20 µg/litre.  Open ocean surface
    waters contain 0.02-0.2 µg Cu/litre, although near-shore seawater may
    have copper concentrations as high as 1.0 µg/litre.  In the ocean,
    copper concentration increases with depth.  These natural copper
    levels can be increased by anthropogenic input; for example, acid mine
    drainage increased the copper concentration up to 600 µg/litre in
    Restronquet creek, United Kingdom, and Chesapeake bay, USA, can have
    copper levels as high as 80 µg/litre as a result of shipping activity.

         The toxic effect of copper on aquatic biota is critically
    dependent on the bioavailability of copper in water, which in turn
    depends on the physicochemical form (i.e. speciation) of the copper.
    The bioavailability is decreased by the complexation and adsorption of
    copper by natural organic matter, iron and manganese hydrated oxides,
    and chelating agents excreted by algae and other aquatic organisms.
    Toxicity can also be affected by pH and hardness.  For these reasons,
    total copper is rarely useful as a predictor of toxicity.  Studies
    have shown that in natural seawater more than 98% of copper is bound
    by organic matter and in rivers a high percentage is often organically
    bound, but the actual percentage depends on the dissolved organic
    concentration of the river water and its pH.  Overview of toxicity data

         Copper exhibits significant toxicity to some aquatic organisms,
    although the degree of toxicity is highly variable and the
    bioavailability of copper dictates its toxicity to a large extent.

         Some algal species are very sensitive to copper.  EC50 values as
    low as 47 µg/litre total dissolved copper have been reported for 96-h
    growth rate experiments, but for other algal species EC50 values up
    to 481 µg/litre have been found.  However, it is possible that many of
    the high EC50 values in the literature are the result of the growth
    rate experiments being carried out in culture media containing

    copper-complexing agents such as silicate, iron, manganese and EDTA,
    which reduce the bioavailability of copper.

         Acutely lethal copper concentrations to aquatic invertebrates
    range from several µg/litre to several mg/litre.  The 48-96-h LC50s
    of copper ranged from 7 to 54 µg/litre for  Daphnia magna, 37 to 183
    µg/litre for amphipods, 58 to 112 µg/litre for gastropods and 50 to
    100 µg/litre for crab larvae.  Sublethal effects and effects on
    longer-term survival have been reported in a variety of invertebrate
    species for copper concentrations from about 1 µg/litre to a few
    hundred µg/litre.  For high bioavailability waters, effect
    concentrations for several sensitive taxa can be < 10 µg Cu/litre.

         Acutely lethal copper concentrations for fish range from a few
    µg/litre to several mg/litre, depending greatly both on the test
    species and exposure conditions.  Acute LC50s less than 50 µg
    Cu/litre for fish generally are associated with test waters with low
    DOC, low hardness, and neutral to slightly acidic pH.  Sublethal
    effects and effects on longer-term survival have been reported from 1
    µg/litre to a few hundred µg/litre, with effects less than 50 µg
    Cu/litre being reported for several species.  Again, lower effect
    concentrations are generally associated with test waters of high

         Because of the variability of toxic effects concentrations among
    different biological taxa and exposure conditions, the expected
    response of aquatic communities will be highly site specific.  Table
    25 provides a general summary of the nature of response expected for
    various concentration ranges at sites with moderate to high
    bioavailability similar to water used in most toxicity tests.

    10.5.2   Terrestrial biota  Overview of exposure data

         Copper in uncontaminated soils in Europe, USA, Canada and
    elsewhere has been measured as total and extractable and with depth in
    soils.  The range of copper concentrations in such soils varies
    between 0.3 and 250 mg/kg (Bowen, 1985; Adriano, 1992) with soil type
    being a factor in determining the levels found.  Peaty and organic
    soils are at the upper end of this range, as are loams; sandy soils
    are at the low end.

         Any anthropogenic addition to the surface of such soils, whether
    by fertilizer or fungicide applications or from highway dust or
    airborne deposition from urban and industrial sources, causes sharp
    increases in the copper levels of such soils.  Copper added to provide
    adequate levels for citrus crops or in orchards and vineyards from
    fungicide and insecticide application causes a buildup in soils
    (150-400 mg Cu/kg).

        Table 25.  Responses expected for various concentration ranges of coppera

    Total dissolved        Effects of high bioavailability in water
    Cu concentration
    range (µg/litre)

    1-10                   significant effects are expected for diatoms and sensitive
                           invertebrates, notably cladocerans.  Effects on fish could be
                           significant in freshwaters with low pH and hardness

    10-100                 significant effects are expected on various species of
                           microalgae, some species of macroalgae, and a range of
                           invertebrates, including crustaceans, gastropods and sea
                           urchins. Survival of sensitive fish will be affected and a variety
                           of fish should show sublethal effects

    100-1000               most taxonomic groups of macroalgae and invertebrates will
                           be severely affected. Lethal levels for most fish species will
                           be reached

    > 1000                 lethal concentrations for the most tolerant organisms are

    a  Sites chosen have moderate to high bioavailability similar to water used in
       most toxicity tests.

         Mining and smelting activities, especially from copper or
    copper-zinc smelters, often cause surface soil levels to exceed 1000
    mg Cu/kg.  Plant foliar levels

         Generally, vegetation rooted in soils reflects the soil copper
    levels in its foliage.  This is dependent upon the bioavailability of
    the copper, and the physiological requirements of species concerned.
    On uncontaminated soils foliar levels vary broadly in the range 6.1-25
    mg/kg.  For grazing animals and for much of the food chain, plant
    foliar levels are of concern.

         On soil contaminated by copper additions (in the range of 150-450
    mg Cu/kg), the foliar levels may reach 80 mg Cu/kg, and in mining and
    smelting area the copper level in foliage can reach 300 mg/kg.
    Specific hyperaccummulator species can have foliar levels to 17 000 mg
    Cu/kg without adverse symptoms.

         On normal forest soils the nonrooted plants can have higher
    copper concentrations. These species include mosses and lichens.  The
    fruiting bodies and mycorrhizal sheaths of soil fungi associated with
    higher plants in forests often accumulate copper to much higher levels
    than the higher plants at the same site, e.g. Lepp (1992) reports
    copper concentrations in fungi up 469 mg/kg while foliage levels of
    5-20 mg Cu/kg were measured at the same site.  Assessment of toxicity of copper in soil

         Leaving aside the question of copper surface mineralization, at
    the normal soil concentrations reported (0.3-250 mg Cu/kg) plants
    rarely if ever show symptoms of toxicity or of adverse growth effects.
    Crops are often more sensitive to copper than the native flora, so
    protection levels for agricultural crops range from 25 mg Cu/kg to
    several hundred mg/kg, depending on the country.  Chronic and or acute
    effects on sensitive species do occur at copper levels occurring in
    some soils as a result of human activities, e.g. copper fertilizer
    addition, fungicide spraying, sludge additions (50-150 mg Cu/kg).

         When soil levels rise above 150 mg Cu/kg we begin to find more
    and more native and agricultural species showing chronic effects.
    Soils in the range 500-1000 mg Cu/kg act in a strongly selective way,
    allowing only survival of copper-tolerant species or strains.  A
    reduction in species diversity occurs.  By the time soil levels reach
    2000 mg Cu/kg a high number of species cannot survive.  By 3500 mg
    Cu/kg areas are largely devoid of vegetation cover.  Exceptions again
    are the old-established copper-tolerant flora on major
    mineralizations, e.g. in Zaire, Zimbabwe and Borneo.

         Effects of copper in soil on terrestrial biota are reported at
    concentrations ranging from approximately 4 to 7000 mg Cu/kg (chapter
    9).  With the exception of one study reporting a decrease in yield for
    snap beans at 15 mg/kg (copper extracted with EDTA) (Walsh et al.,
    1972) the most sensitive end-points were related to soil microbial
    metabolism, measured as enzymatic activity and soil respiration.  On
    the basis of the data reviewed in this assessment, the organic content
    of the soil appears to be a key factor affecting the bioavailability
    of copper, thus strongly influencing its toxicity.


    11.1  Human health

         The lower limit of the AROI is 20 µg Cu/kg per day.  This figure
    is arrived at from the adult basal requirement with an allowance for
    variations in copper absorption, retention and storage (WHO, 1996).
    In infancy, this figure is 50 µg Cu/kg per day.

         The upper limit of the AROI in adults is uncertain, but it is
    most likely in the range of several but not many mg per day in adults
    (more than 2 or 3 mg/day).  This evaluation is based solely on studies
    of gastrointestinal effects of copper-contaminated drinking-water.  A
    more specific value for the upper AROI could not be confirmed for any
    segment of the general population.  We have limited information on the
    level of ingestion of copper from food that would provoke adverse
    health effects.

         The available data on toxicity in animals were considered
    unhelpful in establishing the upper limit of the AROI, owing to
    uncertainty about an appropriate model for humans.  Moreover,
    traditional methodology for safety assessment, based on application of
    uncertainty factor to data in animals, does not adequately address the
    special attributes of essential elements such as copper.

         From available data on human exposures worldwide, but
    particularly in Europe and the Americas, there is greater risk of
    health effects from deficiency of copper intake than from excess
    copper intake.

         To increase the level of public health protection worldwide, it
    is recommended that:

    1.   National and international nutritional guidelines are adhered to
         in order to address potential copper deficiency.
    2.   Increased monitoring of the concentration of copper in
         drinking-water and food should be carried out.
    3.   There should be increased awareness of the possibility that high
         copper exposure of newborns may result in adverse health effects.
    4.   The development of population-based liver disease registries for
         infant and childhood disease should be encouraged.

    11.2  Environmental protection

         Protection of aquatic life in waters with high bioavailability
    will require limiting total dissolved copper to some concentration
    less than 10 µg/litre (see Table 25); however, the appropriate
    concentration limit will depend on the biota and exposure conditions
    at sites of concern and should be set based on further evaluation of
    relevant data.

         At many sites, physicochemical factors limiting bioavailability
    will warrant higher copper limits.  Regulatory criteria should take
    into account the speciation of copper if dischargers can demonstrate
    that the bioavailability of copper in the receiving water can be
    measured reliably.

         When sampling and analysing environmental media for copper, it is
    essential that clean techiques be employed.

         Because copper is an essential element, procedures to prevent
    toxic levels of copper should not incorporate safety factors that
    result in desired concentrations being below natural levels.


    12.1  Health protection

    1.   Determine the bioavailability of dietary copper, particularly in
         vegetarian diets.

    2.   In human populations develop the methodology for identifying
         adverse effects of marginal copper deficiency and of intakes in
         excess of recommended levels.  This should include an evaluation
         of stable isotope technology to define bioavailability and body
         stores of copper.

    3.   Determine the concentrations of copper and the other quality
         parameters of drinking-water that produce toxicity from single
         and chronic exposures (e.g. gastrointestinal effects).

    4.   Characterize the mechanisms that influence copper homoeostasis
         including placental transfer of copper.

    5.   Studies on ICC populations to determine:

         a)   genetic component
         b)   relationship to ICT
         c)   mechanisms related to basic defect
         d)   methods for early diagnosis of ICC and ICT

    12.2  Environmental protection

    1.   More research is needed to validate existing physicochemical
         speciation techniques for copper and to develop improved methods.
         These methods should be calibrated against suitable bioassays.
         There is also a need for the development of more sensitive, rapid
         bioassays for copper.

    2.   Predictive models should be developed for relating
         bioaccumulation and toxic response to copper speciation and other
         physicochemical factors that affect bioavailability and toxicity.

    3.   Insufficient data are available on the toxicity of copper to
         benthic organisms and more studies are needed in this area.

    4.   Considerations should be given to the development of more
         realistic soil toxicity tests that utilize "real" soils; possibly
         of national relevance.  Alternative and more appropriate
         invertebrate test species should be investigated.  Studies
         correlating measures of copper bioavailability to body burden
         should be undertaken.


         The International Agency for Research on Cancer evaluated copper
    8-hydroxyquinoline in 1977 (IARC, 1977) and re-evaluated it in 1987
    (IARC, 1987).  The conclusions were that there are no data on the
    carcinogenicity of copper 8-hydroxyquinoline in humans and
    insufficient data in animals.  It was, therefore, put into Group
    3 - cannot be classified as to its carcinogenic risk to humans.

         At the twenty-sixth meeting of the Joint FAO/WHO Expert Committee
    on Food Additives and Food Contaminants, the previous recommendation
    of 0.5 mg/kg body weight as an acceptable daily load for copper was
    tentatively reconfirmed (WHO, 1982).  A provisional tolerable daily
    intake (PTDI) from all sources was established as 0.5 mg Cu/kg body

         During the revisions of the WHO Drinking-water Guidelines
    (WHO, 1989), copper was re-evaluated.  Using the PTDI for copper
    developed by JECFA (WHO, 1982) a provisional guideline value of 2 mg
    Cu/litre was proposed (WHO, 1993).


    Achar ST, Raju VB, & Shriramachari S (1960) Indian childhood
    cirrhosis. Trop Pediatr, 57: 744-758.

    Adalsteinsson S (1994) Compensatory root growth in winter
    wheat -- effects of copper exposure on root geometry and nutrient
    distribution. J Plant Nutr, 17(9): 1501-1512.

    Adamson M, Reiner B, Olsen JL, Goodman Z, Plotnick L, Bernardini I,
    & Gahl WA (1992) Indian childhood cirrhosis in an American child.
    Gastroenterology, 102(5): 1771-1777.

    Adriano DC (1986) Trace elements in the terrestrial environment.
    New York, Springer-Verlag.

    Adriano DC ed. (1992) Biogeochemistry of trace metals. Boca Raton,
    Florida, Lewis Publishers.

    Agarwal K, Lahori VC, Mehta SK, Smith DG, & Bayai PC (1979)
    Inheritance of Indian childhood cirrhosis. Hum Hered, 29: 82-89.

    Agarwal K, Sharma A, & Talukder G (1990) Clastogenic effects of
    copper sulphate on the bone marrow chromosomes of mice  in vivo.
    Mutat Res, 243: 1-6.

    Ahsanullah M & Florence TM (1984) Toxicity of copper to the marine
    amphipod  Allorchestes compressa in the presence of water- and
    lipid-soluble ligands. Mar Biol, 84: 41-45.

    Al-Akel AS (1987) Behavioural and the physiological changes in
     Oreochromis niloticus due to contamination of copper. Z Angew
    Zool, 74(4): 479-487.

    Alam MK & Maughan OE (1992) The effect of malathion, diazinon, and
    various concentrations of zinc, copper, nickel, lead, iron, and
    mercury on fish. Biol Trace Elem Res, 34(3): 225-236.

    Alam I & Sadiq M (1989) Metal contamination of drinking-water from
    corrosion of distribution pipes. Environ Pollut, 57: 167-178.

    Alexander J & Aaseth J (1980) Biliary excretion of copper and zinc
    in the rat as influenced by diethylmaleate, selenite and
    diethyldithiocarbamate. Biochem Pharmacol, 29: 2129-2133.

    Alfaro B & Heaton FW (1973) Relationships between copper, zinc and
    iron in the plasma, soft tissue and skeleton of the rats during
    copper deficiency. Br J Nutr, 29: 73-85.

    Alikhan MA (1993) Differentiation in copper and nickel accumulation
    in adult female and juvenile Porcellio spinicornis from
    contaminated and uncontaminated sites in northeaster Ontario. Bull
    Environ Contam Toxicol, 50: 922-928.

    Alikhan MA, Bagatto G, & Zia S (1990) The crayfish as a biological
    indicator of aquatic contamination by heavy metals. Water Res,
    24: 1069-1076.

    Aljajeh IA, Mughal S, Al-Tahou B, Ajrawi T, Ismail EA, & Nayak NC
    (1994) Indian childhood cirrhosis-like liver disease in an Arab
    child: A brief report. Virchow Arch, A424: 225-227.

    Allard B, Hakansson K, Karlsson S, & Sigas E (1991) A field study
    of diffusion controlled migration of copper, zinc and cadmium in a
    clay formation. Water Air Soil Pollut, 57/58: 259-268.

    Allen HE & Brisbin TD (1980) Prediction of bioavailability of
    copper in natural waters. Thalassia Jugosl, 16: 331-334.

    Allen HE & Hansen DJ (1996) The importance of trace metal
    speciation to water quality criteria. Water Environ Res, 68(1):

    Allison JD, Brown DS, & Novo-Gradac KJ (1991) MINTEQA2/PRODEFA2, a
    geochemical assessment model for environmental systems: version 3.0
    user's manual. Washington, DC, US Environmental Protection Agency

    Al-Rashid RA & Spangler J (1971) Neonatal copper deficiency. N Engl
    J Med, 285: 841-843.

    Alt ER, Sternlieb I, & Goldfischer S (1990) The cytopathology of
    metal overload. Int Rev Exp Pathol, 31: 165-188.

    Alva AK & Chen EQ (1995) Effects of external copper concentrations
    on uptake of trace elements by citrus seedlings. Soil Sci, 159(1):

    Anderson JR, Aggett FJ, Buseck PR, Germani MS, & Shattuck TW (1988)
    Chemistry of individual aerosol particles from Chandler, Arizona,
    an arid urban environment. Environ Sci Technol, 22: 811-888.

    Anderson BS, Middaugh DP, Hunt JW, & Turpen SL (1991) Copper
    toxicity to sperm, embryos and larvae of topsmelt  Atherinops
    affinis, with notes on induced spawning. Mar Environ Res, 31:

    Anderson BS, Hunt JW, McNulty HR, Turpen SL, & Martin M (1994)
    Off-season spawning and factors influencing toxicity test
    development with topsmelt  Atherinops affinis. Environ Toxicol
    Chem, 13: 479-485.

    Angelone M & Bini C (1992) Trace element concentrations in soils
    and plants of Western Europe. In: Adriano CD ed. Biogeochemistry of
    trace metals. Boca Raton, Florida, Lewis Publishers, pp 19-60.

    Anke M (1991) Trace element intake (zinc, manganese, copper,
    molybdenum, codine, and nickel) of humans in Thuringia and
    Brandenburg of the Federal Republic of Germany. J Trace Elem
    Electrolytes Health Dis, 5: 69-74.

    Ankley GT, Phipps GL, Leonard EN, Benoit DA, Mattson VR, Kosian PA,
    Cotter AM, Dierkkes JR, Hansen DJ, & Mahony JD (1991) Acid-volatile
    sulfide as a factor mediating cadmium and nickel bioavailability in
    contaminated sediments. Environ Toxicol Chem, 10: 1299-1307.

    Aoyagi S & Baker DH (1994) Copper-amino acid complexes are
    partially protected against inhibitory effects of L-cysteine and
    L-ascorbic acid on copper absorption in chicks. J Nutr, 124(3):

    Arillo A, Calamari D, Margiocco C, Melodia F, & Mensi P (1984)
    Biochemical effects of long-term exposure to cadmium and copper on
    rainbow trout  (Salmo gairdneri): validation of water quality
    criteria. Ecotoxicol Environ Saf, 8: 106-117.

    Armstrong CW, Moore LW, Hackler RL, Miller GB, & Stroube RB (1983)
    An outbreak of metal fume fever: Diagnostic use of urinary copper
    and zinc determinations. J Occup Med, 25: 886-888.

    Arnon DI & Stout PR (1939) The essentiality of certain elements in
    minute quantity for plants with special reference to copper. Plant
    Physiol, 14: 371-375.

    Arthur JW & Leonard EN (1970) Effects of copper on
     Gammarus pseudolimnaeus, Physa integra, and  Campeloma decisum
    in soft water. J Fish Res Board Can, 27: 1277-1283.

    Aschengrau A, Zierler S, & Cohen A (1989) Quality of community
    drinking water and the occurrence of spontaneous abortion. Arch
    Environ Health, 44: 283-290.

    Ash CPJ & Lee DL (1980) Lead, cadmium, copper and iron in
    earthworms from roadside sites. Environ Pollut, A22: 59-67.

    Ashkenazi A, Levin S, Djaldetti M, Fishel E, & Benvenisti D (1973)
    The syndrome of neonatal copper deficiency. Pediatrics, 52:

    Ashmead HDW, Graff DJ, & Ashmead HH (1985) Intestinal absorption of
    metal ions and Chelates. Springfield, Illinois, Charles C. Thomas,
    p 68.

    Askari A, Wang Y, Xie Z, Huang WH, Klauning JE, & Sakari A (1990)
    Superoxide dismutase activity of copper deficient cardiac myocites.
    FASEB J, 4: A392.

    Assaad FF & Nielsen JD (1984) A thermodynamic approach for copper
    adsorption on some Danish arable soils. Acta Agric Scand, 34:

    ATSDR (1990) Toxicological profile for copper. Atlanta, Georgia,
    Agency for Toxic Substances and Disease Registry (TP-90-08).

    August D, Janghorbani M, & Young VR (1989) Determination of zinc
    and copper absorption at three dietary Zn-Cu ratios by using stable
    isotope methods in young adult and elderly subjects. Am J Clin
    Nutr, 50: 1457-1463.

    Aulerich RJ & Ringer RK (1976) Feeding copper sulfate: Could it
    have benefits in nutrition of mink? US Fur Rancher, 56(12): 4.

    Aulerich RJ, Ringer RK, Bleavins MR, & Napolitano A (1982) Effects
    of supplemental dietary copper on growth, reproductive performance
    and kit survival of standard dark mink and the acute toxicity of
    copper to mink. J Anim Sci, 55: 337-343.

    Bagatto G, Crowder AA, & Shorthouse JD (1993) Concentrations of
    metals in tissues of lowbush blueberry  (Vaccinium angustifolium)
    near a copper-nickel smelter at Sudbury, Ontario, Canada: a factor
    analytic approach. Bull Environ Contam Toxicol, 51: 600-604.

    Baird DJ, Barber I, Bradley M, Soares AMVM, & Calow P (1991) A
    comparative study of genotype sensitivity to acute toxic stress
    using clones of  Daphnia magna Straus. Ecotoxicol Environ Saf, 21:

    Baker AJM & Brooks RR (1989) Terrestrial higher plants which
    hyperaccumulate metallic elements. A review of their distribution,
    ecology and phytochemistry. Biorecovery, 1: 81-126.

    Baker DH & Czarnecki-Maulden GL (1987) Pharmacologic role of
    cysteine in ameliorating or exacerbating mineral toxicities. J
    Nutr, 117: 1003-1010.

    Baker EK & Harris PT (1991) Copper, lead, and zinc distribution in
    the sediments of the Fly River Delta and Torres Strait. Mar Pollut
    Bull, 22(12): 614-618.

    Baker EK, Harris PT, & Beck RW (1990) Cu and Cd associated with
    suspended particulate matter in Torres Strait. Mar Pollut Bull,
    21(10): 484-486.

    Baker A, Gormally S, Saxena R, Baldwin D, Drumm B, Bonham J,
    Portmann B, & Mowat AP (1995) Copper-associated liver disease in
    childhood. J Hepatol, 23(5): 538-543.

    Baldwin S, Deaker M, & Maher W (1994) Low-volume microwave
    digestion of marine biological tissues for the measurement of trace
    elements. Analyst, 119: 1701-1704.

    Balsberg Pĺhlsson A-M (1989) Toxicity of heavy metals (Zn, Cu, Cd,
    Pb) to vascular plants. Water Air Soil Pollut, 47: 287-319.

    Balthrop JE, Dameron CT, & Harris ED (1982) Comparison of pathways
    of copper metabolism in aorta and liver. Biochem J, 204: 541-548.

    Bankier A (1995) Menkes disease. J Med Genet, 32(3): 213-215.

    Baranowska I, Czernicki K, & Aleksandrowicz R (1995) The analysis
    of lead, cadmium, zinc, copper and nickel content in human bones
    from the upper Silesian industrial district. Sci Total Environ,
    159: 155-162.

    Barclay SM, Aggett PJ, Lloyd DJ, & Duffty P (1991) Reduced
    erythrocyte superoxide dismutase activity in low birth weight
    infants given iron supplements. Pediatr Res, 29: 297-301.

    Barnes G & Frieden E (1984) Ceruloplasmin receptors of
    erythrocytes. Biochem Biophys Res Commun, 125: 157-162.

    Batley GE, Scammell MS, & Brockbank CI (1992) The impact of the
    banning of tributyltin-based antifouling paints on the Sydney rock
    oyster,  Saccostrea commercialis. Sci Total Environ, 122: 301-314.

    Baudouin MF & Scoppa P (1974) Acute toxicity of various metals to
    freshwater zooplankton. Bull Environ Contam Toxicol, 12(6):

    Bayley M, Baatrup E, Heimbach U, & Bjerregaard P (1995) Elevated
    copper levels during larval development cause altered locomotor
    behavior in the adult carabid beetle  Pterostichus cupreus L.
    (Coleoptera: Carabidae). Ecotoxicol Environ Saf, 32: 166-170.

    Bearn AG (1960) A genetical analysis of thirty families with
    Wilson's disease (hepatolenticular degeneration). Ann Hum Genet,
    24: 33-43.

    Bearn AG & Kunkel HG (1964) Localization of Cu64 in serum fractions
    following oral administration: An alteration in Wilson's disease.
    Proc Soc Exp Biol Med, 85: 44-48.

    Beary ES, Paulsen PJ, & Fassett JD (1994) Sample preparation
    approaches for isotope dilution inductively coupled plasma mass
    spectrometric certification of reference materials. J Anal At
    Spectrosc, 9: 1363-1369.

    Beaumont AR, Tserpes G, & Budd MD (1987) Some effects of copper on
    the veliger larvae of the mussel  Mytilus edulis and the scallop
     Pecten maximus (Mollusca, Bivalvia). Mar Environ Res, 21:

    Bechmann RK (1994) Use of life tables and LC50 tests to evaluate
    chronic and acute toxicity effects of copper on the matine copepod
     Tisbe furcata (Baird). Environ Toxicol Chem, 13: 1509-1517.

    Becker W & Kumpulainen J (1991) Contents of essential and toxic
    mineral elements in Swedish market-basket diets in 1987. Br J Nutr,
    66: 151-160.

    Beckett PHT & Davis RD (1977) Upper critical levels of toxic
    elements in plants. New Phytol, 79: 95-106.

    Beckett PHT & Davis RD (1978) The additivity of the toxic effects
    of Cu, Ni and Zn in young barley. New Phytol, 81: 155-173.

    Beckman BR & Zaugg WS (1988) Copper intoxication in chinook salmon
     (Oncorhynchus tshawytscha) induced by natural springwater:
    effects on gill Na+, K+-ATPase, hematocrit, and plasma glucose. Can
    J Fish Aquat Sci, 45: 1430-1435.

    Béguin-Bruhin Y, Escher F, Solms J, & Roth HR (1983) [Threshold
    concentration of copper in drinking-water.] Lebensm. Wiss Technol,
    16: 22-26 (in German).

    Belanger SE, Farris JL, & Cherry DS (1989) Effects of diet, water
    hardness, and population source on acute and chronic copper
    toxicity to  Ceriodaphnia dubia. Arch Environ Contam Toxicol, 18:

    Bello MA, Callejon M, Jimenez JC, Pablos F, & Ternero M (1994)
    Determination of heavy metals in estuarine sediments by acid
    digestion and atomic absorption spectrometry. Toxicol Environ Chem,
    44: 203-210.

    Benedetti I, Albano AG, & Mola L (1989) Histomorphological changes
    in some organs of the brown bullhead,  Ictalurus nebulosus
    LeSueur, following short- and long-term exposure to copper. J Fish
    Biol, 34: 273-280.

    Bengtsson G, Gunnarsson T, & Rundgren S (1983) Growth changes
    caused by metal uptake in a population of  Onychiurus armatus
    (Collembola) feeding on metal polluted fungi. Oikos, 40: 216-225.

    Bengtsson G, Gunnarsson T, & Rundgren S (1986) Effects of metal
    pollution on the earthworm  Dendrobaena rubida (Sav.) in acidified
    soils. Water Air Soil Pollut, 28: 361-383.

    Benoit DA (1975) Chronic effects of copper on survival, growth, and
    reproduction of the bluegill  (Lepomis macrochirus). Trans Am Fish
    Soc, 104: 353-358.

    Berg R & Lundh S (1981) Copper contamination of drinking-water as
    a cause of diarrhea in children. Halsovardskontakt, 1: 6-10.

    Berger B & Dallinger R (1989) Accumulation of cadmium and copper by
    the terrestrial snail  arianta arbustorum L.: Kinetics and
    budgets. Oecologia, 79: 60-65.

    Berggren D (1992) Speciation of copper in soil solutions from
    podzols and cambisols of S. Sweden. Water Air Soil Pollut, 62:

    Berk SG, Gunderson JH, & Derk LA (1985) Effects of cadmium and
    copper on chemotaxis of marine and freshwater ciliates. Bull
    Environ Contam Toxicol, 34: 897-903.

    Besser JM, Ingersol CG, & Giesy JP (1996) Effects of spatial and
    temoral variation of acid-volatile sulfide on the bioavailability
    of copper and zinc in freshwater sediments. Environ Toxicol Chem,
    15: 286-293.

    Bettger WJ, Fish TJ, & O'dell BL (1978) Effects of copper and zinc
    status of rats on erythrocyte stability and superoxide dismutase
    activity. Proc Soc Exp Biol Med, 158(2): 279-282.

    Beyer WN, Pattee OH, Sileo L, Hoffman DJ, & Mulhern BM (1985) Metal
    contamination in wildlife living near two zinc smelters. Environ
    Pollut, A38: 63-86.

    Bhagwat AG & Walia BNS (1980) Indian childhood cirrhosis: A
    commentary. Indian J Pediatr, 48: 433-437.

    Bhave S, Pandit AN, Pradhan AM, Sidhaye DG, Kantarjian A, Williams
    A, Talbot IC, & Tanner MS (1982) Paediatric liver disease in India.
    Arch Dis Child, 57(12): 922-928.

    Bhave SA, Sidhaye DG, Pandit AN, & Tanner MS (1983) Incidence and
    clinical features of Indian childhood cirrhosis. Indian Pediatr,
    20: 741-746.

    Bhave SA, Pandit AN, Singh S, Walia BNS, & Tanner MS (1992) The
    prevention of Indian childhood cirrhosis Ann Trop Paediatr, 12:

    Bhunya SP & Pati PC (1987) Genotoxicity of an inorganic pesticide,
    copper sulphate in mouse  in vivo test system. Cytologia,
    52: 801-808.

    Bidwell RGS (1979) Plant physiology, 2nd ed. New York, MacMillan
    Publishing Company, 726 pp.

    Biesinger KE & Christensen GM (1972) Effects of various metals on
    survival, growth, reproduction, and metabolism of  Daphnia magna.
     J Fish Res Board Can, 29: 1691-1700.

    Bionetics Research Lab (1968) Evaluation of carcinogenic,
    teratogenic and mutagenic activities of selected pesticides and
    industrial chemicals -- Volume 1: Carcinogenic study. Bionetics
    Research Laboratories (Prepared for the National Cancer Institute,
    Bethesda, Maryland, USA) (PB-223-159).

    Blaise C, Forghani R, Legault R, Guzzo J,& Dubow MS (1994) A
    bacterial toxicity assay performed with microplates,
    microluminometry and Microtox reagent. Biotechniques, 16: 932-937.

    Blakley BR & Hamilton DL (1985) Ceruloplasmin as an indicator of
    copper status in cattle and sheep. Can J Comp Med, 49: 405-408.

    Bligh SW, Boyle HA, McEwen AB, Sadler PJ, & Woodham RH (1992) 1H
    NMR studies of reactions of copper complexes with human blood
    plasma and urine. Biochem Pharmacol, 43: 137-145.

    Bloom NS & Crecelius EA (1984) Determination of silver in sea water
    by coprecipitation with cobalt pyrrolidinedithio-carbamate and
    zeeman graphite furnace atomic absorption spectrometry. Anal Chim
    Acta, 156: 139-145.

    Bodar CWM, Zee AVD, Voogt PA, Wynne H, & Zander DI (1989) Toxicity
    of heavy metals to early life stages of  Daphnia magna. Ecotoxicol
    Environ Saf, 17: 333-338.

    Bodek I, Lyman WJ, Reehl WF, & Rosenblatt DH (1988) Environmental
    inorganic chemistry: Properties, processes and estimation methods.
    New York, Pergamon Press, 9 pp (SETAC Special Publications Series).

    Borgmann U & Ralph KM (1984) Copper complexation and toxicity to
    freshwater zooplankton. Arch Environ Contam Toxicol, 13: 403-409.

    Borgmann U (1983) Metal speciation and toxicity of free metal ions
    to aquatic biota. In: Nriagu JO ed. Aquatic toxicology. New York,
    John Wiley & Sons Ltd, pp 47-72.

    Borgmann U & Charlton CC (1984) Copper complexation and toxicity to
     Daphnia in natural waters. J Great Lakes Res, 10: 393-398.

    Borgmann U & Ralph KM (1983) Complexation and toxicity of copper
    and the free metal bioassay technique. Water Res, 17: 1697-1703.

    Borszeki J, Halmos P, Gegus E, & Karpati P (1994) Application of
    pressurized sample preparation methods for the analysis of steels
    and copper alloys. Talanta, 41: 1089-1093.

    Bowen HJM (1985) The natural environment and the biogeochemical
    cycles. In: Hutzinger D ed. Handbook of environmental chemistry.
    New York, Basel, Springer-Verlag, pp 1-26.

    Boyle EA, Sclater FR, & Edmond JM (1977) The distribution of
    dissolved copper in the pacific. Earth Planet Sci Lett, 37: 38-54.

    Bradley SB & Cox JJ (1988) The potential availability of cadmium,
    copper, iron, lead, manganese, nickel and zinc in standard river
    sediment (NBS 1645). Environ Technol Lett, 9: 733-739.

    Bremner I (1987) Involvement of metallothionein in the hepatic
    metabolism of copper: Critical review. J Nutr, 117: 19-29.

    Bremner JM & Douglas LA (1971) Inhibition of urease activity in
    soils. Soil Biol Biochem, 3: 297-307.

    Brenner A & Harris ED (1995) A quantitative test for copper using
    bichinchioninc acid. Anal Biochem, 226: 80-84.

    Brewer GJ, Hill GM, Prasad AS, Cossack ZT, & Rabbani P (1983) Oral
    zinc therapy for Wilson's disease. Ann Intern Med, 99: 314-320.

    Bro S, Sandstrom B, & Heydorn K (1990) Intake of essential and
    toxic trace elements in a random sample of Danish men as determined
    by duplicate portion sampling technique. J Trace Elem Electrolytes
    Health Dis, 4: 147-155.

    Brooks RR, McCleave JA, & Malaisse F (1977) Copper and cobalt in
    African species of  Crotalaria L. Proc R Soc Ser B, 197: 231-236.

    Brooks RR, Reeves RD, Morrison RS, & Malaisse F (1980)
    Hyperaccumulation of copper and cobalt. A review. Bull Soc R Bot
    Belg, 113: 166-172.

    Brooks RR, Baker AJM, & Malaisse F (1992) Copper flowers. Natl
    Geogr Res Explor, 8: 338-351.

    Brown BT & Rattigan BM (1979) Toxicity of soluble copper and other
    metal ions to  Elodea canadensis. Environ Pollut, 20: 303-314.

    Brown KW, Thomas JC, & Slowey JF (1983) The movement of metals
    applied to soils in sewage effluents. Water Air Soil Pollut, 19:

    Bruland KW (1980) Oceanographic distributions of cadmium, zinc,
    nickel, and copper in the North Pacific. Earth Planet Sci Lett, 47:

    Bruland KW, Coale KH, & Mart L (1985) Analysis of seawater for
    dissolved cadmium copper and lead: an intercomparison of
    voltammetric and atomic absorption methods. Mar Chem, 17: 285-300.

    Brungs WA, Geckler JR, & Gast M (1976) Acute and chronic toxicity
    of copper to the fathead minnow in a surface water of variable
    quality. Water Res, 10: 37-43.

    Bryan GW & Hummerstone LG (1973) Brown seaweed as an indicator of
    heavy metals in estuaries in South-West England. J Mar Biol Assoc
    (UK), 53: 705-720.

    Bryan GW & Langston WJ (1992) Bioavailability, accumulation and
    effects of heavy metals in sediments with special reference to
    United Kingdom estuaries: a review. Environ Pollut, 76: 89-131.

    Bubb JM & Lester JN (1994) Anthropogenic heavy metal inputs to
    lowland river systems, a case study. The River Stour, UK Water Air
    Soil Pollut, 78: 279-296.

    Bubb JM, Rudd T, & Lester JN (1991) Distribution of heavy metals in
    the river Yare and its associated broads: II. Copper and cadmium.
    Sci Total Environ, 102: 169-188.

    Bucholtz CF (1816) [Chemical study of the vanilla shoot
     (Siliqua vanillae).] Report Pharm, 2: 253 (in German).

    Buck WB, Osweiler GD, & Van Gelder GA (1976) Clinical and
    diagnostic veterinary toxicology, 2nd ed. Dubuque, Iowa,
    Kendell/Hunt Publishing Co., 380 pp.

    Buckley JA (1983) Complexation of copper in the effluent of a
    sewage-treatment plant and an estimate of its influence on toxicity
    to coho salmon. Water Res, 17(12): 1929-1934.

    Buckley PJM & van den Berg CMG (1986) Copper complexation profiles
    in the Atlantic Ocean: A comparative study using electrochemical
    and ion exchange techniques. Mar Chem, 19: 281-296.

    Buckley JT, Roch M, McCarter JA, Rendell CA, & Matheson AT (1982)
    Chronic exposure of coho salmon to sublethal concentrations of
    copper: I. Effect on growth, on accumulation and distribution of
    copper, and on copper tolerance. Comp Biochem Physiol, 72C: 15-19.

    Buhl KJ & Hamilton SJ (1990) Comparative toxicity of inorganic
    contaminants released by placer mining to early life stages of
    salmonids. Ecotoxicol Environ Saf, 20: 325-342.

    Bull PC, Thomas GR, Rommens JM, Forbes JR, & Cox DW (1993) The
    Wilson disease gene is a putative copper transporting P-type ATPase
    similar to the Menkes gene. Nat Genet, 5: 327-337.

    Burguera JL, Burguera M, & Brunetto MR (1993)  In vivo sample
    uptake and on-line measurement of zinc and copper in whole blood by
    microwave-assisted mineralization and flow injection AAS. At
    Spectrosc, 14: 90-94.

    Burki HR & Okita GT (1969) Effect of oral copper sulfate on
    7,12-dimethyl-benz(alpha)anthracene carcinogenesis in mice. Br J
    Cancer, 23: 591-596.

    Burns LV & Parker GH (1988) Metal burdens in two species of
    fiddleheads growing near the nore smelters at Sudbury, Ontario,
    Canada. Bull Environ Contam Toxicol, 40: 717-723.

    Burton GA (1987) Occurrence of bacterial resistance to arsenite,
    copper, and selenite in adverse habitats. Bull Environ Contam
    Toxicol, 39: 990-997.

    Burton DT & Fisher DJ (1990) Acute toxicity of cadmium, copper,
    zinc, ammonia, 3,3'-dichlorobenzidine, 2,6-dichloro-4-nitroaniline,
    methylene chloride, and 2,4,6-trichlorophenol to juvenile grass
    shrimp and killifish. Bull Environ Contam Toxicol, 44: 776-783.

    Burton KW, Morgan E, & Roig A (1986) Interactive effects of
    cadmium, copper and nickel on the growth of Sitka spruce and
    studies of metal uptake from nutrient solutions. New Phytol, 103:

    Cabrera F, Soldevilla M, Cordon R, & De Arambarri P (1987) Heavy
    metal pollution in the Guadiamar river and Guadalquivir estuary
    (South west Spain). Chemosphere, 16(2-3): 463-468.

    Callahan M, Slimak M, Gabel N, May I, Fowler C, Freed R, Jennings
    P, Durfee R, Whitmore F, Maestri B, Mabey W, Holt B, & Gould C
    (1979) Copper. In: Water-related environmental fate of 129 priority
    pollutants. Washington, DC, US Environmental Protection Agency,
    Office of Water Planning and Standards.

    Calmano W, Hong J, & Förstner U (1993) Binding and mobilization of
    heavy metals in contaminated sediments affected by pH and redox
    potential. Water Sci Technol, 28: 223-235.

    Campanella L, Ferri T, & Petronio BM (1989) Effect of speciation in
    sludges on the adsorption of leached metals from soil. Sci Total
    Environ, 79: 223-231.

    Campbell PGC (1995) Interactions between trace metals and aquatic
    organisms: a critique of the free-ion activity model. In: Tessier
    A & Turner DR ed. Metal speciation and bioavailability in aquatic
    systems New York, John Wiley & Sons Ltd, pp 45-102.

    Camusso M, Tartari G, & Cappelletti E (1989) Seasonal trends of
    copper sedimentation in Lake Orta (Italy). Sci Total Environ,
    87/88: 59-75.

    Cannon HL, Connally GC, Epstein JB, Parker JG, Thornton I, & Wixson
    G (1978) Rocks: geological sources of most trace elements. Report
    to the workshop at South Seas plantation, Captiva Islands, FL, US.
    Geochem Environ, 3: 17-31.

    Carbon C (1996) Good chemistry goes to waste. Chem Aust, April:

    Carlson AR, Nelson H, & Hammermeister D (1986) Development and
    validation of site-specific water quality criteria for copper.
    Environ Toxicol Chem, 5: 997-1012.

    Carlson-Ekvall CEA & Morrison GM (1995) Toxicity of copper in the
    presence of organic substances in sewage sludge. Environ Technol,
    16: 243-251.

    Carlton WW & Price PS (1973) Dietary copper and the induction of
    neoplasms in the rat by acetylaminofluorene and
    dimethylnitrosamine. Food Cosmet Toxicol, 11: 827-840.

    Carry MT, Galiazzo F, Ciriolo MR, & Rotilio G (1991) Evidence of
    co-regulation of Cu, Zn superoxide dismutase and metallothionein
    gene expression in yeast through transcriptional control by copper
    via the ACE1 factor. FEBS Lett, 2278: 263-266.

    Castillo-Duran C & Uauy R (1988) Copper deficiency impairs growth
    of infants recovering from malnutrition. Am J Clin Nutr, 47:

    Castillo-Duran C, Fishberg M, Valenzuela A, Egańa JI, & Uauy R
    (1983) Controlled trial of copper supplementation during the
    recovery from marasmus. Am J Clin Nutr, 37: 898-903.

    Castillo-Duran C, Vial P, & Uauy R (1988) Trace mineral balance
    during acute diarrhea in infants. J Pediatr, 113: 452-457.

    Cavallo F, Gerber M, Marubini E, Richardson S, Barbieri A, Costa A,
    DeCarli H, & Pujol A (1991) Zinc and copper in breast cancer: A
    joint study in Northern Italy and Southern France. Cancer, 67:

    Cavell PA & Widdowson EM (1964) Intakes and excretions of iron,
    copper, and zinc in the neonatal period. Arch Dis Child, 39:

    Centeno MDF, Brendonck L, & Persoone G (1993) Acute toxicity tests
    with  Streptocephalus proboscideus (Crustacea: Branchiopoda:
    Anostraca): influence of selected environmental conditions.
    Chemosphere, 27: 2213-2224.

    Çetinkaya N, Çetinkaya D, & Yüce M (1988) Serum copper, zinc
    levels, and copper: Zinc ratio in healthy women and women with
    gynecological tumors. Biol Trace Elem Res, 18: 29-38.

    Chakoumakos C, Russo RC, & Thurston RV (1979) Toxicity of copper to
    cutthroat trout  (Salmo clarki) under different conditions of
    alkalinity, pH, and hardness. Environ Sci Technol, 13(2): 213-219.

    Chakrabarti CL, Lu Y, Cheng J, Back MH, & Schroeder WH (1993)
    Studies on metal speciation in the natural environment. Anal Chim
    Acta, 267: 47-64.

    Chakrabarti CL, Lu YJ, Gregoire DC, Back MH, & Schroeder WH (1994)
    Kinetic studies of metal speciation using chelex cation exchange
    resin-application to cadmium, copper, and lead speciation in river
    water and snow. Environ Sci Technol, 28: 1957-1967.

    Chan WH, Tank AJS, Chung DHS, & Lusis MA (1986) Concentration and
    deposition of trace metals in Ontario -- 1982. Water Air Soil
    Pollut, 29: 373-389.

    Chandra RK (1976) ICC geneologic data, alpha-foetoprotein MbsAg &
    circulating immune complexes. Trans R Soc Trop Med Hyg, 70:

    Chang F-H & Broadbent FE (1981) Influence of trace metals on carbon
    dioxide evolution from a Yolo soil. Soil Sci, 132: 416-421.

    Chapman GA (1978) Toxicities of cadmium, copper, and zinc to four
    juvenile stages of chinook salmon and steelhead. Trans Am Fish Soc,
    107(6): 841-847.

    Chapman GA & Stevens DG (1978) Acutely lethal levels of cadmium,
    copper, and zinc to adult male coho salmon and steelhead. Trans Am
    Fish Soc, 107(6): 837-840.

    Chawla V, Chandra RK, Verma IC, & Ghai OP (1973) An epidemiological
    approach to Indian Childhood cirrhosis. Indian Pediatr, 10: 73-79.

    Chelly J, Tumer Z, Tonnesen T, Petterson A, Ishikawa-Brush Y,
    Tommerup N, Horn N, & Monaco AP (1993) Isolation of a candidate
    gene for Menkes disease that encodes a potential heavy metal
    binding protein see comments. Nat Genet, 3: 14-19.

    Chen L, Li G, & Ren Y (1990) Reoxygenation injured of cultured
    neonatal rat myocardial cells and protective effects of selenium,
    copper and zinc. Zhonghua Yixue Zashi, 70: 221-223.

    Chen LC, Peoples SM, & Amdur MO (1991) Pulmonary effects of sulfur
    oxides on the surface of copper oxide aerosol. Am Indl Hyg Assoc J,
    52: 187-191.

    Chen J, Geissler C, Parpia B, Li J, & Campbell TC (1992)
    Antioxidant status and cancer mortality in China. Int J Epidemiol,
    21: 625-635.

    Chen R, Wei L, & Huang H (1993) Mortality from lung cancer among
    copper miners. Br J Ind Med, 50: 505-509.

    Cheng J, Chakrabarti CL, Back MH, & Schroeder WH (1994) Chemical
    speciation of Cu, Zn, Pb and Cd in rain water. Anal Chim Acta, 288:

    Chester R & Murphy KJT (1986) Oceanic sources of copper to the
    Atlantic aerosol. Sci Total Environ, 49: 325-338.

    Christensen ER, Scherfig J, & Dixon PS (1979) Effects of manganese,
    copper and lead on  Selenastrum capricornutum and
     Chlorella stigmatophora. Water Res, 13: 79-92.

    Christie P & Beattie JAM (1989) Grassland soil microbial biomass
    and accumulation of potentially toxic metals from long-term slurry
    application. J Appl Ecol, 26: 597-612.

    Chugh KS, Sharma BK, Singhal PC, Das KC, & Datta BN (1977) Acute
    renal failure following copper sulphate intoxication. Postgrad Med
    J, 53(615): 18-23.

    Chung IK & Brinkhuis BH (1986) Copper effects in early stages of
    the kelp,  Laminaria saccharina. Mar Pollut Bull, 17(5): 213-218.

    Chuttani HK, Gupta PS, Gulati S, & Gupta DN (1965) Acute copper
    sulfate poisoning. Am J Med, 39: 849-854.

    Claisse D & Alzieu C (1993) Copper contamination as a result of
    antifouling paint regulations? Mar Pollut Bull, 26(7): 395-397.

    Clark JB (1953) The mutagenic action of various chemicals on
     Micrococcus aureus. Proc Okla Acad Sci, 34: 114-118.

    Clarkson DT & Hanson JB (1980) The mineral nutrition of higher
    plants. Annu Rev Plant Physiol, 31: 239-298.

    Claveri B, Morhain E, & Mouvet C (1994) A methodology for the
    assessment of accidental copper pollution using the aquatic moss
    Rhynchostegium riparioides. Chemosphere, 28: 2001-2010.

    Clements WH, Cherry DS, & Cairns J Jr (1988) Structural alterations
    in aquatic insect communities exposed to copper in laboratory
    streams. Environ Toxicol Chem, 7: 715-752.

    Clements WH, Farris JL, Cherry DS, & Cairns J (1989) The influence
    of water quality on macroinvertebrate community responses to copper
    in outdoor experimental streams. Aquat Toxicol, 14: 249-262.

    Clements WH, Cherry DS, & Cairns J Jr (1990) Macroinvertebrate
    community responses to copper in laboratory and field experimental
    streams. Arch Environ Contam Toxicol, 19: 361-365.

    Coale KH & Bruland KW (1988) Copper complexation in the northeast
    Pacific. Limnol Oceanogr, 33: 1084-1101.

    Coates RJ, Weiss NS, Daling JR, Rettmer RL, & Warnick GR (1989)
    Cancer risk in relation to serum copper levels. Cancer Res, 49:

    Codina JC, Pérez-García A, Romero P, & De Vicente A (1993) A
    comparison of microbial bioassays for the detection of metal
    toxicity. Arch Environ Contam Toxicol, 25: 250-254.

    Cohen JM, Kamphake LJ, Harris EK, Woodward RL (1960) Taste
    threshold concentrations of metals in drinking-water. J Am Water
    Works Assoc, 52: 660-670.

    Cohen NL, Keen CL, Lönnerdal B, &Hurley LS (1985a) Effects of
    varying dietary iron on the expression of copper deficiency in the
    growing rat: anemia, ferroxidase I and II, tissue trace elements,
    ascorbic acid, and exanthine dehydrogenase. J Nutr, 115: 633-649.

    Cohen N, Keen CL, Hurley LS, & Lönnerdal B (1985b) Determinants of
    copper-deficiency anemia in rats. J Nutr, 115: 710-725.

    Collvin L (1985) The effect of copper on growth, food consumption
    and food conversion of perch Perca fluviatilis L. offered maximal
    food rations. Aquat Toxicol, 6: 105-113.

    Collyard SA, Ankley GT, Hoke RA, & Goldenstein T (1994) Influence
    of age on the relative sensitivity of  Hyalella azteca to
    diazinon, alkylphenol ethoxylates, copper, cadmium, and zinc. Arch
    Environ Contam Toxicol, 26: 110-113.

    Combs GE, Ammerman CB, Shirley RL, & Wallace HD (1966) Effect of
    source and level of dietary protein on pigs fed high copper
    rations. J Anim Sci, 25(3): 613-616.

    Cordano A, Baertl J, & Graham GG (1964) Copper deficiency in
    infants. Pediatrics, 34: 324-336.

    Correa JA, González P, Sánchez P, Muńoz J, & Orellana MC (1996)
    Copper-algae interactions: inheritance or adaptation? Environ Monit
    Assess, 40: 41-54.

    Cossu P, Pirastu M, Nucaro A, Figus A, Balestrieri A, Borrone C,
    Giacchino R, Devoto M, Monni G, & Cao A (1992) prenatal diagnosis
    of Wilson's disease by analysis of DNA polymorphism. N Engl J Med,
    326: 57.

    Cotton FA & Wilkinson G (1989) Advanced inorganic chemistry. New
    York, John Wiley & Sons Ltd, pp 755-775.

    Couillard Y, Ross P, & Pinel-Alloul B (1989) Acute toxicity of six
    metals to the rotifer  Brachionus calyciflorus, with comparisons
    to other freshwater organisms. Toxicol Assess, 4: 451-462.

    Cousins RJ (1985) Absorption, transport, and hepatic metabolism of
    copper and zinc: Special reference to metallothionein and
    ceruloplasmin. Phys Rev, 65: 238-309.

    Cowgill UM & Milazzo DP (1991) Comparison of the effect of metallic
    copper and copper nitrate (CU(CNO-3)2.3H2O) on  Ceriodaphnia
    dubia utilizing the three-brood test. Bull Environ Contam Toxicol,
    46: 141-145.

    Cox DW (1995) Genes of the copper pathway. Am J Hum Genet, 56:

    Crecelius EA, Hardy JT, Gibson CI, Schmidt RL, Apts CW, Gurtisen
    JM, & Joyce SP (1982) Copper bioavailability to marine bivalves and
    shrimp: relationship to cupric ion activity. Mar Environ Res, 6:

    Cumings JN (1948) The copper and iron content of brain and liver in
    the normal and in hepato-lenticular degeneration. Brain, 71:

    Curtis MW, Copeland TL, & Ward CH (1979) Acute toxicity of 12
    industrial chemicals to freshwater and saltwater organisms. Water
    Res, 13: 137-141.

    Cusimano RF, Brakke DF, & Chapman GA (1986) Effects of pH on the
    toxicities of cadmium, copper, and zinc to steelhead trout
     (Salmo gairdneri). Can J Fish Aquat Sci, 43: 1497-1503.

    Dabek JT, Hyvonen-Dabek M, Harkonen M, & Adlercreutz H (1992)
    Evidence for increased non-ceruloplasmin copper in early-stage
    human breast cancer serum. Nutr Cancer, 17: 195-201.

    Dallinger R & Wieser W (1977) The flow of copper through a
    terrestrial food chain: 1.Copper and nutrition in isopods.
    Oecologia, 30: 253-264.

    Dallinger R & Wieser W (1984) Patterns of accumulation,
    distribution and liberation of Zn, Cu, Cd and Pb in different
    organs of the land snail,  Helix pomatia L. Comp Biochem Physiol,
    79C(1): 117-124.

    Daly HR, Campbell IC, & Hart BT (1990a) Copper toxicity to
     Paratya australiensis: I. Influence of nitrilotriacetic acid and
    glycine. Environ Toxicol Chem, 9: 997-1006.

    Daly HR, Campbell IC, & Hart BT (1990b) Copper toxicity to
     Paratya australiensis: II. Influence of bicarbonate and ionic
    strength. Environ Toxicol Chem, 9: 1007-1011.

    Daly HR, Jones MJ, Hart BT, & Campbell IC (1990c) Copper toxicity
    to  Paratya australiensis: III. Influence of dissolved organic
    matter. Environ Toxicol Chem, 9: 1013-1018.

    Daly HR, Hart BT, & Campbell IC (1992) Copper toxicity to
     Paratya australiensis: IV. Relationship with ecdysis. Environ
    Toxicol Chem, 11: 881-883.

    Dameron CT & Harris ED (1987a) Regulation of aortic Cu
    Zn-superoxide dismutase with copper: Effects  in vivo. Biochem J,
    248: 663-668.

    Dameron CT & Harris ED (1987b) Regulation of aortic CuZn-superoxide
    dismutase with copper: Caeruloplasmin and albumin reactivate and
    transfer copper to the enzyme in culture. Biochem J, 248: 669-675.

    Dameron CT, Winge DR, George GN, Sansone M, Hu S, & Hamer D (1991)
    A copper-thiolate polynuclear cluster in the ACE1 transcription
    factor. Proc Natl Acad Sci (USA), 88: 6127-6131.

    Dangel RA (1975) Study of corrosion products in the Seattle Water
    Department Tolt Distribution System. Washington, DC, US
    Environmental Protection Agency (EPA-670/2-75-036).

    Daniel GF & Chamberlain AH (1981) Copper immobilization in fouling
    diatoms. Bot Mar, 24: 229-243.

    Danks DM (1988) Copper deficiency in humans. Annu Rev Nutr, 8:

    Dann T (1994) Environment Canada -- PM10 and PM2.5: Concentrations
    at Canadian sites, 1984 to 1993. Ottawa, Environment Canada,
    Environmental Technology Centre, 28 pp (Report series No. PMD 94-3).

    Daramola JA & Oladimeji AA (1989) Accumulation of copper in
     Clarias anguillaris L. and  Oreochromis niloticus L. Water Air
    Soil Pollut, 48: 457-461.

    Dauncey MJ, Shaw JLC, & Urman J (1977) The absorption and retention
    of magnesium, zinc and copper by low birth weight infants fed
    pasteurized human breast milk. Pediatr Res, 11: 991-997.

    Dave G (1984) Effects of copper on growth, reproduction, survival
    and haemoglobin in  Daphnia magna. Comp Biochem Physiol, 78C(2):

    Dave G & Xiu RQ (1991) Toxicity of mercury, copper, nickel, lead,
    and cobalt to embryos and larvae of zebrafish,  Brachydanio rerio.
    Arch Environ Contam Toxicol, 21: 126-134.

    Davidson LA, McOrmond SL, & Harris ED (1994) Characterization of a
    particulate pathway for copper in K562 cells. Biochim Biophys Acta,
    1221: 1-6.

    Davis RD & Beckett PHT (1978) Upper critical levels of toxic
    elements in plants: II. Critical levels of copper in young barley,
    wheat, rape, lettuce and ryegrass, and of nickel and zinc in young
    barley and ryegrass. New Phytol, 80: 23-32.

    De Boer JA (1981) Nutrients. In: Lobban CS & Wynne MJ ed. The
    biology of seaweeds. Oxford, London, Blackwell Scientific
    Publications, pp 356-391.

    Delaguardia M, Carbonell V, Moralesrubio A, & Salvador A (1993)
    On-line microwave-assisted digestion of solid samples for their
    flame atomic spectrometric analysis. Talanta, 40: 1609-1617.

    De Nicola Giudici M & Migliore L (1988) Long term effect of cadmium
    or copper on  Asellus aquaticus (L.) (Crustacea, Isopoda). Vehr.
    Int Verein Limnol, 23: 1660-1662.

    Denizeau F & Marion M (1989) Genotoxic effects of heavy metals in
    rat hepatocytes. Cell Biol Toxicol, 5: 15-25.

    Denneman CAJ & Van Straalen NM (1991) The toxicity of lead and
    copper in reproduction tests using the oribatid mite Platynothrus
    peltifer. Pedobiologia, 35: 305-311.

    Depledge MH (1989) Re-evaluation of metabolic requirements for
    copper and zinc in crustaceans. Mar Environ Res, 27: 115-126.

    DeVevey E, Bitton G, Rossel D, Ramos LD, & Guerrero SM (1993)
    Concentration and bioavailability of heavy metals in sediments in
    Lake Yojoa (Honduras). Bull Environ Contam Toxicol, 50: 253-259.

    Devi VU (1987) Heavy metal toxicity to fiddler crabs,  Uca
    annulipes  Latreille and  Uca triangularis (Milne Edwards):
    tolerance to copper, mercury, cadmium, and zinc. Bull Environ
    Contam Toxicol, 39: 1020-1027.

    de Vries DJ, Sewell RB, & Beart PM (1986) Effects of copper on
    dopaminergic function in the rat corpus striatum. Exp Neurol, 91:

    De Zwart D & Sloof W (1987) Toxicity of mixtures of heavy metals
    and petrochemicals to  Xenopus laevis. Bull Environ Contam
    Toxicol, 38: 345-351.

    Dickinson NM, Turner AP, & Lepp NW (1991) Survival of trees in a
    metal-contaminated environment. Water Air Soil Pollut, 57/58:

    Dieter von HH, Meyer E, & Möller R (1991) [Copper occurrence,
    relevance and detection -- The drinking-water directive:
    introduction and explanations for water supply services and control
    authorities], 3rd ed., pp 472-491 (in German).

    Dinnell PA, Link JM, Stober QJ, Letourneau MW, & Roberts WE (1989)
    Comparative sensitivity of sea urchin sperm bioassays to metals and
    pesticides. Arch Environ Contam Toxicol, 18: 748-755.

    Dirilgen N & Inel Y (1994) Cobalt-copper and cobalt-zinc effects on
    duckweed growth and metal accumulation. J Environ Sci Health, A29:

    Disilvestro RA & Harris ED (1981) A post absorption effect of
    L-ascorbic acid on copper metabolism in chicks. J Nutr, 111:

    Di Toro R, Capotorti MG, Gialanella G, del Giudice MM, Moro R, &
    Perrone L (1987) Zinc and copper status of allergic children. Acta
    Paediatr Scand, 76: 612-617.

    Di Toro DM, Mahony JD, Hansen DJ, Scott KJ, Hicks MB, Mayr SM, &
    Redmond MS (1990) Toxicity of cadmium in sediments: the role of
    acid volatile sulfide. Environ Toxicol Chem, 9: 1487-1502.

    Di Toro DM, Zarba CS, Hansen DJ, Berry WJ, Swartz RC, Cowan CE,
    Pavlou SP, Allen HE, Thomas NA, & Paquin PR (1991) Technical basis
    for establishing sediment quality criteria for nonionic organic
    chemicals by using equilibrium partitioning. Environ Toxicol Chem,
    10: 1541-1583

    Dodds-Smith ME, Johnson MS, & Thompson DJ (1992a) Trace metal
    accumulation by the shrew Sorex araneus: I. Total body burden,
    growth, and mortality. Ecotoxicol Environ Saf, 24: 102-117.

    Dodds-Smith ME, Johnson MS, & Thompson DJ (1992b) Trace metal
    accumulation by the shrew Sorex araneus: II. Tissue distribution in
    kidney and liver. Ecotoxicol Environ Saf, 24: 118-130.

    Dodge EE & Theis TL (1979) Effect of chemical speciation on the
    uptake of copper by Chironomus tentans. Environ Sci Technol, 13:

    Doelman P & Haanstra L (1984) Short-term and long-term effects of
    cadmium, chromium, copper, nickel, lead and zinc on soil microbial
    respiration in relation to abiotic soil factors. Plant Soil, 79:

    Doelman P & Haanstra L (1986) Short- and long-term effects of heavy
    metals on urease activity in soils. Biol Fertil Soils, 2: 213-218.

    Donat JR, Lao KA, & Bruland KW (1994) Speciation of dissolved
    copper and nickel in South San Francisco Bay: a multi-method
    approach. Anal Chim Acta, 284: 547-571.

    Donkin SG & Dusenbery DB (1993) A soil toxicity test using the
    nematode Caenorhabditis elegans and an effective method of
    recovery. Arch Environ Contam Toxicol, 25: 145-151.

    Dörner K, Dziadzka S, Hohn A, Sievers E, Oldigs HD, Schulz-Lell G,
    & Schaub J (1989) Longitudinal manganese and copper balances in
    young infants and preterm infants fed on breast-milk and adapted
    cow's milk formulas. Br J Nutr, 61(3): 559-572.

    Dreosti IE & Record IR (1978) Lysosomal stability, superoxide
    dismutase and zinc deficiency in regenerating rat liver. Br J Nutr,
    40(1): 133-137.

    Drummond JG, Aranyi C, Schiff LJ, Fenters JD, & Graham JA (1986)
    Comparative study of various methods used for determining health
    effects of inhaled sulfates. Environ Res, 41: 514-528.

    Duby P (1980) Extractive metallurgy. In: Kirk-Othmer encyclopedia
    of chemical technology, 3rd ed. New York, John Wiley & Sons Ltd, pp
    739, 767.

    Dumontet S, Dinel H, & Lévesque PEN (1992) The distribution of
    pollutant heavy metals and their effect on soil respiration and
    acid phosphatase activity in mineral soils of the Rouyn-Noranda
    region, Québec. Sci Total Environ, 121: 231-245.

    Dumontet S, Levesque M, & Mathur SP (1990) Limited downward
    migration of polluted metals (Cu, Zn, Ni and Pb) in acidic virgin
    peat soils near a smelter. Water Air Soil Pollut, 49: 329-342.

    Dunn MA, Green MH, & Leach RM (1991) Kinetics of copper metabolism
    in rats: A compartmental model. Am J Physiol, 261: E115-E125.

    Dutka BJ & Kwan KK (1981) Comparison of three microbial toxicity
    screening tests with the Microtox test. Bull Environ Contam
    Toxicol, 27: 753-757.

    Duvigneau P & Denaeyer-De Smet S (1963) Cuivre et végétation au
    Katanga. Bull Soc R Bot Belg, 96: 93-231.

    Ebele S, Oladimeji AA & Daramola JA (1990) Molluscicidal and
    piscisidal properties of copper(II) tetraoxosulfate(VI) on
     Bulinus globosus (Morelet) and  Clarias anguillaris (L.). Aquat
    Toxicol, 17: 231-238.

    Eden A & Green HH (1939) The fate of copper in the blood stream. J
    Comp Pathol Ther, 52: 301-315.

    Effler SW, Litten S, Field SD, Tong-Ngork T, Hale F, Meyer M, &
    Quirk M (1980) Whole lake response to low level copper sulfate
    treatment. Water Res, 14: 1489-1499.

    Ehrenkranz RA, Gettner PA, Nelli CM, Sherwonit EA, Williams JE,
    Ting BTG, Janghorbani M (1989) Zinc and copper nutritional studies
    in very low birth weight infants: comparison of stable isotopic
    extrinsic tag and chemical balance methods. Pediatr Res, 26:

    Eife R, Reiter K, Sigmund B, Schramel P, Dieter HH, & Müller-Hocker
    J (1991) [Childhood liver cirrhosis as a result of copper
    intoxication]. Bundesgesundh. bl, 32: 327-329 (in German).

    Eldad A, Wisoki M, Cohen H, Breiterman S, Chaouat M, Wexler MR, &
    Ben-Bassat H (1995) Phosphorous burns: evaluation of various
    modalities for primary treatment. J Burn Care Rehabil, 16: 49-55.

    Ellgaard EG & Guillot JL (1988) Kinetic analysis of the swimming
    behaviour of bluegill sunfish, Lepomis macrochirus Rafinesque,
    exposed to copper: hypoactivity induced by sublethal
    concentrations. J Fish Biol, 33: 601-608.

    Elliott NG, Swain R, & Ritz DA (1985) The influence of cyclic
    exposure on the accumulation of heavy metals by Mytilus edulis
    planulatus (Lamarck). Mar Environ Res, 15: 17-30.

    Elliott HA, Leberati MR, & Huang CP (1986) Competitive adsorption
    of heavy metals by soil. J Environ Qual, 15: 214-219.

    El-Sharouny HMM, Bagy MM, & El-Shanawany AA (1988) Toxicity of
    heavy metals to Egyptian soil fungi. Int Biodeterior, 24: 49-64.

    Epstein O (1983) Liver copper in health and disease. Postgrad Med
    J, 59(suppl 4): 88-94.

    Erickson RJ, Benoit DA, & Mattson VR (1987) A prototype toxicity
    factors model for site-specific copper water quality criteria.
    Duluth, Minnesota, US Environmental Protection Agency.

    Erickson RJ, Benoit DA, Mattson VR, Nelson HP, & Leonard EN (1996)
    The effects of water chemistry on the toxicity of copper to fathead
    minnows. Environ Toxicol Chem, 15(2): 181-193.

    Evans GW (1973) Copper homeostasis in the mammalian system. Physiol
    Rev, 53: 535-569.

    Evans EG, Evans GF, & Ray DB (1984) Air quality data for metals
    1977 through 1979 from the Naito Air Surveillance Networks.
    Research Triangle Park, North Carolina, US Environmental Protection

    Office of Research and Development Monitoring Laboratory (EPA

    Fabiano M, Baffi F, Povero P, & Frache R (1988) Particulate organic
    matter and heavy metals in Lingurian open sea. Chem Ecol, 3:

    Farooqui A, Kulshreshtha K, Srivastava K, Farooqui SA, Pandey V, &
    Ahmad KJ (1995) Photosynthesis, stomatal response and metal
    accumulation in  Cineraria maritima L. and  Centauria moschata L.
    grown in metal-rich soil. Sci Total Environ, 164(3): 203-207.

    Farquharson C, Duncan A, & Robins SP (1989) The effects of copper
    deficiency on the pyridinium crosslinks of mature collagen in the
    rat skeleton and cardiovascular system. Proc Soc Exp Biol Med, 192:

    FDO (1965) Appraisal of the safety of chemicals in foods, drugs and
    cosmetics. Washington, DC, Editorial Committee of the Association
    of Food and Drug Officials of the United States.

    Felix K, Nagel W, Hartmann HJ, & Weser U (1990) Copper transfer
    through the intestinal wall. Serosal release of metallothionein.
    Biol Metab, 3: 141-145.

    Fergusson JE & Stewart C (1992) The transport of airborne trace
    elements copper, lead, cadmium, zinc and manganese from a city into
    rural areas. Sci Total Environ, 121: 247-269.

    Fernandes JC & Henriques FS (1991) Biochemical, physiological, and
    structural effects of excess copper in plants. Bot Rev, 57(3):

    Ferrando MD & Andreu E (1993) Feeding behavior as an index of
    copper stress in  Daphnia magna and  Brachionus calyciflorus.
    Comp Biochem Physiol, 106C(2): 327-331.

    Ferrando MD, Andreu-Moliner E, & Fernández-Casalderrey A (1992)
    Relative sensitivity of  Daphnia magna and  Brachionus
    calyciflorus  to five pesticides. J Environ Sci Health, B27(5):

    Ferrando MD, Janssen CR, Andreu E, & Persoone G (1993)
    Ecotoxicological studies with the freshwater rotifer
     Brachionus calyciflorus: III. The effects of chemicals on the
    feeding behavior. Ecotoxicol Environ Saf, 26(1): 1-9.

    Fewtrell L, Kay D, Jones F, Baker A, & Mowat A (1996) Copper in
    drinking water -- an investigation into possible health effects.
    Public Health, 110: 175-177.

    Fields M, Ferreti RJ, Smith JC Jr & Reiser S (1984) The interaction
    of type of dietary carbohydrates with copper deficiency. Am J Clin
    Nutr, 39: 289-295.

    Filipek LH, Nordstrom DK, & Ficklin WH (1987) Interaction of acid
    mine drainage with waters and sediments of West Squaw Creek in the
    West Shasta mining district, California. Environ Sci Technol, 21:

    Finley EB & Cerklewski FL (1983) Influence of ascorbic acid
    supplementation on copper status in young adult men. Am J Clin
    Nutr, 37: 553-556.

    Fischer PW, Giroux A, & L'Abbé MR (1983) Effects of zinc on mucosal
    copper binding and on the kinetics of copper absorption. J Nutr,
    113: 462-469.

    Fischer PW, L'Abbé MR, & Giroux A (1990) Effects of age, smoking,
    drinking, exercise and estrogen use on indices of copper status in
    healthy adults. Nutr Res, 10: 1081-1090.

    Fischer JG, Tackett RL, Howerth EW, & Johnson MA (1992) Copper and
    selenium deficiencies do not enhance the cardiotoxicity in rats due
    to chronic doxorubicin treatment. J Nutr, 122: 2128-2137.

    Fisher D (1992) Copper. In: Sullivan JB & Krieger GR ed. Hazardous
    materials toxicology: Clinical principles of environmental health.
    Baltimore, Maryland, Williams & Wilkins, pp 860-864.

    Fjeldstad H, Hvatum OO, & Bjorndalen JE (1988) Heavy metal
    pollution of ombrotrophic bogs in the Kristiansand area,
    Vest-Agder, Norway. Nor J Agric Sci, 2(2): 161-177.

    Flemming CA & Trevors JT (1988) Effect of copper on nitrous oxide
    reduction in freshwater sediment. Water Air Soil Pollut, 40:

    Fleurent E & Levi L (1920) Sur la présence du cuivre dans
    l'organisme végétal et animal. Bull Soc Chim France, 27: 440.

    Florence TM (1989) Electrochemical techniques for trace element
    speciation in waters. In: Batley GE ed. Trace element speciation:
    Analytical methods and problems. Boca Raton, Florida, CRC Press, pp

    Florence TM, Morrison GM, & Stauber JL (1992) Determination of
    trace element speciation and the role of speciation in aquatic
    toxicity. Sci Total Environ, 125: 1-13.

    Flynn A, Franzmann AW, & Arneson PD (1976) Molybdenum-sulfur
    interactions in the utilization of marginal dietary copper in
    Alaskan moose  (Alces alces gigas). In: Chappel WR & Petersen KK
    ed. Molybdenum in the environment -- Volume 1: The biology of
    molybdenum. New York, Marcel Dekker, Inc., pp 115-124.

    Flynn A, Franzmann AW, Arneson PD, & Oldemeyer JL (1977)
    Indications of copper deficiency in a subpopulation of Alaskan
    moose. J Nutr, 107(7): 1182-1189.

    Förstner U & Wittmann GTW (1979) Metal pollution in the aquatic
    environment. Berlin, Springer-Verlag.

    Förstner U & Wittmann GTW (1981) Metal pollution in the aquatic
    environment, 2nd ed. New York, Basel, Springer-Verlag.

    Fraser WD, Taggart DP, Fell GS, Lyon TDB, Wheatley D, Garden OJ, &
    Shenken A (1989) Changes in iron, zinc, and copper concentrations
    in serum and in their binding to transport proteins after
    cholecystectomy and cardiac surgery. Clin Chem, 35(11): 2243-2247.

    Freedman JH, Ciriolo MR, & Peisach J (1989) The role of glutathione
    in copper metabolism and toxicity. J Biol Chem, 264: 5598-5605.

    Frenckell-Insam BAK & Hutchinson TC (1993) Occurrence of heavy
    metal tolerance and co-tolerance in  Deschampsia cespitosa (L.)
    Beauv. from European and Canadian populations. New Phytol, 125:

    Freundt KJ & Ibrahim HA (1991) Influence of Pb, Cd, Zn, Mn, Cu, Hg,
    or Be salts on the glutathione S-transferases of the rat liver.
    Bull Environ Contam Toxicol, 46: 618-624.

    Friberg L, Nordberg GF, & Vouk VB (1979) Handbook on the toxicology
    of metals. Amsterdam, Elsevier/North Holland Biomedical Press.

    Frieden E & Hsieh HS (1976) The biological role of ceruloplasmin
    and its oxidase activity. Adv Exp Med Biol, 74: 505-529.

    Frommer DJ (1981) Urinary copper excretion and hepatic copper
    concentrations in liver disease. Digestion, 21: 169-178.

    Frostegĺrd Ĺ, Tunlid A, & Bĺĺth E (1993) Phospholipid fatty acid
    composition, biomass, and activity of microbial communities from
    two soil types experimentally exposed to different heavy metals.
    Appl Environ Microbiol, 59: 3605-3617.

    Frydman M, Bonne-Tamir B, Farrer LA, Conneally PM, Magazanik A,
    Ashbel S, & Goldwitch Z (1985) Assignment of the gene for Wilson's
    disease to chromosome 13: linkage to the esterase D locus. Proc
    Natl Acad Sci (USA), 82(6): 1819-1821.

    Gabuchyan VV (1987) Impairment mechanism of the reproductive
    function ni cuprum chloride-exposed white male rats. Gig Tr Prof
    Zabol, 31(9): 28-31.

    Gadh R, Tandon SN, Mathur RP, & Singh OV (1993) Speciation of
    metals in Yamuna river sediments. Sci Total Environ, 136: 229-242.

    Ganezer KS, Hjart ML, & Carnes WH (1976) Tensile properties of
    tendon in copper deficient swine. Proc Soc Exp Biol Med, 153:

    Garcia-Sanchez F, Navas-Diaz A, & Medinilla J (1990) Mineralization
    procedure for determination of copper in aerosols using photometric
    method based on copper-BPKQH complex. J Assoc Off Anal Chem, 73:

    Gardner M & Ravenscroft J (1991) The behaviour of copper
    complexation in rivers and estuaries: two studies in North-East
    England. Chemosphere, 23: 695-713.

    Gauss JD, Woods PE, Winner RW, & Skillings JH (1985) Acute toxicity
    of copper to three life stages of Chironomus tentans as affected by
    water hardness-alkalinity. Environ Pollut, A37: 149-157.

    Geckler JR, Horning WB, Neiheisel TM, Pickering QH, Robinson EL, &
    Stephan CE (1976) Validity of laboratory tests for predicting
    copper toxicity in streams. Duluth, Minnesota, US Environmental
    Protection Agency (EPA-600/3-76-116).

    Gerdes AM, Tonnesen T, Pergament E, Sander C, Baerlocher KE, Wartha
    R, Guttler F, & Horn N (1988) Variability in clinical expression of
    Menkes syndrome. Eur J Pediatr, 148(2): 132-135.

    Germani MS, Small M, Zoller WH, & Moyers JL (1981) Fractionation of
    elements during copper smelting. Environ Sci Technol, 15: 299-305.

    Gettier SW, Martens DC, & Kornegay ET (1988) Corn response to six
    annual Cu-enriched pig manure applications to three soils. Water
    Air Soil Pollut, 40: 409-418.

    Giesy JP, Alberts JJ, & Evans DW (1986) Conditional stability
    constants and binding capacities for copper (II) by dissolved
    organic carbon isolated from surface waters of the southeastern
    United States. Environ Toxicol Chem, 5: 139-154.

    Giesy JP, Newell A, & Leversee GJ (1983) Copper speciation in soft
    acid humic water: effect on copper bioaccumulation by and toxicity
    to  Simocephalus serrulatus. Sci Total Environ, 28: 23-36.

    Gintenreiter S, Ortel J, & Nopp HJ (1993) Effects of different
    dietary levels of cadmium, lead, copper, and zinc on the vitality
    of the forest pest insect  Lymantria dispar L. (Lymantriidae,
    Lepid.). Arch Environ Contam Toxicol, 25: 62-66.

    Gleason RP (1968) Exposure to copper dust. Am Ind Hyg Assoc J, 29:

    Gold LS, Sawyer CB, Magaw R, Backman GM, de Veciana M, Levinson R,
    Hooper NK, Havender WR, Bernstein L, Peto R, Pike MC, & Ames BN
    (1984) A carcinogenic potency database of the standardized results
    of animal bioassays. Environ Health Perspect, 58: 9-319.

    Goldstein S & Czapski G (1986) The role and mechanism of metal ions
    and their complexes in enhancing damage in biological systems or in
    protecting these systems from the toxicity of O2-. J Free Radic
    Biol Med, 2(1): 3-11.

    Gollan JL & Deller DJ (1973) Studies on the nature and excretion of
    biliary copper in man. Clin Sci, 44: 9-15.

    Gooneratne SR, Chaplin RK, Trent AM, & Christensen DA (1989) Effect
    of tetrathiomolybdate administration on the excretion of copper,
    zinc, iron and molybdenum in sheep bile. Br Vet J, 145: 62-72.

    Gormally SM, Baker A, Portmann B, Mowat A, & Drumm B (1994) High
    water copper content associated with Indian childhood cirrhosis in
    European children. Gastroenterology, 106: A900 (abstract).

    Gorzelska K (1989) Locally generated atmospheric trace metal
    pollution in Canadian Arctic as reflected by chemistry of snowpack
    samples from the Mackenzie delta region. Atmos Environ, 22:

    Goyer RA (1991) Toxic effects of metals. In: Andur MO, Doull J, &
    Klaassen CD ed. Casarett and Doull's toxicology: The basic science
    of poisons, 4th ed. Oxford, New York, Pergamon Press, chapter 10,
    pp 623-680.

    Graham JH, Timmer LW, & Fardelmann D (1986) Toxicity of fungicidal
    copper in soil to citrus seedlings and vesicular-arbuscular
    mycorrhizal fungi. Phytopathology, 76: 66-70.

    Gralla EB, Thiele DJ, Silar P, & Valentine JS (1991) ACE1, a copper
    dependent transcription factor, activates expression of the yeast
    copper, zinc superoxide dismutase gene. Proc Natl Acad Sci (USA),
    88: 8558-8562.

    Grant A, Hateley JG, & Jones NV (1989) Mapping the ecological
    impact of heavy metals on the estuarine polychaete Nereis
    diversicolor using inherited metal tolerance. Mar Pollut Bull,
    20(5): 235-238.

    Grant LD, Elias R, Nicholson W, Goyer R, & Olem H (1990) Indirect
    health effects associated with acidic deposition. In: State of
    science and technology. National Acid Precipitation Assessment
    Program (NAPAP), pp 23-33 (Report No. 23).

    Greene FL, Lamb LS, Barwick M, & Pappas NJ (1987) Effect of dietary
    copper on colonic tumor production and aortic integrity in the rat.
    J Surg Res, 42: 503-512.

    Greger JL & Mulvaney J (1985) Absorption and tissue distribution of
    zinc, iron and copper by rats fed diets containing lactalbumin, soy
    and supplemental sulfur-containing amino acids. J Nutr, 115:

    Greger JL, Zaikis SC, Abernathy RP, Bennett OA, & Huffman J (1978)
    Zinc, nitrogen, copper, iron, and manganese balance in adolescent
    females fed two levels of zinc. J Nutr, 108(9): 1449-1456.

    Gregory JR, Collins DL, Davies PSW, Hughes JM, & Clarke PC (1995)
    National diet and nutrition survey: children aged 1 ´ to 4 ´ years
    -- Volume 1: Report of the diet and nutrition survey. London, Her
    Majesty's Stationary Office (HMSO), pp 177-201.

    Gross JB Jr, Miers BM, Kost LJ, Kuntz SM, & La Russo NF (1989)
    Biliary copper excretion by hepatocyte lysosomes in the rat. Mayor
    excretory pathway in experimental copper overload. J Clin Invest,
    83: 30-39.

    Groudev SN & Groudeva VI (1993) Microbial communities in four
    industrial copper dump leaching operations in Bulgaria. FEMS
    Microbiol Rev, 11: 261-267.

    Guerihault B (1920) Sur la présence du cuivre dans les plantes et
    particuličrement dans les matičres alimentaires d'origine végétale.
    C R Soc Biol, 171: 196.

    Haanstra L & Doelman P (1984) Glutamic acid decomposition as a
    sensitive measure of heavy metal pollution in soil. Soil Biol
    Biochem, 16: 595-600.

    Haanstra L & Doelman P (1991) An ecological dose-response model
    approach to short- and long-term effects of heavy metals on
    arylsulphatase activity in soil. Biol Fertil Soils, 11: 18-23.

    Haas RH, Robinson A, Evans K, Lascelles PT, & Dubowitz V (1981) An
    X-linked disease of the nervous system with disordered copper
    metabolism and features differing from Menkes disease. Neurology,
    31(7): 852-859.

    Hackel H, Miller K, Elsner P, & Burg G (1991) Unusual combined
    sensitization to palladium and other metals. Contact Dermatitis,
    24: 131-132.

    Haddad DS, Al-Alousi LA, & Kantarjian AH (1991) The effect of
    copper loading on pregnant rats and their offspring. Funct Dev
    Morphol, 1: 17-22.

    Hĺkansson K, Karlsson S, & Allard B (1989) Effects of pH on the
    accumulation and redistribution of metals in a polluted stream bed
    sediment. Sci Total Environ, 87/88: 43-57.

    Hall HC (1921) La dégénérescence hépato-lenticulaire: Maladie de
    Wilson-pseudosclérose. Paris, Editions Masson, pp 190-192.

    Hall A (1981) Copper accumulation in copper-tolerant and
    non-tolerant populations of the marine fouling alga  Ectocarpus
    siliculosus  (Dillw.) Lyngbze. Bot Mar 24: 223-228.

    Hall A, Fielding AH, & Butler M (1979) Mechanism of copper
    tolerance in the marine fouling alga  Ectocarpus siliculosus:
    Evidence for an exclusion mechanism. Mar. Biol., 54: 195-199

    Hall WS, Bushong SJ, Hall LW, Lenkevich MJ, & Pinkney AE (1988)
    Monitoring dissolved copper concentrations in Chesapeake Bay, USA.
    Environ Monit Assess, 11(1): 33-42.

    Hall LW, Unger MA, Ziegenfuss MC, Sullivan JA, & Bushong SJ (1992)
    Butyltin and copper monitoring in a northern Chesapeake Bay marina
    and river system in 1989: an assessment of tributyltin legislation.
    Environ Monit Assess, 22(1) 15-38.

    Hamer DH (1986) Metallothionein. Annu Rev Biochem, 55: 913-951.

    Hamilton SJ & Buhl KJ (1990) Safety assessment of selected
    inorganic elements to fry of chinook salmon  (Oncorhynchus
    tshawytscha).  Ecotoxicol Environ Saf, 20: 307-324.

    Hamilton EI, Minski MJ, & Cleary JJ (1972) The concentration and
    distribution of some stable elements in healthy human tissues from
    the United Kingdom. Sci Total Environ, 1: 341-361.

    Han B-C & Hung T-C (1990) Green oysters caused by copper pollution
    on the Taiwan coast. Environ Pollut, 65: 347-362.

    Han B-C, Jeng W-L, Tsai Y-N, & Jeng M-S (1993) Depuration of copper
    and zinc by green oysters and blue mussels of Taiwan. Environ
    Pollut, 82: 93-97.

    Hansen IV, Weeks JM, & Depledge MH (1995) Accumulation of copper,
    zinc, cadmium and chromium by the marine sponge  Halichondria
    panicea Pallas and the implications for biomonitoring. Mar Pollut
    Bull, 31(1-3): 133-138.

    Hanson MJ & Stefan HG (1984) Side effects of 58 years of copper
    sulfate treatment of the Fairmont Lakes, Minnesota. Water Resour
    Bull, 20(6): 889-900.

    Haraldsson C & Westerlund S (1988) Trace metals in the water
    columns of the Black Sea and Framvaren Fjord. Mar Chem, 23(3-4):

    Harless E (1847) [About the blue blood of some non-vertebrate
    animals and its copper content.] Muller's Arch Anat Physiol, 1847:
    148 (in German).

    Harris ED (1991) Copper transport: An overview. Proc Soc Exp Biol
    Med, 196: 130-140.

    Harris ED (1995) The iron-copper connection: the link to
    ceruloplasmin grows stronger. Nutr Rev, 53: 170-173.

    Harris ED & DiSilvestro RA (1981) Correlation of lysyl oxidase
    activation with the p-phenylenediamine oxidase activity
    (ceruloplasmin) in serum. Proc Soc Exp Biol Med, 166(4): 528-531.

    Harris ZL & Gitlin JD (1996) Genetic and molecular basis for copper
    toxicity. Am J Clin Nutr, 63(suppl): 836S-841S.

    Harris ED & Percival SS (1991) A role for ascorbic acid in copper
    transport. Am J Clin Nutr, 54: 1193S-1197S.

    Harris ED, Gonnerman WA, Savage JE, & O'Dell BL (1974) Connective
    tissue amine oxidase. II. Purification and partial characterization
    of lysyl oxidase from chick aorta. Biochim Biophys Acta, 341(2):

    Harris ED, Blount JE, & Leach RM (1980) Localization of
    lysyloxidase in hen oviduct: implications in egg shell membrane
    formation and composition. Science, 208: 55-56.

    Harrisson JWE, Levin SE, & Trabin B (1954) The safety and fate of
    potassium sodium copper chlorophyllin and other copper compounds.
    J Am Pharm Assoc, 43: 722-737.

    Hart EB, Steenbock H, Waddell J, & Elvehjem CA (1928) Iron in
    nutrition: VII. Copper as a supplement to iron for hemoglobin
    building in the rat. J Biol Chem, 77: 797-812.

    Hart BT, Currey NA, & Jones MJ (1992) Biogeochemistry and effects
    of copper, manganese and zinc added to enclosures in Island
    Billabong, Magela Creek, northern Australia. Hydrobiologia, 230:

    Hartmann HA & Evenson MA (1992) Deficiency of copper can cause
    neuronal degeneration. Med Hypotheses, 38: 75-85.

    Hartter DE & Barnea A (1988) Brain tissue accumulates 67 copper by
    two ligand-dependent saturable processes. A high affinity, low
    capacity and a low affinity, high capacity process. J Biol Chem,
    263(2): 799-805.

    Hartwell SI, Jin JH, Cherry DS, & Cairns J (1989) Toxicity versus
    avoidance response of golden shiner,  Notemigonus crysoleucas, to
    five metals. J Fish Biol, 35: 447-456.

    Haschke F, Ziegler EE, Edwards BB, & Fomon SJ (1986) Effect of iron
    fortification of infant formula on trace mineral absorption. J
    Pediatr Gastroenterol Nutr, 5: 768-773.

    Hasegawa R, Nakaji Y, Kurokawa Y, & Tobe M (1989) Acute toxicity
    tests on 113 environmental chemicals. Research Institute of the
    Tohoku University, pp 10-16 (Scientific Report No. 36).

    Hatakeyama S (1988) Chronic effects of Cu on reproduction of
     Polypedilumium nubifer (Chironomidae) through water and food.
    Ecotoxicol Environ Saf, 16: 1-10.

    Havens KE (1994a) An experimental comparison of the effects of two
    chemical stressors on a freshwater zooplankton assemblage. Environ
    Pollut, 84: 245-251.

    Havens KE (1994b) Structural and functional responses of a
    freshwater plankton community to acute copper stress. Environ
    Pollut, 86: 259-266.

    Hayton BA, Broome HE, & Lilenbaum RC (1995) Copper
    deficiency-induced anemia and neutropenia secondary to intestinal
    malabsorption. Am J Hematol, 48(1): 45-47.

    Haywood S (1980) The effect of excess dietary copper on the liver
    and kidney of the male rat. J Comp Pathol, 90: 217-232.

    Haywood S (1985) Copper toxicosis and tolerance in the rat: I.
    Changes in copper content of the liver and kidney. J Pathol, 145:

    Haywood S & Comerford B (1980) The effect of excess dietary copper
    on plasma enzyme activity and on the copper content of the blood of
    the male rat. J Comp Pathol, 90: 233-238.

    Haywood S & Loughran M (1985) Copper toxicosis and tolerance in the
    rat: II. Tolerance -- a liver protective adaptation. Liver, 5:

    Heath AG (1991) Effect of water-borne copper on physiological
    responses of bluegill (Lepomis macrochirus) to acute hypoxic stress
    and subsequent recovery. Comp Biochem Physiol, 100C: 559-564.

    Hébert CD (1993) NTP technical report on toxicity studies of cupric
    sulfate (CAS No. 7758-99-8) administered in drinking water and feed
    to F344/N rats and B6C3F1 mice. Research Triangle Park, North
    Carolina, United States Department of Health and Human Services,
    National Toxicology Program, 94 pp (NTP Toxicity Report Series No.
    29; NIH Publication 93-3352).

    Hébert CD, Elwell MR, Travlos GS, Fitz CJ, & Bucher JR (1993)
    Subchronic toxicity of cupric sulfate administered in drinking
    water and feed to rats and mice. Fundam Appl Toxicol, 21: 461-475.

    Hedberg T, Vik AE, & Ferguson J ed. (1996) Report from the
    International Seminar and Workshop on Internal Corrosion in Water
    Distribution Systems, Göteborg, 22-27 May 1995. Göteborg, Sweden,
    Göteborg University.

    Hedtke SF (1984) Structure and function of copper-stressed aquatic
    microcosms. Aquat Toxicol, 5: 227-244.

    Helios Rybicka E, Wilson MJ, & Mchard WJ (1994) Chemical and
    mineralogical forms and mobilization of copper and lead in soils
    from a Cu-smelting area in Poland. J Environ Sci Health, A29(3):

    Heller RM, Kirchner SG, O'Neill JA Jr, Hough AJ Jr, Howard L,
    Kramer SS, & Green HL (1978) Skeletal changes of copper deficiency
    in infants receiving prolonged total parenteral nutrition. J
    Pediatr, 92(6): 947-949.

    Helz GR, Hugget RJ, & Hill JM (1975) Behavior of Mn, Fe, Cu, Cd,
    and Pb discharged from a wastewater treatment plant into an
    estuarine environment. Water Res, 9: 631-636.

    Henry CL & Harrison RB (1992) Fate of trace metals in sewage sludge
    compost. In: Domy CA ed. Biogeochemistry of trace metals. Boca
    Raton, Florida, Lewis Publishers, pp 195-216.

    Heresi G, Castillo-Durán C, Muńoz C, Arevalo M, & Schlesinger L
    (1985) Phagocytosis and immunoglobulins levels in hypocupremic
    infants. Nutr Res, 5: 1327-1334.

    Hill R & Williams HL (1965) The effects on intensively reared lambs
    of diets containing excess copper. Vet Rec, 77(36): 1043-1045.

    Hirano S, Ebihara H, Sakai S, Kodama N, & Suzuki KT (1993)
    Pulmonary clearance and toxicity of intratracheally instilled
    cupric oxide in rats. Arch Toxicol, 67: 312-317.

    Hirano S, Sakai S, Ebihara H, Kodama N, & Suzuki KT (1990)
    Metabolism and pulmonary toxicity of intratracheally instilled
    cupric sulfate in rats. Toxicology, 64(3): 223-233.

    Holak W (1983) Determination of copper, nickel, and chromium in
    foods. J Assoc Off Anal Chem, 66: 620-624.

    Holdbrook JT, Smith JC Jr, & Reiser S (1989) Dietary fructose or
    starch: effects on copper, zinc, iron, manganese, calcium, and
    magnesium balances in humans. Am J Clin Nutr, 49: 1290-1294.

    Holmgren GGS, Meyer MW, Chaney RL, & Daniels RB (1993) Cadmium,
    lead, zinc, copper, and nickel in agricultural soils of the United
    States of America. J Environ Qual, 22: 335-348.

    Holtzman NA, Charache P, Cordano A, & Graham GG (1970) Distribution
    of serum copper in copper deficiency. John Hopkins Med J, 126:

    Holwerda DA (1991)Cadmium kinetics in freshwater clams: V.
    Cadmium-copper interactions in metal accumulation by  Anadonta
    cygnea and characterization of the metal-binding protein. Arch
    Environ Contam Toxicol, 21: 432-437.

    Hong S, Candelone J-P, Patterson CC, & Boultron CF (1996) History
    of ancient copper smelting pollution during Roman and Medieval
    times recorded in Greenland ice. Science, 272: 246-247.

    Hoogenraad TU & van den Hamer CJA (1983) Three years of continuous
    oral zinc therapy in tour patients with Wilson's disease. Acta
    Neurol Scand, 67: 356-364.

    Hopkin SP (1993) Deficiency and excess of copper in terrestrial
    isopods. In: Dallinger R & Rainbow PS ed. Ecotoxicology of metals
    in invertebrates. Boca Raton, Florida, Lewis Publishers, pp

    Hopkin R & Kain JM (1978) The effects of some pollutants on the
    survival, growth and respiration of  Laminaria hyperborea. Estuar
    Coast Mar Sci, 7(6): 531-554.

    Hopkin SP, Hardisty GN, & Martin MH (1986) The woodlouse
     Porcellio scaber as a 'biological indicator' of zinc, cadmium,
    lead and copper pollution. Environ Pollut, B11: 271-290.

    Hopkin SP, Jones DT, & Dietrich D (1993) The isopod Porcellio
    scaber as a monitor of the bioavailability of metals in terrestrial
    ecosystems: towards a global "woodlouse watch" scheme. Sci Total
    Environ, 1(suppl): 357-365.

    Hopper SH & Adams HS (1958) Copper poisoning from vending machines.
    Public Health Rep, 73: 910-914.

    Horning WB & Neiheisel TW (1979) Chronic effect of copper on the
    bluntnose minnow, Pimephales notatus (Rafinesque). Arch Environ
    Contam Toxicol, 8: 545-552.

    Howarth RS & Sprague JB (1978) Copper lethality to rainbow trout in
    waters of various hardness and pH. Water Res, 12: 455-462.

    Howeler RH (1983) [Study of some tropical plants for the diagnosis
    of nutritional problems.] Cali, Colombia, International Centre for
    Tropical Agriculture, 28 pp (in Spanish).

    Howell JS (1958) The effect of copper acetate on
     p-dimethylaminoazobenzene carcinogenesis in the rat. Br J Cancer,
    12: 594-610.

    Huber MC, Winter REK, & Bolla RI (1989) Effect of copper sulfate
    and lead acetate on infection of pines with  Bursaphelenchus
    xylophilus.  J Nematol, 21: 1-9.

    Hunt CE & Carlton WW (1965) Cardiovascular lesions associated with
    experimental copper deficiency in the rabbit. J Nutr, 87: 385-393.

    Hunt CE, Carlton WW, & Newberne PM (1970) Interrelationships
    between copper deficiency and dietary ascorbic acid in the rabbit.
    Br J Nutr, 24: 61-69.

    Hunter BA & Johnson MS (1982) Food chain relationships of copper
    and cadmium in contaminated grassland ecosystems. Oikos, 38:

    Hunter BA, Johnson MS, & Thompson DJ (1987a) Ecotoxicology of
    copper and cadmium in a contaminated grassland ecosystem: I. Soil
    and vegetation contamination. J Appl Ecol, 24: 573-586.

    Hunter BA, Johnson MS, & Thompson DJ (1987b) Ecotoxicology of
    copper and cadmium in a contaminated grassland ecosystem: II.
    Invertebrates. J Appl Ecol, 24: 587-599.

    Hunter BA, Johnson MS, & Thompson DJ (1987c) Dynamics of metal
    accumulation in the grasshopper Chorthippus brunneus in
    contaminated grasslands. Arch Environ Contam Toxicol, 16: 711-716.

    Hurley LS & Keen CL (1988) Fetal and neonatal development in
    relation to maternal trace element nutrition: manganese, zinc, and
    copper. In: Berger H ed. Vitamins and minerals in pregnancy and
    lactation. New York, Raven Press Ltd, pp 215-230 (Nestle Nutrition
    Workshop Series, Volume 16).

    Hutchinson TH, Williams TD, & Eales GJ (1994) Toxicity of cadmium,
    hexavalent chromium and copper to marine fish larvae
     (Cyprinodon variegatus) and copepods  (Tisbe battagliai). Mar
    Environ Res, 38: 275-290.

    IARC (1977) Copper 8-hydroxygquinoline. In: Some fumigants, the
    herbicides 2,4-D and 2,4,5-T chlorinated dibenzodioxins and
    miscellaneous industrial chemicals. Lyon, International Agency for
    Research on Cancer, pp 103-110 (IARC Monographs on the Evaluation
    of the Carcinogenic Risk of Chemicals to Humans, Volume 15).

    IARC (1987) Overall evaluations of carcinogenicity: an updating of
    IARC monographs volumes 1 to 42. Lyon, International Agency for
    Research on Cancer, p 61 (IARC Monographs on the Evaluation of the
    Carcinogenic Risk of Chemicals to Humans, Supplement 7).

    IAT (Institute of Animal Technicians) (1963) In: Short DJ &
    Woodnott DP ed. Manual of laboratory animal practice techniques.
    London, Crosby Lockwood & Son Ltd.

    ICME (1995) Persistence, bis-accumulation and toxicity of metals
    and metal compounds. Ottawa, Canada, International Council on
    Metals and the Environment, 93 pp.

    ICSG (1996) World refinery production of copper. Lisbon, Portugal,
    The International Copper Study Group, pp 13-15 (table 5) (Copper
    Bulletin, Volume 3).

    ILO (1991) Occupational exposure limits for airborne toxic
    substances, 3rd ed. Geneva, International Labour Organisation
    (Occupational Safety and Health Series, No. 37).

    Ingersoll CG & Winner RW (1982) Effect on  Daphnia pulex (De Geer)
    of daily pulse exposures to copper or cadmium. Environ Toxicol
    Chem, 1: 321-327.

    IPCS (1994) Environmental health criteria 170: Assessing human
    health risks of chemicals: Derivation of guidance values for
    health-based exposure limits. Geneva, World Health Organization,
    International Programme on Chemical Safety, 73 pp.

    ISO (1986) Water quality -- determination of cobalt, nickel,
    copper, zinc, cadmium and lead -- flame atomic absorption
    spectrometric methods, 1st ed. Geneva, International Standards
    Organization, 11 pp.

    Isolda A & Hayasaka SS (1991) Effect of herbicide residues on
    microbial processes in pond sediment. Arch Environ Contam Toxicol,
    20: 81-86.

    Iwata M, Hirano A, & French JH (1979) Degeneration of the
    cerebellar system in X-chromosome-linked copper malabsorption. Ann
    Neurol, 5(6): 542-549.

    Iyer VN & Szybalski W (1958) Two simple methods for the detection
    of chemical mutagens. App Microbiol, 6: 23-29.

    Jacob RA, Skala JR, Omaye ST, & Turnlund JR (1987) Effect of
    varying ascorbic acid intakes on copper absorption and
    ceruloplasmin levels of young men. J Nutr, 117: 2109-2115.

    Jacobsen T & Slotfeldt-Ellingsen D (1983) Phytic acid and metal
    availability: A study of Ca and Cu binding. Cereal Chem, 60:

    Jain VK & Mohan G (1991) Serum zinc and copper in myocardial
    infarction with particular reference to prognosis. Biol Trace Elem
    Res, 31: 317-322.

    Jain SK, Vasudevan P, & Jha NK (1989) Removal of some heavy metals
    from polluted water by aquatic plants: studies on duckweed and
    water velvet. Biol Wastes, 28: 115-126.

    Janes N & Playle RC (1995) Modeling silver binding to the gill of
    rainbow trout  (Oncorhynchus mykiss). Environ Toxicol Chem, 14:

    Janssen MPM, Ooszrthoff C, Heijmans GJSM, & van der Voet H (1995)
    The toxicity of metal salts and the population growth of the
    ciliate  Colpoda cucculus. Bull Environ Contam Toxicol, 54:

    Jantsch W, Kulig K, & Rumack BH (1985) Massive copper sulfate
    ingestion resulting in hepatotoxicity. J Toxicol Clin Toxicol,
    22: 585-588.

    Jarvis SC (1978) Copper uptake and accumulation by perennial
    ryegrass grown in soil and solution culture. J Sci Food Agric, 29:

    Jeng SL & Yang CP (1995) Determination of lead, cadmium, mercury
    and copper concentrations in duck. Poultry Sci, 74: 187-193.

    Jenne EA (1987) Sediment quality criteria for metals: IV. Surface
    complexation and acidity constants for modeling cadmium and zinc
    adsorption onto iron oxides. Prepared for the US Environmental
    Protection Agency, Office of Water Regulations and Standards,
    Criteria and Standards Division, Washington, DC.

    Johansson C & Moberg LE (1991) Area ratio effects on metal ion
    release from amalgam in contact with gold. Scand J Dent Res, 99:

    Johansson A, Camner P, Jarstrand C, & Wiernik A (1983) Rabbit
    alveolar macrophages after inhalation of soluble cadmium, cobalt,
    and copper: A comparison with the effects of soluble nickel.
    Environ Res, 31: 340-354.

    Johansson A, Curstedt T, Robertson B, & Camner P (1984) Lung
    morphology and phospholipids after experimental inhalation of
    soluble cadmium, copper, and cobalt. Environ Res, 34(2): 295-309.

    Johnson MW & Gentile JH (1979) Acute toxicity of cadmium, copper,
    and mercury to larval American lobster  Homarus americanus. Bull
    Environ Contam Toxicol, 22: 258-264.

    Johnson PE & Korynta ED (1992) Effects of copper, iron, and
    ascorbic acid on manganese availability to rats. Proc Soc Exp Biol
    Med, 199: 470-480.

    Johnson MA & Murphy CL (1988) Adverse effects of high dietary iron
    and ascorbic acid on copper status in copper-deficient and
    copper-adequate rats. Am J Clin Nutr, 47: 96-101.

    Johnson CA, Sigg L, & Zobrist J (1987) Case studies on the chemical
    composition of fogwater: Influence of local gaseous emissions.
    Atmos Environ, 21: 2365-2374.

    Johnson RK, Ericsson L, & Wiederholm T (1992) Ordination of
    profundal zoobenthos along a trace metal pollution gradient in
    northern Sweden. Water Air Soil Pollut, 65(3/4): 339-351.

    Jorhem L & Sundstrom B (1993) Levels of lead, cadmium, zinc,
    copper, nickel, chromium, manganese, and cobalt in food on the
    Swedish market. 1983-1990. J Food Compos Anal, 6: 223-241.

    Josephs HW (1931) Treatment of anemia of infancy with iron and
    copper. Bull John Hopkins Hosp, 49: 246-258.

    Jungmann J, Reins H-A, Lee J, Romeo A, Hassett R, Kosman D, &
    Jentsch S (1993) MAC1, is a nuclear regulatory protein related to
    Cu-dependent transcription factors involved in CU/Fe utilization
    and stress resistance in yeast. EMBO J, 12: 5051-5056.

    Juste C & Mench M (1992) Long-term application of sewage sludge and
    its effects on metal uptake by crops. In: Domy CA ed.
    Biogeochemistry of trace metals. Boca Raton, Florida, Lewis
    Publishers, pp 159-192.

    Kabata-Pendias A & Pendias H (1984) Trace elements in soils and
    plants. Boca Raton, Florida, CRC Press, Inc., 315 pp.

    Kaitala S (1988) Multiple toxicity and accumulation of heavy metals
    in two bivalve mollusc species. Water Sci Technol, 20(6/7): 23-32.

    Kalac P, Niznanská M, Bevilaqua D, & Stasková I (1996)
    Concentrations of mercury, copper, cadmium and lead in fruiting
    bodies of edible mushrooms in the vicinity of a mercury smelter and
    a copper smelter. Sci Total Environ, 177: 251-258.

    Kaler SG, Gallo LK, Proud VK, Percy AK, Mark Y, Segal NA, Goldstein
    DS, Holmes CS, & Gahl WA (1994) Occipital horn syndrome and a mild
    Menkes phenotype associated with splice site mutations at the MNK
    locus. Nat Genet, 8: 195-202.

    Kanematsu N, Hara M, & Kada T (1980) Rec assay and mutagenicity
    studies on metal compounds. Mutat Res, 77: 109-116.

    Karlberg A-T, Boman A, & Wahlberg JE (1983) Copper -- a rare
    sensitizer. Contact Dermatitis, 9: 134-139.

    Kasama T & Tanaka H (1988) Effects of copper administration on
    fetal and neonatal mice. J Nutr Sci Vitam, 34: 595-605.

    Kataoka M & Tavassoli M (1984) Ceruloplasmin receptors in liver
    cell suspensions are limited to the endothelium. Exp Cell Res, 155:

    Kataoka M & Tavassoli M (1985) Identification of ceruloplasmin
    receptors on the surface of human blood monocytes, granulocytes,
    and lymphocytes. Exp Hematol, 13: 806-810.

    Kawahara D, Oshima H, Kosugi H, Nakamura M, Sugai T, & Tamaki T
    (1993) Further epidemiologic study of occupational contact
    dermatitis in the dental clinic. Contact Dermatitis, 28: 114-115.

    Kay SH, Haller WT, & Garrard LA (1984) Effects of heavy metals on
    water hyacinths  (Eichhornia crassipes (Mart.) Solms). Aquat
    Toxicol, 5: 117-128.

    Keinholz EW (1977) Effects of environmental molybdenum levels upon
    wildlife. In: Chappel WR & Petersen KK ed. Molybdenum in the
    environment -- Volume 2 : The geochemistry, cycling and industrial
    uses of molybdenum. New York, Marcel Dekker, Inc., pp 731-737.

    Kelley DS, Daudu PA, Taylor PC, Mackey BE, & Turnlund JR (1995)
    Effects of low-copper diets on human immune response. Am J Clin
    Nutr, 62: 412-416.

    Khangarot BS (1992) Copper-induced hepatic ultrastructural
    alterations in the snake-headed fish, Channa punctatus. Ecotoxicol
    Environ Saf, 23: 282-293.

    Khangarot BS & Ray PK (1987) Sensitivity of toad tadpoles,
     Bufo melanostictus (Schneider), to heavy metals. Bull Environ
    Contam Toxicol, 38: 523-527.

    Khangarot BS & Ray PK (1989) Sensitivity of midge larvae of
     Chironomus tentans Fabricius (Diptera Chironomidae) to heavy
    metals. Bull Environ Contam Toxicol, 42: 325-330.

    Kim N & Fergusson J (1993) Concentrations and sources of cadmium,
    copper, lead and zinc in house dust in Christchurch, New Zealand.
    Sci Total Environ, 138: 1-21.

    King LD (1988) Retention of metals by several soils of the
    southeastern United States. J Environ Qual, 17: 239-246.

    King KA, Leleux J, & Mulhern BM (1984) Molybdenum and copper levels
    in white-tailed deer near uranium mines. J Wildl Manage, 48(1):

    Kinsman GD, Howard AN, Stone DL, & Mullins PA (1990) Studies in
    copper status and atheroesclerosis. Biochem Soc Trans, 18:

    Kirk RS & Lewis JW (1993) An evaluation of pollutant induced
    changes in the gills of rainbow trout using scanning electron
    microscopy. Environ Technol, 14: 577-585.

    Kjaer A, Laursen K, Thormann L, Borggaard O, & Lebech PE (1993)
    Copper release from copper intrauterine devices removed after up to
    8 years of use. Contraception, 47: 349-358.

    Klapheck S, Fliegner W, & Zimmer I (1994) Hydroxymethyl
    phytochelatins (gamma glutamylcysteine)(n)-serine are metal induced
    peptides of the poaceae. Plant Physiol, 104(4): 1325-1332.

    Klein WJ, Metz EN, & Price AR (1972) Acute copper intoxication.
    Arch Intern Med, 129: 578-582.

    Klein D, Schloz P, Drasch GA, Muller-Hocker J, & Summer KH (1991)
    Metallothionein, copper and zinc in fetal and neonatal human liver:
    changes during development. Toxicol Lett, 56(1-2): 61-67.

    Klevay LM (1975) Coronary heart disease: the zinc/copper
    hypothesis. Am J Clin Nutr, 28: 764-774.

    Klevay LM (1988) Dietary cholesterol lowers liver copper in
    rabbits. Biol Trace Elem Res, 38: 47-54.

    Klevay LM (1992) Re: Serum copper and the risk of acute myocardial
    infarction: a prospective population study in Eastern Finland. Am
    J Epidemiol, 135: 832-834.

    Klevay LM, Inman L, Johnson LK, Lawler M, Mahalko JR, Milne DB,
    Lusaski HC, Bolonchuk W, & Sandstead HH (1984) Increased
    cholesterol in plasma in a young man during experimental copper
    depletion. Metabolism, 33: 1112-1118.

    Klevay LM, Canfield WK, & Gallagher SK (1986) Decreased glucose
    tolerance in two men during experimental copper depletion. Nutr Rep
    Int, 33: 371-382.

    Knobeloch L, Ziarnik M, Howard J, Theis B, Farmer D, Anderson H, &
    Proctor M (1994) Gastrointestinal upsets associated with ingestion
    of copper-contaminated water. Environ Health Perspect, 102:

    Kodama H (1993) Recent developments in Menkes disease. J Inherit
    Metab Disease, 16: 791-799.

    Kok FJ, Van Duijn CM, Hofman A, Van Der Voit GB, De Wolff FA, Paays
    CHC, & Valkenburg HA (1988) Serum copper and zinc and the risk of
    death from cancer and cardiovascular disease. Am J Epidemiol, 128:

    Kolb M, Rach P, Schafer J, & Wild A (1992) Investigations of
    oxidative UV photolysis: I. Sample preparation for the voltammetric
    determination of Zn, Cd, Pb, Cu, Ni and Co in waters. Fresenius J
    Anal Chem, 342: 341-349.

    Konar SK & Mullick S (1993) Problems of safe disposal of petroleum
    products, detergents, heavy metals and pesticides to protect
    aquatic life. Sci Total Environ 2 (suppl): 989-1000.

    Kosalwat P & Knight AW (1987a) Acute toxicity of aqueous and
    substrate-bound copper to the midge,  Chironomus decorus. Arch
    Environ Contam Toxicol, 16(3): 275-282.

    Kosalwat P & Knight AW (1987b) Chronic toxicity of copper to a
    partial life cycle of the midge,  Chironomus decorus. Arch Environ
    Contam Toxicol, 16(3): 283-290.

    Kraak MHS, Lavy D, Peeters WHM, & Davids C (1992) Chronic
    ecotoxicity of copper and cadmium to the zebra mussel  Dreissena
    polymorpha. Arch Environ Contam Toxicol, 23: 363-369.

    Kraak MHS, Toussaint M, Lavy D, & Davids C (1994) Short-term
    effects of metals on the filtration rate of the zebra mussel
     Dreissena polymorpha. Environ Pollut, 84: 139-143.

    Kressner MJ, Stockert RJ, Morell AG, & Sternlieb I (1984) Origins
    of biliary copper. Hepathology, 4(5): 867-870.

    Krishnakumar PK, Asokan PK, & Pillai VK (1990) Physiological and
    cellular responses to copper and mercury in the green mussel
     Perna viridis (Linnaeus). Aquat Toxicol, 18(3): 163-174.

    Krolczyk AJ, Bear CE, Lai PFH, & Schimmer BP (1995) Effects of
    mutations in camp-dependent protein kinase on chloride efflux in
    caco-2 human colonic carcinoma cells. J Cell Physiol, 162: 64-73.

    Kruckeberg AL & Wu L (1992) Copper tolerance and copper
    accumulation of herbaceous plants colonizing California copper
    mines. Ecotoxicol Environ Saf, 23: 307-319.

    Kumar D (1984) Genetics of Indian childhood cirrhosis. Trop Geogr
    Med, 36(4): 313-316.

    Kumpulainen J, Mutanen M, Paaki M, & Lehto J (1987) Validity of
    calculation method in estimating mineral element concentration. Var
    Foda, 39(1): 75-82.

    Ladefoged O & Sturup S (1995) Copper deficiency in cattle, sheep
    and horses caused by excess molybdenum from fly ash: a case report.
    Vet Hum Toxicol, 37: 63-65.

    Lanno RP, Slinger SJ, & Hilton JW (1985) Maximum tolerable and
    toxicity levels of dietary copper in rainbow trout  (Salmo
    gairdneri  Richardson). Aquaculture, 49: 257-268.

    Larcher W (1995) Physiological plant ecology -- Ecophysiology and
    stress physiology of functional groups, 3rd ed. Berlin,

    LeBlanc GA (1982) Laboratory investigation into the development of
    resistance of  Daphnia magna (Straus) to environmental pollutants.
    Environ Pollut, A27: 309-322.

    LeBlanc GA (1985) Effects of copper on the competitive interactions
    of two species of cladocera. Environ Pollut, A37: 13-25.

    Lecyk M (1980) Toxicity of CuSO4 in mice embryonic development.
    Zool Pol, 28: 101-105.

    Lee YH & Stuebing RB (1990) Heavy metal contamination in the river
    toad,  Bufo juxtasper (Inger), near a copper mine in East
    Malaysia. Bull Environ Contam Toxicol, 45: 272-279.

    Lee SH, Lancey R, Montaser A, Madani N, & Linder MC (1993)
    Ceruloplasmin and copper transport during the latter part of
    gestation in the rat. Proc Soc Exp Biol Med, 203: 428-439.

    Lehman AJ (1951) Chemicals in foods -- A report to the Association
    of Food and Drug Officials on current developments: Part II.
    Pesticides. Q Bull Assoc Food Drug Off, 15: 122-133.

    Lehmann RG & Harter RD (1984) Assessment of copper-soil bond
    strength by desorption kinetics. Soil Sci Soc Am J, 48: 769-772.

    Leland HV, Fend SV, Dudley TL, & Carter JL (1989) Effects of copper
    on species composition of benthic insects in a Sierra Nevada,
    California, stream. Freshw Biol, 21: 163-179.

    Lepp NW (1992) Uptake and accumulation of metals in bacteria and
    fungi. In: Advances in trace substances research: Biogeochemistry
    of trace metals. Boca Raton, Florida, Lewis Publishers, pp 283-289.

    Levinson B, Gitschier J, Vulpe C, Whitney S, Yang S, & Packman S
    (1993) Are X-linked cutis laxa and Menkes disease allelic? Nat
    Genet, 3: 6.

    Levy Y, Zeharia A, Grunebaum M, Nitzan M, & Steinherz R (1985)
    Copper deficiency in infants fed cow milk. J Pediatr, 106: 786-788.

    Lewis MA (1983) Effect of loading density on the acute toxicities
    of surfactants, copper, and phenol to  Daphnia magna Straus. Arch
    Environ Contam Toxicol, 12: 51-55.

    Lide DR & Frederikse HPR (1993) CRC handbook of chemistry and
    physics, 74th ed. Boca Raton, Florida, CRC Press.

    Lighthart B, Baham J, & Volk VV (1983) Microbial respiration and
    chemical speciation in metal-amended soils. J Environ Qual, 12(4):

    Lim CT & Choo KE (1979) Wilson's disease in a 2 year old child. J
    Singap Paediatr Soc, 21: 99-102.

    Lin W, Rice MA, & Chien PK (1992) The effects of copper, cadmium
    and zinc on particle filtration and uptake of glycine in the
    Pacific oyster  Crassostrea gigas. Comp Biochem Physiol, 103C(1):

    Lin H-C & Dunson WA (1993) The effect of salinity on the acute
    toxicity of cadmium to the tropical, estuarine, hermaphroditic
    fish,  Rivulus marmoratus: a comparison of Cd, Cu, and Zn
    tolerance with  Fundulus heteroclitus. Arch Environ Contam
    Toxicol, 25: 41-47.

    Linder McC(1991) The biochemistry of copper. New York, Plenum

    Linder MC & Hazegh-Azam M (1996) Copper biochemistry and molecular
    biology. Am J Clin Nutr, 63: 797s-811s.

    Linder MC, Wooten L, Cerveza P, Cotton S, Shulze R, & Lomeli N
    (1998) Copper transport. Am J Clin Nutr, 67(suppl 5): 965/S-971/S.

    Liu C-CF & Medeiros DM (1986) Excess diet copper increases systolic
    blood pressure in rats. Biol Trace Elem Res, 9: 15-24.

    Liu Y, Liu J, Iszard MB, Andrews GK, Palmiteer RD, & Klaassen CD
    (1995) Transgenic mice that over-express metallothionein-I are
    protected from cadmium lethality and toxicity. Toxicol Appl
    Pharmacol, 135: 222-228.

    Llewellyn GC, Floyd EA, Hoke GD, Weekley LB, & Kimbrough TD (1985)
    Influence of dietary aflatoxin, zinc, and copper on bone size,
    organ weight, and body weight in hamsters and rats. Bull Environ
    Contam Toxicol, 35: 149-156.

    Lo GS, Settle SL, & Steinke FH (1984) Bioavailability of copper in
    isolated soybean protein using the rat as an experimental model.
    J Nutr, 114: 332-340.

    Lobban C, Harrison P, & Duncan M (1985) The physiological ecology
    of seaweeds. Cambridge, UK, Cambridge University Press.

    Logan JI, Harveyson KB, Wisdon GB, Highes AE, & Archbold GPR (1994)
    Hereditary caeruloplasmin deficiency, dementia and diabetes
    mellitus. Q J Med, 87: 663-670.

    Logue JN, Koontz MD, & Hattwick MAW (1982) A historical prospective
    mortality study of workers in copper and zinc refineries. J Occup
    Med, 24: 398-408.

    Lönnerdal B, Bell JG, & Keen CL (1985) Copper absorption from human
    milk, cow's milk and infant formulas using a suckling rat model. Am
    J Clin Nutr, 42: 836-844.

    Lowe TP, May TW, Brumbaugh WG, & Kane DA (1985) National
    contaminant, biomonitoring program: Concentration of seven elements
    in freshwater fish 1978-1981. Arch Environ Contam Toxicol, 14:

    Lowy SL, Fisler JS, Drenick EJ, Hunt IF, & Swendseid ME (1986) Zinc
    and copper nutriture in obese men receiving very low calorie diets
    of soy or collagen protein. Am J Clin Nutr, 43(2): 272-287.

    Lu PL, Huang KS, & Jiang SJ (1993) Determination of traces of
    copper, cadmium and lead in biological and environmental samples by
    flow-injection isotope dilution inductively coupled plasma mass
    spectrometry. Anal Chim Acta, 284: 181-188.

    Lukaski HC, Klevay LM, & Milne DB (1988) Effects of copper on human
    autonomic cardiovascular function. Eur J Appl Physiol, 58: 74-80.

    Lundborg M & Camner P (1984) Lysozyme levels in rabbit lung after
    inhalation of nickel, cadmium, cobalt, and copper chlorides.
    Environ Res, 34: 335-342.

    Lussi A, Hotz P, & Schoenberg V (1992) [The release of mercury and
    copper from  in vivo aged amalgam fillings.] Schweiz. Mon.schr
    Zahnmed, 102: 411-415 (in German).

    Lydy MJ & Wissing TE (1988) Effect of sublethal concentrations of
    copper on the critical thermal maxima (CTMax) of the fantail
     (Etheostoma flabellare) and johnny (E. nigrum) darters. Aquat
    Toxicol, 12: 311-322.

    Lyle WH, Payton JE, & Hui M (1976) Haemodialysis and copper fever.
    Lancet, I: 1324-1325.

    Lynch SM & Strain JJ (1990) Effects of skim milk powder, whey or
    casein on tissue trace element status and antioxidant enzyme
    activities in rats fed control and copper-deficient diets. Nutr
    Res, 10: 449-460.

    Lyon DB (1984) Studies on the solubility of Ca, Mg, Zn, and Cu in
    cereal products. Am J Clin Nutr, 39: 190-195.

    Lyon TDB & Fell GS (1990) Isotopic composition of copper in serum
    by inductively coupled plasma mass spectrometry. J Anal At
    Spectrom, 5: 135-137.

    Lyon TDB, Fell GS, Hutton RC, & Eaton AN (1988) Evaluation of
    inductively coupled argon plasma mass spectrometry (ICP-MS) for
    simultaneous multi-element trace analysis in clinical chemistry.
    J Anal At Spectrom, 3: 265-271.

    Lyon TDB, Fell GS, Gaffney D, McGaw BA, Russell RI, Park RHR,
    Beattie AD, Curry G, Crofton RJ, Gunn I, Sturniolo GS, D'Inca R, &
    Patriarca M (1995) Use of a stable copper isotope (65Cu) in the
    differential diagnosis of Wilson's disease. Clin Sci, 88: 727-732.

    Lyon TDB, Fletcher S, Fell GS, & Patriarca M (1996) Measurement and
    application of stable copper isotopes to investigations of human
    metabolism. Microchem J, 54: 236-243.

    Ma W (1984) Sublethal toxic effects of copper on growth,
    reproduction and litter breakdown activity in the earthworm
     Lumbricus rubellus,  with observations on the influence of
    temperature and soil pH. Environ Pollut, A33: 207-219.

    Ma W (1988) Toxicity of copper to lumbricid earthworms in sandy
    agricultural soils amended with Cu-enriched organic waste
    materials. Ecol Bull, 39: 53-56.

    McArdle HJ (1995) The metabolism of copper during pregnancy -- a
    review. Food Chem, 54: 79-84.

    McArdle HJ & Erlich R (1991) Copper uptake and transfer to the
    mouse fetus during pregnancy. J Nutr, 121: 208-214.

    McCormick RJ, Ovecka GD, & Medeiros DM (1989) Myofibrillar and
    nonmyofibrillar myocardial proteins of copper-deficient rats. J
    Nutr, 119: 1683-1690.

    McCullough AJ, Flemming R, & Thistle JL (1983) Diagnosis of
    Wilson's disease presenting as fulminant hepatic failure.
    Gastroenterology, 84: 161.

    MacDonald JM, Shields JD, & Zimmer-Faust RK (1988) Acute toxicities
    of eleven metals to early life-history stages of the yellow crab
     Cancer anthonyi. Mar Biol, 98: 201-207.

    McHargue JS (1925) The occurrence of copper, manganese, zinc,
    nickel, and cobalt in soils, plants, and animals, and their
    possible function as vital factors. J Agric Res, 30: 193-196.

    McHargue JS (1926) Mineral constituents of the cotton plant. J Am
    Soc Agron, 18: 1076-1083.

    McHargue JS (1927a) The proportion and significance of copper, iron
    and zinc in some mollusks and crustaceans. Trans Ky Acad Sci, 2:

    McHargue JS (1927b) Significance of the occurrence of manganese,
    copper, zinc, nickel, and cobalt Kentucky blue grass. Ind Eng Chem
    Res, 19: 274-276.

    Mackey DJ & Higgins HW (1988) The copper-complexing capacity of
    seawater. Sci Total Environ, 75: 151-167.

    McKim JM & Benoit DA (1971) Effects of long-term exposures to
    copper on survival, growth, and reproduction of brook trout
     (Salvelinus fontinalis). J Fish Res Board Can, 28: 655-662.

    McKim JM, Eaton JG, & Holcombe GW (1978) Metal toxicity of embryos
    and larvae of eight species of freshwater fish: II. Copper. Bull
    Environ Contam Toxicol, 19: 608-616.

    McLachlan J (1973) Growth media -- marine. In: Stein JR ed.
    Handbook of phycological methods, culture methods and growth
    measurements. Cambridge, UK, Cambridge University Press, pp 25-51.

    McLaren JW, Lam JWH, Berman SS, Akatsuka K, & Azeredo MA (1993)
    On-line method for the analysis of sea-water for trace elements by
    inductively coupled plasma mass spectrometry. J Anal At Spectrom,
    8: 279-285.

    McLaughlin JK, Chen JQ, Dosemeci M, Chen RA, Rexing SH, Wu Z, Hearl
    FJ, McCawley MA, & Blot WJ (1992) A nested case-control study of
    lung cancer among silica exposed workers in China. Br J Ind Med,
    49: 167-171.

    McLeese DW & Ray S (1986) Toxicity of CdCl2, CdEDTA, CuCl2, and
    CuEDTA to marine invertebrates. Bull Environ Contam Toxicol, 36:

    McMaster D, McCrum E, Patterson CC, Kerr MM, O'Reilly D, Evans AE,
    & Love AH (1992) Serum copper and zinc in random samples of the
    population of Northern Ireland. Am J Clin Nutr, 56(2): 440-446.

    McNulty HR, Anderson BS, Hunt JW, Turpen SL, & Singer MM (1994)
    Age-specific toxicity of copper to larval topsmelt
     Atherinops affinis. Environ Toxicol Chem, 13: 487-492.

    MacRae RK, Smith DE, Swoboda-Colberg N, Meyer JS, & Bergmann HL (in
    press) Copper binding affinity of rainbow trout
     (Oncorhynchus mykiss) and brook trout  (Salvelinus fontinalis) 
    gills. Environ Toxicol Chem.

    Madoni P, Esteban G, & Gorbi G (1992) Acute toxicity of cadmium,
    copper, mercury, and zinc to ciliates from activated sludge plants.
    Bull Environ Contam Toxicol, 49: 900-905.

    Madoni P, Davoli D, & Gorbi G (1994) Acute toxicity of lead,
    chromium, and other heavy metals to ciliates from activated sludge
    plants. Bull Environ Contam Toxicol, 53: 420-425.

    Maessen O, Freedman B, & McCurdy R (1985) Metal mobilization in
    home well water systems in Nova Scotia. J Am Water Works Assoc, 77:

    Maggiore G, Giacomo D, Sessa F, & Burgio GR (1987) Idiopathic
    hepatic copper toxicosis in a child. J Pediatr Gastroenterol Nutr,
    6: 908.

    Malaisse F, Grégoire J, Brooks RR, Morrison RS, & Reeves RD (1978)
    Aeolanthus biformifolius: a hyperaccumulator of copper from Zaďre.
    Science, 199: 887-888.

    Malaisse F, Grégoire J, Morrison RS, Brooks RR, & Reeves RD (1979)
    Copper and cobalt in vegetation of Fungurume, Shaba Province,
    Zaďre. Oikos, 33: 472-478.

    Malea P, Haritonidis S, & Stratis I (1994) Bioaccumulation of
    metals by Rhodophyta species at Antikyra Gulf (Greece) near an
    aluminium factory. Bot Mar, 37: 505-513.

    Malecki MR, Neuhauser EF, & Loehr RC (1982) The effect of metals on
    the growth and reproduction of  Eisenia foetida (Oligochaeta,
    Lumbricidae). Pedobiologia, 24: 129-137.

    Malhotra KM, Shukla GS, & Chandra SV (1982) Neurochemical changes
    in rats coexposed to lead and copper. Arch Toxicol, 49: 331-336.

    Malvankar PL & Shinde VM (1991) Ion-pair extraction and
    determination of copper(II) and zinc(II) in environmental and
    pharmaceutical samples. Analyst, 116: 1081-1084.

    Manzler AD & Schreiner AW (1970) Copper-induced acute hemolytic
    anemia: A new complication of hemodialysis. Ann Intern Med, 73:

    Marceau N & Aspin N (1973a) The intracellular distribution of the
    radiocopper derived from ceruloplasmin and from albumin. Biochim
    Biophys Acta, 328(2): 338-350.

    Marceau N & Aspin N (1973b) The association of the copper derived
    from ceruloplasmin with cytocuprein. Biochim Biophys Acta, 328(2):

    Marceau N, Aspin N, & Sass-Kortsak A (1970) Absorption of copper 64
    from gastrointestinal tract of the rat. Am J Physiol, 218: 377-383.

    MARCO (1989) Copper. Birmingham, UK, Market Analysis and Research

    Marigomez JA, Angulo E, & Saez V (1986) Feeding and growth
    responses to copper, zinc, mercury and lead in the terrestrial
    gastropod  Arion ater (Linne). J Molluscan Stud, 52: 68-78.

    Marschner H (1986) Mineral nutrition of higher plants. New York,
    London, Academic Press.

    Martin NA (1986) Toxicity of pesticides to  Allolobophora
    caliginosa  (Oligochaeta: Lumbricidae). N Z J Agric Res, 29:

    Martin TR & Holdich DM (1986) The acute lethal toxicity of heavy
    metals to peracarid crustaceans (with particular reference to
    fresh-water asellids and gammarids). Water Res, 20(9): 1137-1147.

    Martin M, Hunt JW, Anderson BS, Turpen SL, & Palmer FH (1989)
    Experimental evaluation of the mysid  Holmesimysis costata as a
    test organism for effluent toxicity testing. Environ Toxicol Chem,
    8: 1003-1012.

    Martin LA, McNemar A, & O'Brien EL (1994) Menkes kinky hair
    disease. Am J Matern Child Nurs, 19(3): 162-164.

    Martínez J, Soto Y, Vives-Rego J, & Bianchi M (1991)Toxicity of Cu,
    Ni and alkylbenzene sulfonate (LAS) on the naturally occurring
    bacteria in the Rhone river plume. Environ Toxicol Chem, 10:

    Marzin DR & Phi HV (1985) Study of the mutagenicity of metal
    derivatives with  Salmonella typhimurium TA 102. Mutat Res, 155:

    Mash DC, Pablo J, Flynn DD, Efange SM, & Weiner WJ (1990)
    Characterization and distribution of transferrin receptors in the
    rat brain. J Neurochem, 55: 1972-1979.

    Masters BA, Kelly EJ, Quaife CJ, Brinster RL, & Palmiter RD (1994)
    Targeted disruption of metallothionein I and II genes increases
    sensitivity to cadmium. Proc Natl Acad Sci (USA), 91: 584-588.

    Matejovic I & Durackova A (1994) Comparison of microwave digestion,
    wet and dry mineralisation, and solubilisation of plant samples by
    flow-injection isotope dilution inductively coupled plasma mass
    spectrometry. Commun Soil Sci Plant Anal, 25: 1277-1288.

    Mathur SP, Hamilton HA, & Levesque MP (1979) The mitigating effect
    of residual fertilizer copper on the decomposition of an organic
    soil  in situ. Soil Sci Soc Am J, 43: 200-203.

    Matsui S (1980) Evaluation of a  Bacillus subtilis rec-assay for
    the detection of mutagens which may occur in water environments.
    Water Res, 14: 1613-1619.

    Maund SJ, Taylor EJ, & Pascoe D (1992) Population responses of the
    freshwater amphipod crustacean  Gammarus pulex (L.) to copper.
    Freshw Biol, 28: 29-36.

    Mayer FL (1987) Acute toxicity handbook of chemicals to estuarine
    organisms. Gulf Breeze, Florida, US Environmental Protection
    Agency, Environmental Research Laboratory, 274 pp

    Mayer FL & Ellersieck MR (1986) Manual of acute toxicity:
    Interpretation and data base for 410 chemicals and 66 species of
    freshwater animals. Washington, DC, US Department of Interior, Fish
    and Wildlife Service, 506 pp (Resource Publication 160).

    Mbofung CMF & Subbarau VV (1990) Trace element (zinc, copper, iron
    and magnesium) concentrations in human placenta and their
    relationship to birth weight of babies. Nutr Res, 10: 359-366.

    Meador JP (1991) The interaction of pH, dissolved organic carbon,
    and total copper in the determination of ionic copper and toxicity.
    Aquat toxicol, 19: 13-32.

    Medeiros DM, Milton A, Brunett E, & Stacy L (1991) Copper
    supplementation effects on indicators of copper status and serum
    cholesterol in adult males. Biol Trace Elem Res, 30(1): 1935.

    Meissner W (1817) Sur la présence du cuivre dans les cendres des
    végétaux. Ann Chim Phys, 4: 106.

    Mercer JF, Livingston J, Hall B, Paynter JA, Begy C,
    Chandrasekharappa S, Lockhart P, Grimes A, Bhave M, & Siemieniak D
    (1993) Isolation of a partial candidate gene for Menkes disease by
    positional cloning see comments. Nat Genet, 3: 20-25.

    Mersch J, Morhain E, & Mouvet C (1993) Laboratory accumulation and
    depuration of copper and cadmium in the freshwater mussel
     Dreissena polymorpha and the aquatic moss
     Rhynchostegium riparioides. Chemosphere, 27(8): 1475-1485.

    Mesuere K & Fish W (1989) Behavior of runoff-derived metals in a
    detention pond system. Water Air Soil Pollut, 47(´): 125-138.

    Metaxas A & Lewis AG (1991) Copper tolerance of  Skeletonema
    costatum and  Nitzschia thermalis. Aquat Toxicol, 19: 265-280.

    Michalska AE & Choo KH (1993) Targeting and germ-line transmission
    of a null mutation at the metallothionein I and II loci in mouse.
    Proc Natl Acad Sci (USA), 90: 8088-8092.

    Midorikawa T, Tanoue E, & Sugimura Y (1992) Interaction between
    dissolved organic matter in seawater and copper. Sci Total Environ,
    117/118: 499-507.

    Migon C (1993) Riverine and atmospheric inputs of heavy metals to
    the Ligurian Sea. Sci Total Environ, 138: 289-299.

    Migon C, Morelli J, Nicolas E, & Copin-Montegut G (1991) Evaluation
    of total atmospheric deposition of Pb, Cd, Cu and Zn to the
    Ligurian Sea. Sci Total Environ, 105: 135-148.

    Miller TG & Mackay WC (1980) The effects of hardness, alkalinity
    and pH of test water on the toxicity of copper to rainbow trout
     (Salmo gairdneri). Water Res, 14: 129-133.

    Mills ES (1930) The treatment of idiopathic (hypochromic) anemia
    with iron and copper. Can Med Assoc J, 22: 175-178.

    Mills CF, Dalgarno AC, & Wenham G (1976) Biochemical and
    pathological changes in tissues of Friesian cattle during the
    experimental induction of copper deficiency. Br J Nutr, 35(3):

    Milne DB & Johnson PE (1993) Assessment of copper status: Effect of
    age and gender on reference ranges in healthy adults. Clin Chem,
    39: 883-887.

    Milne DB, Johnson PE, Klevay LM, & Sandstead HH (1990) Effect of
    copper intake on balance, absorption, and status indices of copper
    in men. Nutr Res, 10: 975-986.

    Minnich MM & McBride MB (1986) Effect of copper activity on carbon
    and nitrogen mineralization in field-aged copper-enriched soils.
    Plant Soil, 91: 231-240.

    Mittal SR (1972) Oxyhaemoglobinuria following copper sulphate
    poisoning: A case report and a review of the literature. Forensic
    Sci, 1: 245-248.

    Miyajima H, Nishimura Y, Mizoguchi K, Sakamota M, Shimizu T, &
    Honda N (1987) Familial apoceruloplasmin deficiency associated with
    blefarospasm and retinal degeneration. Neurology, 37: 761-767.

    Moffett JM & Zika RG (1987) Photochemistry of copper complexes in
    sea water. In: Zika RG & Cooper WJ ed. Photochemistry of
    environmental aquatic systems. Washington, DC, American Chemical
    Society, pp 116-131 (ACS Symposium Series No. 327).

    Mohan P, Failla M, Bremner I, Arthur-Smith A, & Kerzner B (1995)
    Biliary copper excretion in the neonatal rat: role of glutathione
    and metallothionein. Hepatology, 21: 1051-1057.

    Moore MN (1978) The distribution of dissolved copper in the eastern
    Atlantic Ocean. Earth Planet Sci Lett, 41: 461.

    Moore MV & Winner RW (1989) Relative sensitivity of
     Ceriodaphnia dubia laboratory tests and pond communities of
    zooplankton and benthos to chronic copper stress. Aquat Toxicol,
    15: 311-330.

    Moore PG, Rainbow PS, & Hayes E (1991) The beach-hopper
     Orchestia gammarellus (Crustacea: Amphipoda) as a biomonitor for
    copper and zinc: North Sea trials. Sci Total Environ, 106: 221-238.

    Morgan JE & Morgan AJ (1988) Earthworms as biological monitors of
    cadmium, copper, lead and zinc in metalliferous soils. Environ
    Pollut, 54: 123-138.

    Mori M, Hattori A, Sawaki M, Tsuzuki N, Sawada N, Oyamada M,
    Sugawara N, & Enomoto K (1994) The LEC rat: a model for human
    hepatitis, liver cancer, and much more. Am J Pathol, 144(1):

    Morita H, Ikeda S, Yamamoto K, Morita S, Yoshida K, Nomoto S, Kato
    M, & Yanagisawa N (1995) Hereditary ceruloplasmin deficiency with
    hemosiderosis: a clinicopathological study of a Japanese family.
    Ann Neurol, 37(5): 646-656.

    Moriya M, Ohta T, Watanabe K, Miyazawa T, Kato K, & Shirasu Y
    (1983) Further mutagenicity studies on pesticides in bacterial
    reversion assay systems. Mutat Res, 116: 185-216.

    Morrison GMP & Florence TM (1989) Comparison of physicochemical
    speciation procedures with metal toxicity to  Chlorella
    pyrenoidosa.  Copper complexation capacity. Electroanalysis, 1:

    Moser H & Wieser W (1979) Copper and nutrition in  Helix pomatia 
    (L.). Oecologia, 42: 241-251.

    Mount DI (1968) Chronic toxicity of copper to fathead minnows
     (Pimephales promelas, Rafinesque). Water Res, 2: 215-223.

    Mount DI & Norberg TJ (1984) A seven-day life-cycle cladoceran
    toxicity test. Environ. Toxicol Chem, 3: 425-434.

    Mount DI & Stephen C (1969) Chronic toxicity of copper to fathead
    minnow  (Pimephales promelas) in soft water. J Fish Res Board Can,
    26: 2449-2457.

    Mount DR, Barth AK, Garrison TD, Barten KA, & Hockett JR (1994)
    Dietary and waterborne exposure of rainbow trout
     (Oncorhynchus mykiss) to copper, cadmium, lead and zinc using a
    live diet. Environ Toxicol Chem, 13: 2031-2041.

    Müller T, Feichtinger H, Berger H, & Müller W (1996) Endemic
    Tyrolean infantile cirrhosis: an ecogenetic disorder. Lancet,
    347(9005): 877-880.

    Müller-Höcker J, Weiss M, & Meyer J (1987) Fatal copper storage
    disease of the liver in a German infant resembling Indian childhood
    cirrhosis. Virchows Arch A411: 379-385.

    Müller-Höcker J, Meyer U, Wiebecke B, & Hubner G (1988) Copper
    storage disease of the liver and chronic dietary copper
    intoxication in two further German infants mimicking Indian
    childhood cirrhosis. Pathol Res Pract, 183: 39-45.

    Muńoz MJ, Carballo M, & Tarazona JV (1991) The effect of sublethal
    levels of copper and cyanide on some biochemical parameters of
    rainbow trout along subacute exposition. Comp Biochem Physiol,
    100C: 577-582.

    Murphy EA (1993) Effectiveness of flushing on reducing lead and
    copper levels in school drinking water. Environ Health Perspect,
    101: 240-241.

    Murthy RC, Lal S, Saxena DK, Shukla GS, Ali MM, & Chandra SV (1981)
    Effect of manganese and copper interaction on behavior and biogenic
    amines in rats fed a 10% casein diet. Chem-Biol Interact, 37:

    Mussalo-Rauhamaa H, Salmela SS, Leppanen A, & Pyysalo H (1986)
    Cigarettes as a source of some trace and heavy metals and
    pesticides in man. Arch Environ Health, 41: 49-55.

    Napolitano M, Gialanell G, Grossi GG, Durante M, Zhang YX, Lanzone
    A, & Mancuso S (1994) Trace elements in amniotic fluid in different
    physiopathologic conditions. Trace Elem Med, 11: 96-98.

    Naveh Y, Hazzani A, & Berant M (1981) Copper deficiency with cow's
    milk diet. Pediatrics, 68: 397-400.

    Nayak NC & Ramalingaswamy V (1975) Indian childhood cirrhosis. Clin
    Gastroenterol, 4: 333-339.

    Nebeker AV, Cairns MA, & Wise CM (1984a) Relative sensitivity of
     Chironomus tentans life stages to copper. Environ Toxicol Chem,
    3: 151-158.

    Nebeker AV, Savonen C, Baker RJ, & McCrady JK (1984b) Effects of
    copper, nickel and zinc on the life cycle of the caddisfly
     Clistoronia magnifica (Limnephilidae). Environ Toxicol Chem, 3:

    Nebeker AV, Stinchfield A, Savonen C, & Chapman GA (1986) Effects
    of copper, nickel and zinc on three species of Oregon freshwater
    snails. Environ Toxicol Chem, 5: 807-811.

    Nectoux M & Bounias M (1988) Toxicologie du sulfate cuivrique chez
    l'abeille: I. Nouveaux paramčtres algébriques de létalité comme
    alternative ŕ la DL50. C R Séances Soc Biol, 182: 544-555.

    Nell JA & Chvojka R (1992) The effect of bis-tributyltin oxide
    (TBTO) and copper on the growth of juvenile Sydney rock oysters
     Saccostrea commercialis (Iredale and Roughley) and Pacific
    oysters  Crassostrea gigas Thunberg. Sci Total Environ, 125:

    Nelson DA, Miller JE, & Calabrese A (1988) Effect of heavy metals
    on bay scallops, surf clams, and blue mussels in acute and
    long-term exposures. Arch Environ Contam Toxicol, 17: 595-600.

    Nemcsók JG & Hughes GM (1988) The effect of copper sulphate on some
    biochemical parameters of rainbow trout. Environ Pollut, 49: 77-85.

    Neubecker TA & Allen HE (1983) The measurement of complexation
    capacity and conditional stability constants for ligands in natural
    waters--A review. Water Res, 17: 1-14.

    Neuhauser EF, Malecki MR, & Loehr RC (1984) Growth and reproduction
    of the earthworm  Eisenia fetida after exposure to sublethal
    concentrations of metals. Pedobiologia, 27: 89-97.

    Neuhauser EF, Loehr RC, Milligan DL, & Malecki MR (1985) Toxicity
    of metals to the earthworm  Eisenia fetida. Biol Fertil Soils,
    1: 149-152.

    Neumann PZ & Sass-Kortsak A (1967) The state of copper in human
    serum: evidence for an amino acid-bound fraction. J Clin Invest,
    46(4): 646-658.

    NFA (Australian National Food Authority) (1992) The 1992 Australian
    market basket survey. Canberra, Australian Government Publishing
    Service, 96 pp.

    NFA (Australian National Food Authority) (1993) The 1992 Australian
    market survey -- A total diet survey of pesticides and
    contaminants. Canberra, Australian National Food Authority, 96 pp.

    Nielsen FH, Gallagher SK, Johnson LK, & Nielsen EJ (1992) Boron
    enhances and mimics some effects of estrogen therapy in
    postmenopausal women. J Trace Elem Exp Med, 5: 237-246.

    NIOSH (1981a) Health hazard evaluation report No. HHE-80-084-927,
    General Electric Company, Lynn, Massachusetts. Springfield,
    Virginia, US Department of Commerce, National Technical Information
    Service (PB83-102848).

    NIOSH (1981b) Health hazard evaluation report No. HHE-78-132-818,
    Copper Division Southwire Company, Inc. Carrollton. Springfield,
    Virginia, US Department of Commerce, National Technical Information
    Service (PB82-188632).

    NIOSH (1987) Manual of analytical methods, 3rd ed. Cincinnati,
    Ohio, National Institute for Occupational Safety and Health (DHHS
    Publication No. 84-100).

    NIOSH (1993) Registry of toxic effects of chemical substances.
    Cincinnati, Ohio, National Institute for Occupational Safety and
    Health (Silverplatter, Chem-Bank, July 1993).

    NIPHEP (1989) Integrated criteria document copper. Bilthoven, The
    Netherlands, National Institute of Public Health and Environmental
    Protection, 91 pp (Appendix to Report No. 758474 009).

    Nishioka H (1975) Mutagenic activities of metal compounds in
    bacteria. Mutat Res, 31: 185-189.

    Nor YM (1987) Ecotoxicity of copper to aquatic biota: a review.
    Environ Res, 43(1): 274-282.

    Nor YM & Cheng HH (1986) Chemical speciation and bioavailability of
    copper: uptake and accumulation by Eichornia. Environ Toxicol Chem,
    5: 941-947.

    Nordlind K & Liden S (1992) Patch test reactions to metal salts in
    patients with oral mucosal lesions associated with amalgam
    restorations. Contact Dermatitis, 27(3): 157-160.

    NRC (National Research Council) (1980) Mineral tolerance of
    domestic animals. Washington, DC, National Academy of Sciences.

    Nriagu JO (1979a) Global inventory of natural and anthropogenic
    emissions of trace metals to the atmosphere. Nature (Lond), 279:

    Nriagu JO ed. (1979b) Copper in the environment: Part 1. Ecological
    cycling. New York, John Wiley & Sons Ltd, pp 43-75.

    Nriagu JO (1989) A global assessment of natural sources of
    atmospheric trace metals. Nature (Lond), 338: 47-49.

    Odermatt A, Suter H, Krapf R, & Solioz M (1993) Primary structure
    of two P-type ATPases involved in copper homeostasis in
     Enterococcus hirae. J Biol Chem, 268: 12775-12779.

    O'Donohue JW, Reid MA, Varghese A, Portmann B, & Williams R (1993)
    Case report: Micronodular cirrhosis and acute liver failure due to
    chronic copper self-intoxication. Eur J Gastroenterol Hepatol, 5:

    OECD (1995) Report of the OECD Workshop on Environmental
    Hazard/Risk Assessment. Paris, Organisation for Economic
    Co-operation and Development, 96 pp (OECD Environment Monographs
    No.15; OCDE/GD(95)134).

    Oestreicher P & Cousins RJ (1985) Copper and zinc absorption in the
    rat: mechanism of mutual antagonism. J Nutr, 115: 159-166.

    Ohi R & Lilly JR (1980) Copper kinetics in infantile hepatobiliary
    disease. J Pediatr Surg, 15: 509-512.

    Oikari A, Kukkonen J, & Virtanen V (1992) Acute toxicity of
    chemicals to  Daphnia magna in humic waters. Sci Total Environ,
    117/118: 367-377.

    Olin KL, Walter RM, & Keen CL (1994) Copper deficiency affects
    selenoglutathione peroxidase and selenodeiodinase activities and
    antioxidant defense in weanling rats. Am J Clin Nutr, 59: 654-658.

    Olivier P & Marzin D (1987) Study of the genotoxic potential of 48
    inorganic derivatives with the SOS chromotest. Mutat Res, 189:

    Olsen KB, Wang J, Setiadji R, & Lu JM (1994) Fiels screening of
    chromium, zinc, copper, and lead in sediments by stripping
    analysis. Environ Sci Technol, 28: 2074-2079.

    OMME (1992) Air monitoring programme. Toronto, Canada, Province of
    Ontario, Ministry of Environment and Energy (Unpublished data).

    Omoto E & Tavassoli M (1990) Purification and partial
    characterization of ceruloplasmin receptors from rat liver
    endothelium. Arch Biochem Biophys, 282: 34-38.

    O'Neill NC & Tanner MS (1989) Uptake of copper from brass vessels
    by bovine milk and its relevance to Indian childhood cirrhosis. J
    Pediatr Gastroenterol Nutr, 9: 167-172.

    Oris JT, Winner RW, & Moore MV (1991) A four-day survival and
    reproduction toxicity test for  Ceriodaphnia dubia. Environ
    Toxicol Chem, 10: 217-224.

    Ortel J, Gintenreiter S, & Nopp H (1993) The effects of host metal
    stress on a parasitoid in an insect/insect relationship
     (Lymantria dispar L., Lymantriidae Lepid.- Glyptapanteles
    liparidis Bouchč, Braconidae Hym.). Arch Environ Contam Toxicol,
    24: 421-426.

    Ottley CJ & Harrison RM (1993) Atmospheric dry deposition flux of
    metallic species to the North Sea. Atmos Environ, 27A: 685-695.

    Ouseph PP (1992) Dissolved and particulate trace metals in the
    Cochin estuary. Mar Pollut Bull, 24(4): 186-192.

    Overvad K, Wang DY, Olsen J, Allen DS, Thorling EB, Bulbrook RD, &
    Hayward JL (1993) Copper in human mammary carcinogenesis: a
    case-cohort study. Am J Epidemiol, 137: 409-414.

    Owen CA Jr (1965) Metabolism of radio copper (Cu64) in the rat. Am
    J Physiol, 209: 900-904.

    Owen CA Jr (1982) Biochemical aspects of copper: Copper deficiency
    and toxicity. In: Physiological aspects of copper. Park Ridge, New
    Jersey, Noyes Publications.

    Ozoh PTE (1992a) The effect of temperature and salinity on copper
    body-burden and copper toxicity to Hediste (Nereis) diversicolor.
    Environ Monit Assess, 21: 11-17.

    Ozoh PTE (1992b) The importance of adult Hediste (Nereis)
    diversicolor in managing heavy metal pollution in shores and
    estuaries. Environ Monit Assess, 21: 165-171.

    Ozoh PTE (1992c) The effects of salinity, temperature and sediment
    on the toxicity of copper to juvenile  Hediste (Nereis)
    diversicolor (O.F. Muller). Environ Monit Assess, 21(1): 1-10.

    Ozoh PTE (1994) The effect of salinity, temperature and time in the
    accumulation and depuration of copper in ragworm, Hediste (Nereis)
    diversicolor (O.F. Muller). Environ Monit Assess, 29: 155-166.

    Ozoh PTE & Jones NV (1990) The effects of salinity and temperature
    on the toxicity of copper to 1-day and 7-day-old larvae of Hediste
    (Nereis) diversicolor (O.F. Muller). Ecotoxicol Environ Saf, 19:

    Pagenkopf GK (1983) Gill surface interaction model for trace metal
    toxicity to fishes: Role of complexation, pH, and water hardness.
    Environ Sci Technol, 17(6): 342-347.

    Palanques A. & Diaz JI (1994) Anthropogenic heavy metal pollution
    in the sediments of the Barcelona continental shelf (northwestern
    Mediterranean). Mar Environ Res, 38: 17-31.

    Palmiter RD (1993) Constitutive expression of metallothionein -- III
    (MT-III), but not MT-I, inhibits growth when cells become zinc
    deficient. Toxicol Appl Pharmacol, 135: 139-146.

    Palmiter RD, Sandgren EP, Koeller DM, & Brinster RL (1993) Distal
    regulatory elements from the mouse methallothionein locus stimulate
    gene expression in transgenic mice. Mol Cell Biol, 13: 5266-5275.
    Pandit AN & Bhave SA (1983) Copper and Indian childhood cirrhosis.
    Indian Pediatr, 20: 893-899.

    Pantani C, Ghetti PF, Cavacini A, & Muccioni P (1990) Acute
    toxicity of equitoxic binary mixtures of some metals, surfactants
    and pesticides to the freshwater amphipod  Gammarus italicus
    Goedm. Environ Technol, 11: 1143-1146.

    Pantani C, Spreti N, Novelli AA, Ghirardini AV, & Ghetti PF (1995)
    Effect of particulate matter on copper and surfactants' acute
    toxicity to  Echinogammarus tibaldii (Crustacea, Amphipoda).
    Environ Technol, 16: 263-270.

    Paoletti MG, Iovane E, & Cortese M (1988) Pedofauna bioindicators
    and heavy metals in five agroecosystems in north-east Italy. Rev
    Ecol Biol Sol, 25: 33-58.

    Parekh SR & Patel BD (1972) Epidemiologic survey of Indian
    childhood cirrhosis. Indian Pediatr, 9: 43-49.

    Parmelee RW, Wentsel RS, Phillips CT, Simini M, & Checkai RT (1993)
    Soil microcosm for testing the effects of chemical pollutants on
    soil fauna communities and trophic structure. Environ Toxicol Chem,
    12: 1477-1486.

    Parrish CS & Uchrin CG (1990) Runoff-induced metals in Lakes Bay,
    New Jersey. Environ Toxicol Chem, 9: 559-567.

    Paulson PC, Pratt JR, & Cairns J (1983) Relationship of alkaline
    stress and acute copper toxicity in the snail  Goniobasis livescens
     (Menke). Bull Environ Contam Toxicol, 31: 719-726.

    Pedder SCJ & Maly EJ (1985) The effect of lethal copper solutions
    on the behavior of rainbow trout,  Salmo gairdneri. Arch Environ
    Contam Toxicol, 14: 501-507.

    Pennington JA, Young BE, Wilson DB, Johnson RD, & Vanderveen JE
    (1986) Mineral content of food and total diets: the selected
    minerals in food surveys 1982 to 1984. J Am Diet Assoc, 86(7):

    Pennington JA, Young BE, & Wilson DB (1989) Nutritional elements in
    US diets: results from the total diet study, 1982 to 1986. J Am
    Diet Assoc, 89(5): 659-664.

    Percival SS (1995) Neutropenia caused by copper deficiency:
    possible mechanisms of action. Nutr Rev, 53: 59-66.

    Percival SS & Harris ED (1988) Specific binding of ceruloplasmin to
    hemin-induced K562 cells. J Trace Elem Exp Med, 1: 63-70.

    Percival SS & Harris ED (1990) Copper transport from ceruloplasmin:
    characterization of the cellular uptake mechanism. Am J Physiol,
    258C: 140-146.

    Percival SS & Harris ED (1991) Regulation of Cu, Zn superoxide
    dismutase with copper. Caeruloplasmin maintains levels of
    functional enzyme activity during differentiation of K562 cells.
    Biochem J, 274: 153-158.

    Peres I & Pihan JC (1991a) Copper LC50 to  Cyprinus carpio. 
    Influence of hardness, seasonal variation, proposition of maximum
    acceptable toxicant concentration. Environ Technol, 12: 161-167.

    Peres I & Pihan JC (1991b) Study of accumulation of copper by carp
     (Cyprinus carpio L.) - adaptation analysis of bioconcentration by
    the gills. Environ Technol, 12: 169-177.

    Petering HG, Murthy L, Stemmer KL, Finelli VN, & Menden EE (1986)
    Effects of copper deficiency on the cardiovascular system of the
    rat. Biol Trace Elem Res, 9: 251-270.

    Peterson RE & Bollier ME (1955) Spectrophotometric determination of
    serum copper with biscyclohexanoeoxalyldihydrazone. Anal Chem,
    27: 1195-1197.

    Peterson DF, Koo SI, & Lee CC (1990) Interactive effects of dietary
    (Cu) and carbohydrates on serum cholesterol (CH) and minerals.
    FASEB J, 4: A534.

    Petrukhin K, Fischer SG, & Pirastu M (1993) Mapping, cloning and
    genetic characterisation of the region containing the Wilson
    disease gene. Nat Genet, 5: 338.

    Petruzzelli G, Szymura I, Lubrano L, & Cervelli S (1988) Retention
    of Cu and Cd by soil influenced by different adsorbents.
    Agrochimica, 32: 240-243.

    Petruzzelli G, Lubrano L, Petronio BM, Gennaro MC, Vanni A, &
    Liberatori A (1994) Soil sorption of heavy metals as influenced by
    sewage sludge addition. J Environ Sci Health, A29: 31-50.

    Pettersson R & Sandstrom B (1995) Copper. In: Oskarsson A ed. Risk
    evaluation of essential trace elements: essential versus toxic
    levels of intake. Copenhagen, Nordic Council of Ministers, pp
    149-167 (Report NORD 1995:18).

    Phelps HL, Hardy JT, Pearson WH, & Apts CW (1983) Clam burrowing
    behaviour: inhibition by copper-enriched sediment. Mar Pollut Bull,
    14(12): 452-455.

    Phillips DJH (1977) The use of biological indicator organisms to
    monitor trace metal pollution in marine and estuarine environments:
    A review. Environ Pollut, 13: 281-317.

    Phipps GL, Mattson VR, & Ankley GT (1995) Relative sensitivity of
    three freshwater benthic macroinvertebrates to ten contaminants.
    Arch Environ Contam Toxicol, 28: 281-286.

    Pickering QH & Lazorchak JM (1995) Evaluation of the robustness of
    the fathead minnow,  Pimephales promelas, larval survival and
    growth test, US EPA method 1000.0. Environ Toxicol Chem, 14(4):

    Pickering Q, Brungs W, & Gast M (1977) Effect of exposure time and
    copper concentration on reproduction of the fathead minnow
     (Pimephales promelas). Water Res, 11: 1079-1083.

    Pimentel JC & Marques F (1969) "Vineyard sprayer's lung": a new
    occupational disease. Thorax, 24: 678-688.

    Pimentel JC & Menezes AP (1975) Liver granulomas containing copper
    in vineyard sprayer's lung: A new etiology of hepatic
    granulomatosis. Am Rev Respir Dis, 111: 189-195.

    Pimentel JC & Menezes AP (1977) Liver disease in vineyard sprayers.
    Gastroenterology, 72: 275-283.

    Pissarek HP (1974) [Investigation of the anatomical changes in oats
    and sunflower, caused by copper deficiency.] Z Pflanz Ernähr
    Bodenk, 137: 224-234 (in German).

    Plamenac P, Santic Z, Nikulin A, & Serdarevic H (1985) Cytologic
    changes of the respiratory tract in vineyard spraying workers. Eur
    J Respir Dis, 67: 50-55.

    Playle RA (1997) Physiological and toxicological effects of metals
    at gills of freshwater fish. In: Bergmann HL & Dorward-King EJ ed.
    Reassessment of metals criteria for aquatic life protection
    -- Priorities for research and implementation: Proceedings of the
    Pelleston Workshop on Reassessment of Metals Criteria for Aquatic
    Life Protection, Pensacola, Florida, 10-14 February 1996, pp
    101-105 (SETAC Technical Publication Series).

    Playle RC, Gensemer RW, & Dixon DG (1992) Copper accumulation on
    gills of fathead minnows: influence of water hardness, complexation
    and pH of the gill micro-environment. Environ Toxicol Chem, 11:

    Playle RA, Dixon DG, & Burnison K (1993a) Copper and cadmium
    binding to fish gills: modification by dissolved organic carbon and
    synthetic ligands. Can J Fish Aquat Sci, 50: 2667-2677.

    Playle RA, Dixon DG, & Burnison K (1993b) Copper and cadmium
    binding to fish gills: estimates of metal-gill stability constants
    and modelling of metal accumulation. Can J Fish Aquat Sci, 50:

    Pocino M, Malavé I, & Baute L (1990) Zinc administration restores
    the impaired immune response observed in mice receiving excess
    copper by oral route. Immunopharmacol Immunotoxicol, 12: 697-713.

    Pocino M, Baute L, & Malave I (1991) Influence of the oral
    administration of excess copper on the immune response. Fundam Appl
    Toxicol, 16: 249-256.

    Pradhan AM, Talbot IC, & Tanner MS (1983) Indian childhood
    cirrhosis and other cirrhosis of Indian children. Pediatr Res, 17:

    Prasad AS, Brewer GJ, Schoomaker EB, Rabbani P (1978) Hypocupremia
    induced by zinc therapy. J Am Med Assoc, 1978: 2166-2168.

    Prasad MPR, Krishna TP, Pasricha S, Krishnaswamy K, & Quereshi MA
    (1992) Esophageal cancer and diet a case-control study. Nutr
    Cancer, 18: 85-93.

    Pratt WB, Omdahl JL, & Sorenson JRJ (1985) Lack of effects of
    copper gluconate supplementation. Am J Clin Nutr, 42: 681-682.

    Prins HW & Van den Hamer CJ (1981) Comparative studies of copper
    metabolism in liver and kidney of normal and mutated brindled mice
    -- with special emphasis on metallothionein. Comp Biochem Physiol,
    C70(2): 255-260.

    Prohaska JR & Failla ML (1993) Copper and immunity In: Klurfeld DM
    ed. Human nutrition: A comprehensive treatise -- Volume 8.
    Nutrition and immunology. New York, Plenum Press, pp 309-332.

    Prohaska JR, Bailey WR, & Cox DA (1985) Failure of iron injection
    to reverse copper dependent anemia in mice. In: Mills CF, Bremner
    I, & Chesters JK ed. Trace elements in man and animals. Slough,
    Bucks, UK, Commonwealth Agricultural Bureau, pp 27-32.

    Prothro MY (1993) Guidance on interpretation and implementation of
    aquatic life metals criteria. Washington, DC, US Environmental
    Protection Agency, Office of Water Policy and Technical Guidance.

    Punsar S, Erametese O, Karvonen MJ, Ryahanen A, Hilska P, & Vornamo
    H (1975) A search in two Finnish male cohorts for epidemiologic
    evidence of a water factor. J Chron Dis, 28: 259-287.

    Rad MR, Kirchrath L, & Hollenberg CP (1994) A putative p-type
    cu2+-transporting atpase gene on chromosome II of saccharomyces
    cerevisiae. Yeast, 10: 1217-1225.

    Ragan HA, Natch S, Lee GR, Bishop CR, & Cartwright GE (1969) Effect
    of ceruloplasmin on plasma iron in copper deficient swine. Am J
    Physiol, 217: 1320-1323.

    Rahimi A & Bussler W (1973) [Physiological conditions for the
    development of copper deficiency symptoms.] Z Pflanz Bodenk,
    136: 25-32 (in German).

    Rahimi A & Bussler W (1974) [Copper deficiency in higher plants and
    its histochemical detection.] Landwirtsch Forsch Sonderh, 30:
    101-111 (in German).

    Rainbow PS (1988) The significance of trace metal requirements in
    decapods. Symp Zool Soc Lond, 59: 291-313.

    Rainbow PS & Abdennour C (1989) Copper and hemocyanin in the
    mesopelagic decapod crustacean  Systellaspis debilis. Oceanol
    Acta, 12(1): 91-94.

    Rainbow PS & White SL (1989) Comparative strategies of heavy metal
    accumulation by crustaceans: zinc, copper and cadmium in a decapod,
    an amphipod and a barnacle. Hydrobiologia, 174: 245-262.

    Rainbow PS, Moore PG, & Watson D (1989) Talitrid amphipods
    (Crustacea) as biomonitors for copper and zinc. Estuar Coast Shelf
    Sci, 28: 567-582.

    Rajalekshmi P & Mohandas A (1993) Effect of heavy metals on tissue
    glycogen levels in the freshwater mussel,  Lamellidens corrianus 
    (Lea). Sci Total Environ, 1: 617-630.

    Rand GM & Petrocelli SR (1985) Fundamentals of aquatic toxicology.
    New York, Hemisphere Publishing Corporation, 666 pp.

    Räsänen L, Hattula ML, & Arstila AU (1977) The mutagenicity of MCPA
    and its soil metabolites, chlorinated phenols, catechols and some
    widely used slimicides in Finland. Bull Environ Contam Toxicol, 18:

    Rauser WE & Winterhalder EK (1985) Evaluation of copper, nickel,
    and zinc tolerances in four grass species. Can J Bot, 63: 58-63.

    Redpath KJ & Davenport J (1988) The effect of copper, zinc and
    cadmium on the pumping rate of  Mytilus edulis L. Aquat Toxicol,
    13(3): 217-226.

    Reed RH & Moffat L (1983) Copper toxicity and copper tolerance in
     Enteromorpha compressa (L.) Grev. J Exp Mar Biol Ecol, 69(1):

    Rehwoldt R, Menapace LW, Nerrie B, & Alessandrello D (1972) The
    effect of increased temperature upon the acute toxicity of some
    heavy metal ions. Bull Environ Contam Toxicol, 8: 91-96.

    Rehwoldt R, Lasko L, Shaw C, & Wirhowski E (1973) The acute
    toxicity of some heavy metal ions toward benthic organisms. Bull
    Environ Contam Toxicol, 10: 291-294.

    Reilly A & Reilly C (1973) Copper-induced chlorosis in  Becium
    homblei (De Wild) Duvign. and Plancke. Plant Soil, 38: 671-674.

    Reilly C (1967) Accumulation of copper by some Zambian plants.
    Nature (Lond), 215: 667-668.

    Reiser S, Smith JC, Mertz W, Holbrook JT, Scholfield DJ, Powell AS,
    Canfield WK, & Canary JJ (1985) Indices in copper status in humans
    consuming a typical American diet containing either fructose or
    starch. Am J Clin Nutr, 42 242-251.

    Reiser S, Powell A, Yang C-Y, & Canary JJ (1987) Effect of copper
    intake on blood cholesterol and its lipoprotein distribution in
    men. Nutr Rep Int, 36(3): 641-649.

    Remoudaki E, Bergametti G, & Losno R (1991) On the dynamic of the
    atmospheric input of copper and manganese into the western
    Mediterranean Sea. Atmos Environ, 25A: 733-744.

    Reunanen A, Knekt P, & Aaran RK (1992) Serum ceruloplasmin level
    and the risk of myocardial infarction and stroke. Am J Epidemiol,
    136: 1082-1090.

    Reuther W & Smith PF (1952) Iron chlorosis in Florida citrus groves
    in relation to certain soil constituents. Proc Fla State Hortic
    Soc, 65: 62-69.

    Reuther W & Smith PF (1953) Effects of high copper content of sandy
    soil on growth of citrus seedlings. Soil Sci, 75: 219-224.

    Richmond J, Strehlow CD, & Chalkley SR (1993) Dietary intake of Al,
    Ca, Cu, Fe, Pb, and Zn in infants. Br J Biomed Sci, 50: 178-186.

    Ringwood AH (1992) Comparative sensitivity of gametes and early
    developmental stages of a sea urchin species  (Echinometra mathaei)
    and a bivalve species  (Isognomon californicum) during metal
    exposures. Arch Environ Contam Toxicol, 22: 288-295.

    Ritchie HD, Luecke RW, Baltzer BV, Miller ER, Ullrey DE, & Hoefer
    JA (1963) Copper and zinc interrelationships in the pig. J Nutr,
    79: 117-123.

    Robbins CT (1983) Wildlife feeding and nutrition. New York, London,
    Academic Press, 343 pp.

    Robbins KR & Baker DH (1980) Effect of sulfur amino acid level and
    source on the performance of chicks fed high levels of copper.
    Poult Sci, 59: 1246-1253.

    Robson AD & Reuter DJ (1981) Diagnosis of copper deficiency and
    toxicity. In: Loneragan JF, Robson AD, & Graham RD ed. Copper in
    soils and plants: Proceedings of the Golden Jubilee International
    Symposium, Murdoch University, Perth, Western Australia. London,
    New York, Sydney, Academic Press, pp 287-312.

    Rodriguez A, Soto G, Torres S, Venegas G, & Castillo-Duran C (1985)
    Zinc and copper in hair and plasma of children with chronic
    diarrhea. Acta Paediatr Scand, 74: 770-774.

    Rogers JE & Li SW (1985) Effect of metals and other inorganic ions
    on soil microbial activity: soil dehydrogenase assay as a simple
    toxicity test. Bull Environ Contam Toxicol, 34: 858-865.

    Román DA & Rivera L (1992) The behaviour of a Cu (II) ion selective
    electrode in seawater; copper consumption capacity and copper
    determinations. Mar Chem, 38: 165-184.

    Romo-Kröger CM & Llona F (1993) A case of atmospheric contamination
    at the slopes of the Los Andes mountain range. Atmos Environ, 27A:

    Romo-Kröger CM, Morales JR, Dinator MI, Llona F, & Eaton LC (1994)
    Heavy metals in the atmosphere coming from a copper smelter in
    Chile. Atmos Environ, 28: 705-711.

    Rose GA & Parker GH (1983) Metal content of body tissues, diet
    items, and dung of ruffed grouse near the copper-nickel smelters at
    Sudbury, Ont. Can J Zool, 61: 505-511.

    Rowe DW, McGoodwin EB, Martin GR, & Gahn D (1977) Decreased
    lysyloxidase activity in aneurysm-rione, mottled mouse. J Biol
    Chem, 252: 939-942.

    Rucker RB, Parker HE, & Rogler JC (1969) Effect of copper
    deficiency on chick bone collagen and selected bone enzymes. J
    Nutr, 98: 57-63.

    Ruiz R, Romero F, & Besga G (1991) Selective solubilization of
    heavy metals in torrential river sediments. Toxicol Environ Chem,
    33: 1-6.

    Ruoling C & Mengxuan H (1990) A cohort study of cancer mortality in
    copper miners. In Sakurai H ed. Occupational epidemiology.
    Amsterdam, Elsevier Science Publishers BV, Biomedical Division, pp

    Saari JT & Johnson WT (1990) Time course of hematocrit and heart
    weight changes in dietary copper deficiency. FASEB J, 4: A391.

    Sahoo DK, Kar RN, & Das RP (1992) Bioaccumulation of heavy metal
    ions by  Bacillus circulans. Bioresour Technol, 41: 177-179.

    Salim S, Farquharson J, Arneil GC, Cockburn F, Forbes GI, Logan RW,
    Sherlock JC, & Wilson TS (1986) Dietary copper intake in
    artificially fed infants. Arch Dis Child, 61(11): 1068-1075.

    Salomons W & Eagle AM (1990) Hydrology, sedimentology and the fate
    and distribution of copper in mine-related discharges in the Fly
    River system, Papua New Guinea. Sci Total Environ, 97/98: 315-334.

    Salonen JT, Salonen R, Korpela H, Suntioinen S, & Tuomilehto J
    (1991) Serum copper and the risk of acute myocardial infarction: A
    prospective population study in men in Eastern Finland. Am J
    Epidemiol, 134: 268-276.

    Samanidou V & Fytianos K (1990) Mobilization of heavy metals from
    river sediments of northern Greece by complexing agents. Water Air
    Soil Pollut, 52: 217-225.

    Samanidou V, Papadoyannis I, & Vasilikiotis G (1991) Mobilization
    of heavy metals from river sediments of northern Greece, by humic
    substances. J Environ Sci Health, A26: 1055-1068.

    Sanders JR & McGrath SP (1988) Experimental measurements and
    computer predictions of copper complex formation by soluble soil
    organic matter. Environ Pollut, 49: 63-76.

    Sarzeau A (1830) Sur la présence du cuivre dans les végétaux et
    dans le sang. J. Pharm Sci Accessoires, 16: 505-518.

    Sato M & Bremner I (1984) Biliary excretion of metallothionein and
    a possible degradation product in rats injected with copper and
    zinc. Biochem J, 223: 475-479.

    Saward D, Stirling A, & Topping G (1975) Experimental studies on
    the effects of copper on a marine food chain. Mar Biol, 29:

    Sayer MDJ, Reader JP, & Morris R (1989) The effect of calcium
    concentration on the toxicity of copper, lead and zinc to yolk-sac
    fry of brown trout,  Salmo trutta L., in soft, acid water. J Fish
    Biol, 35: 323-332.

    Scanferlato VS & Cairns J (1990) Effect of sediment-associated
    copper on ecological structure and function of aquatic microcosms.
    Aquat Toxicol, 18: 23-34.

    Scarano G, Morelli E, Seritti A, & Zirino A (1990) Determination of
    copper in seawater by anodic stripping voltammetry using
    ethylenediamine. Anal Chem, 62: 943-948.

    Schafer EW Jr & Bowles WA Jr (1985) Acute oral toxicity and
    repellency of 933 chemicals to house and deer mice. Arch Environ
    Contam Toxicol, 14: 111-129.

    Schäfer H, Wenzel A, Fritsche U, Röderer G, & Traunspurger W (1993)
    Long-term effects of selected xenobiotica on freshwater green
    algae: development of a flow-through test system. Sci Total
    Environ, 1(suppl): 735-740.

    Schäfer H, Hettler H, Fritsche U, Pitzen G, Roderer G, & Wenzel A
    (1994) Biotests using unicellular algae and ciliates for predicting
    long-term effects of toxicants. Ecotoxicol Environ Saf, 27: 64-81.

    Schecher WD & McAvoy DC (1992) MINEQL+: a software environment for
    chemical equilibrium modeling. Comput Environ Urban Syst, 16: 65-76

    Scheinberg IH & Sternlieb I (1994) Is non-Indian childhood
    cirrhosis caused by excess dietary copper? Lancet, 344: 1002-1004.

    Schiatz EH (1949) Metal fume fever produced by copper dust. In:
    Proceedings of 9th International Congress on Industrial Medicine,
    London, 13-17 September 1948. Bristol, UK, John Wright & Sons, pp

    Schilsky ML & Sternlieb I (1993) Animal models of copper toxicosis.
    Adv Vet Sci Comp Med, 37: 357-377.

    Schilsky ML (1994) Identification of the Wilson's gene: clues for
    disease pathogenesis and the potential for molecular diagnosis.
    Hepatology, 20: 529.

    Schilsky ML, Scheinberg IH, & Sternlieb I (1991) Prognosis of
    Wilsonian chronic active hepatitis. Gastroenterology, 100: 762.

    Schilsky ML, Scheinberg IH, & Sternlieb I (1994a) Liver
    transplantation for Wilson's disease: indications and outcome.
    Hepatology, 9: 583.

    Schilsky ML, Stockert RJ, & Sternlieb I (1994b) Pleiotropic effect
    of the LEC mutation: a rodent model of Wilson's disease. Am J
    Physiol, 266: G907.

    Schimmelpfennig W, Dieter HH, & Tabert M (1996) Cirrhosis of the
    liver in early childhood and copper content of drinking and well
    water, respectively: Multi-centric retrospective clinical study on
    frequency, distribution and etiology in Germany. Berlin, Institute
    for Water, Soil and Air Hygiene of the Federal Environmental Agency
    of Germany.

    Schmidt JM (1988) Determination of the toxic limit concentration
    and the 50% inhibitory concentration of sodium chloride, copper
    sulfate, dodecylhydrogensulfate-sodium salt and calcium cyanamide
    on the primary root seeds of  Lupius albus and  Cier aretinum. Z
    Wasser Abwasser Forch, 21: 107-109.

    Schmitt CJ & Brumbaugh WG (1990) National contaminant biomonitoring
    program: Concentrations of arsenic, cadmium, copper, lead, mercury,
    selenium, and zinc in US freshwater fish, 1976-1984. Arch Environ
    Contam Toxicol, 19: 731-747.

    Schock MR & Neff CH (1988) Trace metal contamination from brass
    fittings. J Am Water Works Assoc, 7: 47-56.

    Schoenemann HM, Failla ML, & Steele NC (1990) Consequences of
    severe copper deficiency are independent of dietary carbohydrate in
    young pigs. Am J Clin Nutr, 52: 147-154.

    Schramel P, Müller-Höcker J, Meyer U, Wei M, & Eife R (1988)
    Nutritional copper intoxication in three German infants with severe
    liver cell damage (features of Indian childhood cirrhosis). J Trace
    Elem Electrolytes Health Dis, 2: 85-89.

    Schrauzer GN, White DA, & Schneider CJ (1977) Cancer mortality
    correlation studies: IV: Associations with dietary intakes and
    blood levels of certain trace elements, notably Seantagonists.
    Bioinorg Chem, 7: 35-56.

    Schreiber DR, Gordon AS, & Millero FJ (1985) The toxicity of copper
    to the marine bacterium  Vibrio alginolyticus. Can J Microbiol,
    31: 83-87.

    Schroeder WH, Dobson M, Kane DM, & Johnson ND (1987) Toxic trace
    elements associated with airborne particulate matter: A review. J
    Air Pollut Control Assoc, 37: 1267-1285.

    Schubauer-Berigan MK, Dierkes JR, Monson PD & Ankley GT (1993)
    pH-dependent toxicity of Cd, Cu, Ni and Zn to  Ceriodaphnia dubia,
      Pimephales promelas, Hyalella azteca and  Lumbriculus
    variegatus. Environ Toxicol Chem, 12: 1261-1266.

    Schubert WK & Lahey ME (1959) Copper and protein depletion
    complicating hypoferic anemia of infancy. Pediatrics, 24: 710-733.

    Schultz CL & Hutchinson TC (1988) Evidence against a key role for
    metallothionein-like protein in the copper tolerance mechanism of
     Deschampsia cespitosa (L) Beauv. New Phytol, 110(2): 163-171.

    Scott J, Gollan JL, Samourian S, & Sherlock S (1978) Wilson's
    disease, presenting as chronic active hepatitis. Gastroenterology,
    74(4): 645-651.

    Scudder BC, Carter JL, & Leland HV (1988) Effects of copper on
    development of the fathead minnow,  Pimephales promelas
    Rafinesque. Aquat Toxicol, 12: 107-124.

    Scudlark JR, Conko KM, & Church TM (1994) Atmospheric wet
    deposition of trace elements to Chesapeake Bay: CBAD study year 1
    results. Atmos Environ, 28: 1487-1498.

    Seim WK, Curtis LR, Glenn SW, & Chapman GA (1984) Growth and
    survival of developing steelhead trout  (Salmo gairdneri)
    continuously or intermittently exposed to copper. Can J Fish Aquat
    Sci, 41: 433-438.

    Sela M, Tel-Or E, Fritz E, & Huttermann A (1988) Localization and
    toxic effects of cadmium, copper, and uranium in Azolla. Plant
    Physiol, 88: 30-36.

    Semple AB, Parry WH, & Phillips DE (1960) Acute copper poisoning:
    An outbreak traced to contaminated water from a corroded geyser.
    Lancet, 2: 700-701.

    Sethi S, Grover S, & Khodaskar MB (1993) Role of copper in Indian
    childhood cirrhosis. Ann Trop Paediatr, 13: 3-6.

    Shacklette HT & Boerngen JG (1984) Element concentrations in soils
    and other surficial materials of the conterminous United States.
    Washington, DC, US Geological Survey, 105 pp (US Geological Survey
    Professional Paper No. 1270).

    Shanaman JE (1972) Report of one year chronic oral toxicity of
    copper gluconate (W10219A) in beagle dogs. Morris Plains, New
    Jersey, Warner Lambert Research Institute (Report No. 955-0353).

    Shanaman JE, Wazeter FX, & Goldenthal EI (1972) One-year chronic
    oral toxicity of copper gluconate, W/02/09A, in beagle dogs. Morris
    Plains, New Jersey, Warner-Lambert Research Institute (Research
    Report No. 955-0353).

    Shaner SW & Knight AW (1985) The role of alkalinity in the
    mortality of  Daphnia magna in bioassays of sediment-bound copper.
    Comp Biochem Physiol, 82C: 273-277.

    Shanmukhappa H & Neelakantan K (1990) Influence of humic acid on
    the toxicity of copper, cadmium and lead to the unicellular alga,
     Synechosystis aquatilis. Bull Environ Contam Toxicol, 44:

    Sharda B & Bhandari B (1984) Copper concentration in plasma, cells,
    liver, urine, hair and nails in hepalobiliary disorders in
    children. Indian Pediatr, 21: 167-171.

    Sharma VK & Millero FJ (1988) Oxidation of copper(I) in seawater.
    Environ Sci Technol, 22: 768-771.

    Shaw JCL (1992) Copper deficiency in term and preterm infants. In:
    Formon SJ & Zlotkin S ed. Nutritional anaemias. New York, Raven
    Press Ltd, pp 105-119 (Nestlé Nutrition Workshop Series, Volume

    Shenkin A, Fraser WD, Mclelland JD, Fell GS, & Garden OJ (1987)
    Maintenance of vitamin and trace element status in intravenous
    nutrition using a complete nutritive mixture. J Parenter Enter
    Nutr, 11(3): 238-242.

    Shibu MP, Balchand AN, & Nambisan PNK (1990) Trace metal speciation
    in a tropical estuary -- significance of environmental factors. Sci
    Total Environ, 97/98: 267-287.

    Shike M, Roulet M, Kurian R, Whitwell J, Stewart S, & Jeejeebhoy KN
    (1981) Copper metabolism and requirements in total parental
    nutrition. Gastroenterology, 81: 290-297.

    Shiller AM & Boyle EA (1987) Variability of dissolved trace of
    metals in the Mississippi River. Geochim Cosmochim Acta, 51:

    Sideris EG, Charalambous SC, Tsolomyty A, & Katsaros N (1988)
    Mutagenesis, carcinogenesis and the metal elements -- DNA
    interaction. Prog Clin Biol Res, 259: 13-25.

    Silverberg BA, Stokes PM, & Ferstenberg LB (1976) Intranuclear
    complexes in copper tolerant green algae. J Cell Biol, 69: 210-214.

    Sinha S & Chandra P (1990) Removal of Cu and Cd from water by
     Bacopa monnieri L. Water Air Soil Pollut, 51: 271-276.

    Skornik WA & Brain JD (1983) Relative toxicity of inhaled metal
    sulfate salts for pulmonary macrophages. Am Rev Respir Dis, 128:

    Slooff W, Cleven RFMJ, Janus JA, & Ros JPM (1989) Integrated
    criteria document copper. Bilthoven, The Netherlands, National
    Institute of Public Health and Environmental Protection, 147 pp
    (Report No. 758474009).

    Small M, Germaini MS, Small AM, Zoller WH, & Moyers JL (1981)
    Airborne plume study of emissions from the processing of copper
    ores in southeastern Arizona. Environ Sci Technol, 15: 293-299.

    Smith CH & Bidlack WR (1980) Interrelationships of dietary ascorbic
    acid and iron on the tissue distribution of ascorbic acid, iron and
    copper in female guinea-pigs. J Nutr, 110: 1398-1408.

    Smith MJ & Heath AG (1979) Acute toxicity of copper, chromate,
    zinc, and cyanide to freshwater fish: effect of different
    temperatures. Bull Environ Contam Toxicol, 22: 113-119.

    Smith GJ & Rongstad OJ (1982) Small mammal heavy metal
    concentrations from mined and control sites. Environ Pollut, 28A:

    Smith JD, Jordan RM, & Nelson ML (1975) Tolerance of ponies to high
    levels of dietary copper. J Anim Sci, 41: 1645-1649.

    Smith KL, Hann AC, & Harwood JL (1986) The subcellular localization
    of absorbed copper in  Fucus. Physiol Plant, 66(4): 692-698.

    Smyth HF, Carpenter CP, Weil CS, Pozzani UC, Striegel JA, & Nycum
    JS (1969) Range-finding toxicity data: list VII. Am Ind Hyg Assoc
    J, 30: 470-476.

    Snell TW & Persoone G (1989a) Acute toxicity bioassays using
    rotifers: I. A test for brackish and marine environments with
     Brachionus plicatilis. Aquat Toxicol, 14: 65-80.

    Snell TW & Persoone G (1989b) Acute toxicity bioassays using
    rotifers: II. A freshwater test with  Brachionus rubens. Aquat
    Toxicol, 14: 81-92.

    Snell TW, Moffat BD, Janssen C, & Persoone G (1991) Acute toxicity
    tests using rotifers: IV. Effects of cyst age, temperature, and
    salinity on the sensitivity of  Branchionus calyciflorus.
    Ecotoxicol Environ Saf, 21: 308-317.

    Soares HMVM, Teresa M, & Vasconcelos SD (1994) Study of the
    lability of copper(II)-fulvic acid complexes by ion selective
    electrodes and potentiometric stripping analysis. Anal Chim Acta,
    293: 261-270.

    Solioz M, Odermatt A, & Krapf R (1994) Copper pumping ATPases:
    common concepts in bacteria and man. FEBS Lett, 346: 44-47.

    Solomons NW (1979) On the assessment of zinc and copper nutriture
    in man. Am J Clin Nutr, 32: 856-871.

    Sorenson JRJ (1989) Copper complexes offer a physiological approach
    to the treatment of chronic diseases. Prog Med Chem, 26: 437-568.

    Sosnowski SL, Germond DJ, & Gentile JH (1979) The effect of
    nutrition on the response of field populations of the calanoid
    copepod  Acartia tonsa to copper. Water Res, 13: 449-452.

    Spitalny KC, Brondum J, Vogt RL, Sargent HE, & Kappel S (1984)
    Drinking-water-induced copper intoxication in a Vermont family.
    Pediatrics, 74: 1103-1106.

    Spurgeon DJ, Hopkin SP, & Jones DT (1994) Effects of cadmium,
    copper, lead and zinc on growth, reproduction and survival of the
    earthworm  Eisenia fetida (Savigny): Assessing the environmental
    impact of point-source metal contamination in terrestrial
    ecosystems. Environ Pollut, 84: 123-130.

    Srivastava AK, Gupta BN, Bihari V, Mathur N, Gaur JS, Mahendra PN,
    Kumar P, & Bharti RS (1992) Clinical studies in workers engaged in
    maintenance of watermark moulds in a paper mill. Int Arch Occup
    Environ Health, 64: 141-145.

    Stack T, Aggett PJ, Aitken E, & Lloyd DJ (1990) Routine L-ascorbic
    acid supplementation does not alter iron, copper, and zinc balance
    in low-birth-weight infants fed a cow's milk formula. J Pediatr
    Gastroenterol Nutr, 10: 351-356.

    Stauber JL (1995) Toxicity testing using marine and freshwater
    unicellular algae. Australasian J Ecotoxicol, 1: 15-24.

    Stauber JL & Florence TM (1985a) The influence of iron on copper
    toxicity to the marine diatom,  Nitzchia closterium (Ehrenberg) W.
    Smith. Aquat Toxicol, 6: 297-305.

    Stauber JL & Florence TM (1985b) Interactions of copper and
    manganese: a mechanism by which manganese alleviates copper
    toxicity to the marine diatom,  Nitzchia closterium (Ehrenberg) W.
    Smith. Aquat Toxicol, 7: 241-254.

    Stauber JL & Florence TM (1987) Mechanism of toxicity of ionic
    copper and copper complexes to algae. Mar Biol, 94: 511-519.

    Steele CW (1989) Effects of sublethal exposure to copper on diel
    activity of sea catfish,  Arius felis. Hydrobiologia, 178:

    Stein RS, Jenkins D, & Korns ME (1976) Death after use of cupric
    sulfate as emetic. J Am Med Assoc, 235: 801.

    Steinkuhler C, Sapora O, Carri MT, Nagel W, Marcocci L, Ciriolo MR,
    Weser U, & Rotilio G (1991) Increase of Cu, Zn-superoxide dismutase
    activity during differentiation of human K562 cells involves
    activation by copper of a constantly expressed copper-deficient
    protein. J Biol Chem, 266: 24580-24587.

    Stenhammar L (1979) [Copper poisoning: A differential diagnosis of
    diarrhoea in children.] Lakartidningen, 76(30-31): 2618-2620 (in

    Stephenson RR (1983) Effects of water hardness, water temperature,
    and size of the test organism on the susceptibility of the
    freshwater shrimp,  Gammarus pulex (L.) to toxicants. Bull Environ
    Contam Toxicol, 31: 459-466.

    Stephenson MD & Leonard GH (1994) Evidence for the decline of
    silver and lead and the increase of copper from 1977 to 1990 in the
    coastal marine waters.

    Stern RV & Frieden E (1993) Partial purification of the rat
    erythrocyte ceruloplasmin receptor monitored by an electrophoresis
    mobility shift assay. Anal Biochem, 212: 221-228.

    Sternlieb I (1980) Copper and the liver. Gastroenterology, 78:

    Sternlieb I (1990) Perspectives on Wilson's disease. Hepatology,
    12: 1234.

    Sternlieb I (1993) The outlook for the diagnosis of Wilson's
    disease. J Hepatol, 17: 263.

    Stevens MD, DiSilvestro RA, & Harris ED (1984) Specific receptor
    for ceruloplasmin in membrane fragments from aortic and heart
    tissues. Biochemistry, 23: 261-266.

    Stevenson FJ (1986) Cycles of soil: Carbon, nitrogen, phosphorus,
    sulfur, micronutrients. New York, John Wiley & Sons Ltd.

    Stewart C, Norton DA, & Fergusson JE (1991) Historical monitoring
    of heavy metals in kahikatea ring wood in Christchurch, New
    Zealand. Sci Total Environ, 105: 171-190.

    Stiff MJ (1971) The chemical states of copper in polluted fresh
    water and a scheme of analysis to differential them. Water Res, 5:

    Stoner GD, Shimkin MB, Troxell MC, Thompson TL, Terry LS (1976)
    Test for carcinogenicity of metallic compounds by the pulmonary
    tumour response in strain A mice. Cancer Res, 36: 1744-1747.

    Stouthart XJHX, Haans JLM, Lock RAC, & Wendelaar Bonga SE (1996)
    Effects of water pH on copper toxicity to early life stages of the
    common carp  (Cyprinus carpio). Environ Toxicol Chem, 15(3):

    Strain WH, Hershey CO, McInnes S, Breslau D, Hershey LA, McKinney
    BM, Varnes AW, & Khourey CJ (1984) Hazards to groundwater from acid
    rain. Trace Subst Environ Health, 18: 178-184.

    Streit B (1984) Effects of high copper concentrations on soil
    invertebrates (earthworms and oribatid mites): experimental results
    and a model. Oecologia, 64: 381-388.

    Stromgren T & Nielsen MV (1991) Spawning frequency, growth and
    mortality of  Mytilus edulis larvae, exposed to copper and diesel
    oil. Aquat Toxicol, 21: 171-180.

    Sturgeon P & Brubaker C (1956) Copper deficiency in infants. Am J
    Dis Child, 92: 254-265.

    Suciu I, Prodan L, Lazar V, Ilea E, Cocîrla A, Olinici L, Paduraru
    A, Zagreanu O, Lengyel P, Gyrffi L, & Andru D (1981) Research on
    copper poisoning. Med Lav, 3: 190-197.

    Sunda WG & Guillard RRL (1976) Relationship between cupric ion
    activity and toxicity of copper to phytoplankton. J Mar Res, 34:

    Suttle NF & Mills CF (1966) Studies of the toxicity of copper to
    pigs. I. Effects of oral supplements of zinc and iron salts on the
    development of copper toxicosis. Br J Nutr, 20: 135-149.

    Sutton AM, Harvie A, Cockburn A, Farquharson J, & Logan RW (1985)
    Copper deficiency in the preterm infant of very low birth weight:
    four cases and a reference range for plasma copper. Arch Dis Child,
    60(7): 644-651.

    Suzuki N, Iwata Y, & Imura H (1987) Determination of several trace
    metals in seaweed by neutron activation analysis after
    diethydithiocarbamate extraction and polystyrene-foam collection.
    Int J Environ Anal Chem, 30: 289-297.

    Sweet CW, Vermette SJ, & Landsberger S (1993) Sources of toxic
    trace elements in urban air in Illinois. Environ Sci Technol, 27:

    Symeonidis L, McNeilly T, & Bradshaw AD (1985) Differential
    tolerance of three cultivars of Agrostis capillaris L. to cadmium,
    copper, lead, nickel and zinc. New Phytol, 101: 309-315.

    Tachibana K (1952) Pathological transition and functional
    vicissitude of liver during formation of cirrhosis by copper.
    Nagoya J Med Sci, 15: 108-114.

    Taggart DPP, Shenkin A, & Fell GS (1986) Observations on serum
    iron, zinc, copper, and magnesium in intravenously fed patients
    with chronic sepsis. Clin Nutr, 5: 139-144.

    Tan WT, Tan GS, & Khan ISAN (1988) Solubilities of trace copper and
    lead species and the complexing capacity of river water in the
    Linggi River Basin. Environ Pollut, 52: 221-235.

    Tanaka Y, Hatano S, Nishi Y, & Usui T (1980) Nutritional copper
    deficiency in a Japanese infant on formula. J Pediatr, 96: 255-257.

    Tanner MS, Postmann B, Mowat AP, Williams B, Pandit A, Mills C, &
    Bremner I (1979) Increased hepatic copper concentration in Indian
    Childhood cirrhosis. Lancet, 1: 1203-1205.

    Tanner MS, Kantarjian AH, Bhave SA, & Pandit AN (1983) Early
    introduction of copper-contaminated animal milk feeds as a possible
    cause of Indian childhood cirrhosis. Lancet, 2: 992-995.

    Tanner MS, Bhave SA, Pradhan AM, & Pandit AN (1987) Clinical trials
    of penicillamine in Indian childhood liver cirrhosis. Arch Dis
    Childh, 62: 118-124.

    Tanzi RE, Petrukhin K, & Cherov I (1993) The Wilson gene is a
    copper transporting ATPase with homology to the Menkes disease
    gene. Nat Genet, 5: 344.

    Tavassoli M, Kishimoto T, & Kataoka M (1986) Liver endothelium
    mediates the hepatocyte's uptake of ceruloplasmin. J Cell Biol,
    102: 1298-1303.

    Taylor GJ & Crowder AA (1984) Copper and nickel tolerance in
     Typha latifolia clones from contaminated and uncontaminated
    environments. Can J Bot, 62: 1304-1308.

    Taylor GJ & Foy CD (1985) Differential uptake and toxicity of ionic
    and chelated copper in  Triticum aestivum. Can J Bot, 63:

    Taylor D, Maddock BG, & Mance G (1985) The acute toxicity of nine
    'grey list' metals (arsenic, boron, chromium, copper, lead, nickel,
    tin, vanadium and zinc) to two marine fish species: dab
     (Limanda limanda) and grey mullet  (Chelon labrosus). Aquat
    Toxicol, 7: 135-144.

    Taylor EJ, Maund SJ, & Pascoe D (1991) Toxicity of four common
    pollutants to the freshwater macroinvertebrates  Chironomus
    riparius  Meigen (Insecta: Diptera) and  Gammarus pulex (L.)
    (Crustacea: Amphipoda). Arch Environ Contam Toxicol, 21: 371-376.

    Thiel H & Finck A (1973) [Determination of limiting values of
    optimum copper supply of oat and barley plants.] Z Pflanz Bodenk,
    134: 107-125 (in German).

    Thomas GR, Forbes JR, Roberts EA, Walshe JM, & Cox DW (1995) The
    Wilson disease gene -- spectrum of mutations and their
    consequences. Nat Genet, 9: 210-217.

    Thompson JJ & Houk RS (1986) Inductively coupled plasma mass
    spectrometric detection for multielement flow injection analysis
    and elemental speciation by reversed-phase liquid chromatography.
    Anal Chem, 58: 2541-2548.

    Thorn JM, Aggett PJ, Delves HT, & Clayton BE (1978) Mineral and
    trace metal supplement for use with synthetic diets based on
    comminuted chicken. Arch Dis Child, 53(12): 931-938.

    Thornalley PJ & Vasak M (1985) Possible role for metallothionein in
    protection against radiation-induced oxidative stress. Kinetics and
    mechanism of its reaction with superoxide and hydroxyl radicals.
    Biochim Biophys Acta, 827(1): 36-44.

    Tijero J, Guardiola E, Mirada F, & Cortijo M (1991) Effect of
    Cu2+, Ni2+ and Zn2+ on an anaerobic digestion system. J Environ
    Sci Health, 26: 799-811.

    Timmermans KR & Walker PA (1989) The fate of trace metals during
    the metamorphosis of chironomids (Diptera, Chironomidae). Environ
    Pollut, 62: 73-85.

    Tinker D, Romero-Chapman N, Reiser K, Hyde D, & Rucker C (1990)
    Elastin metabolism during recovery from impaired cross-linking
    formation. Arch Biochem Biophys, 278: 326-332.

    Tinwell H & Ashby J (1990) Inactivity of copper sulphate in a mouse
    bone-marrow micronucleus assay. Mutat Res, 245: 223-226.

    Tomlin C ed. (1994) A world compendium -- The pesticide manual,
    incorporating the agrochemicals handbook. London, Crop Protection

    Town RM & Powell HKJ (1993) Ion-selective electrode potentiometric
    studies on the complexation of copper(II) by soil-derived humic
    acid and fulvic acid. Anal Chim Acta, 279: 221-233.

    Tubbing DMJ, Admiraal W, & Katako A (1995) Successive changes in
    bacterioplankton communities in the River Rhine after copper
    additions. Environ Toxicol Chem, 14(9): 1507-1512.

    Tuddenham WM & Dougall PA (1978) Copper. In: Kirk-Othmer
    encyclopedia of chemical technology, 3rd ed. New York, John Wiley
    & Sons Ltd, pp 819-869.

    Turnlund JR, Swanson CA, & King JC (1983) Copper absorption and
    retention in pregnant women fed diets based on animal and plant
    proteins. J Nutr, 113: 2346-2352.

    Turnlund JR, King JC, Keyes WR, Gong B, & Michel MC (1984) A stable
    isotope study of zinc absorption in young men: effects of phytate
    and alpha-cellulose. Am J Clin Nutr, 40: 1071-1077

    Turnlund JR, Keyes WR, Anderson HL, & Acord LL (1989) Copper
    absorption and retention in young men at three levels of dietary
    copper by use of the stable isotope 65Cu. Am J Clin Nutr, 49:

    Turnlund JR, Keens CL, & Smith RG (1990) Copper status and urinary
    and salivary copper in young men at three levels of dietary copper.
    Am J Clin Nutr, 51: 658-664.

    Turnlund JR, Keyes WR, Hudson CA, Betschart AA, Kretsch MJ, &
    Sauberlich HE (1991) A stable-isotope study of zinc, copper, and
    iron absorption and retention by young women fed vitamin
    B-6-deficient diets. Am J Clin Nutr, 54: 1059-1064.

    Tyler LD & McBride MB (1982) Mobility and extractability of
    cadmium, copper, nickel, and zinc in organic and mineral soil
    columns. Soil Sci, 134: 198-205.

    Tyrala EE (1986) Zinc and copper balances in preterm infants.
    Pediatrics, 77: 513-517.

    Uauy R, Castillo-Duran C, Fisberg M, Fernandez N, & Valenzuela A
    (1985) Red cell superoxide dismutase activity as an index of human
    copper nutrition. J Nutr, 115: 1650-1655.

    Underwood EJ (1977) Trace elements in human and animal nutrition,
    4th ed. New York, London, Academic Press, 545 pp.

    Ünlü E & Gümgüm B (1993) Concentrations of copper and zinc in fish
    and sediments from the Tigris River in Turkey. Chemosphere, 26(11):

    US EPA (1984) Ambient water quality criteria for copper.
    Washington, DC, US Environmental Protection Agency, 84 pp (EPA

    US EPA (1986) Test methods for evaluating solid waste, 3rd ed.
    Washington, DC, US Environmental Protection Agency, Office of Solid
    Waste (Report SW-846).

    US EPA (1991) Maximum contaminant levels, goals and national
    primary drinking-water regulations for lead and copper, final rule.
    Fed Reg, 56: 110.

    US EPA (1992) Interim guidance on interpretation and implementation
    of aquatic life criteria for metals. Washington, DC, US
    Environmental Protection Agency.

    US EPA (1995) Sampling ambient water for trace metals at EPA water
    quality criteria levels: Method 1669. Washington, DC, US
    Environmental Protection Agency.

    V-Balogh K (1988) Heavy metal pollution from a point source
    demonstrated by mussel  (Unio pictorum L.) at Lake Balaton,
    Hungary. Bull Environ Contam Toxicol, 41: 910-914.

    Van Campen DR & Mitchell EA (1965) Absorption of Cu64, Zn65, Mo99,
    and Fe59 from ligated segments of the rat gastrointestinal tract.
    J Nutr, 86: 120-124.

    Van den Berg CMG (1984) Organic and inorganic speciation of copper
    in the Irish Sea. Mar Chem, 14: 201-212.

    Van den Berg GJ & Beynen AC (1992) Influence of ascorbic acid
    supplementation on copper metabolism in rats. Br J Nutr, 68:

    Van den Berg CMG, Nimmo M, Daly P, & Turner DR (1990) Effects of
    the detection window on the determination of organic copper
    speciation in estuarine waters. Anal Chim Acta, 232: 149-159.

    Van den Berg GJ, Yu S, Lemmens AG, & Beynen AC (1994) Ascorbic acid
    feeding of rats reduces copper absorption, causing impaired copper
    status and depressed biliary copper excretion. Biol Trace Elem Res,
    41: 47-58.

    Van Gestel CAM, Van Dis WA, Van Breemen EM, & Sparenburg PM (1989)
    Development of a standardized reproduction toxicity test with the
    earthworm species  Eisenia fetida Andrei using copper,
    pentachlorophenol, and 2,4-dichloroaniline. Ecotoxicol Environ Saf,
    18: 305-312.

    Van Gestel CAM, Van Dis WA, Dirven-van Breemen EM, Sparenburg PM,
    & Baerselman R (1991) Influence of cadmium, copper, and
    pentachlorophenol on growth and sexual development of  Eisenia
    Andrei (Oligochaeta; Annelida). Biol Fertil Soils, 12: 117-121.

    van Leeuwen CJ, Büchner JL, & van Dijk H (1988) Intermittent flow
    system for population toxicity studies demonstrated with  Daphnia
    and copper. Bull Environ Contam Toxicol, 40: 496-502.

    van Tilborg WJM (1996) 'A further look at zinc' refuted. The
    Netherlands, VTBC, 107 pp (Report No. 9601).

    Vardia HK, Rao PS, & Durve VS (1988) Effect of copper, cadmium and
    zinc on fish-food organisms,  Daphnia lumholtzi and
     Cypris subglobosa. Proc Indian Acad Sci (Anim Sci), 97(2):

    Venugopal B & Luckey TD (1978) Metal toxicity in mammals -- 2. New
    York, Plenum Press.

    Vermeiren K, Vandecasteele C, & Dams R (1990) Determination of
    trace amounts of cadmium, lead, copper and zinc in natural waters
    by inductively coupled plasma atomic emission spectrometry with
    thermospray nebulisation, after enrichment on Chelex-100. Analyst,
    115: 17-22.

    Verriopoulos G & Dimas S (1988) Combined toxicity of copper,
    cadmium, zinc, lead, nickel, and chrome to the copepod  Tisbe
    holothuriae.  Bull Environ Contam Toxicol, 41: 378-384.

    Verweij W, Glazewski R, & DeHaan H (1992) Speciation of copper in
    relation to its bioavailability. Chem Speciation Bioavailab, 4:

    Viarengo A, Canesi L, Pertica M, Poli G, Moore MN, & Orunesu M
    (1990) Heavy metal effects on lipid peroxidation in the tissues of
     Mytilus galloprovincialis Lam. Comp Biochem Physiol, 97C(1):

    Viksna A, Mwiruki G, Jagner D, & Selin E (1995) Intercomparison
    between energy-dispersive X-ray fluorescence and stripping
    potentiometry for the determination of copper levels in human
    serum. X-Ray Spectrom, 24: 76-80.

    Viren JR & Silvers A (1994) Unit risk estimates for airborne
    arsenic exposure: an updated view based on recent data from two
    copper smelter cohorts. Regul Toxicol Pharmacol, 20: 125-138.

    Visviki I & Rachlin JW (1991) The toxic action and interactions of
    copper and cadmium to the marine alga  Dunaliella minuta, in both
    acute and chronic exposure. Arch Environ Contam Toxicol, 20:

    Visviki I & Rachlin JW (1994a) Acute and chronic exposure of
     Dunaliella salina and  Chlamydomonas bullosa to copper and
    cadmium: Effects on growth. Arch Environ Contam Toxicol, 26:

    Visviki I & Rachlin JW (1994b) Acute and chronic exposure of
     Dunaliella salina and  Chlamydomonas bullosa to copper and
    cadmium: Effects on ultrastructure. Arch Environ Contam Toxicol,
    26: 154-162.

    Vogt G & Quinitio ET (1994) Accumulation and excretion of metal
    granules in the prawn,  Penaeus monodon, exposed to water-borne
    copper, lead, iron and calcium. Aquat Toxicol, 28: 223-241.

    Vohra P, Gray GA, & Kratzer FH (1965) Phytic acid-metal complexes.
    Proc Soc Exp Biol Med, 120: 447-449.

    Vollkopf U & Barnes K (1995) Rapid multielement analysis of urine.
    At Spectrosc, 1995: 19-21.

    Vranken G, Tiré C, & Heip C (1988) The toxicity of paired metal
    mixtures to the nematode  Monhystera disjuncta (Bastian, 1865).
    Mar Environ Res, 26: 161-179.

    Vulpe C, Levinson B, Whitney S, Packman S, & Gitschier J (1993)
    Isolation of a candidate gene for Menkes disease and evidence that
    it encodes a copper-transporting ATPase. Nat Genet, 3(1): 7-13
    (erratum in Nat Genet, 3(3):273).

    Wainwright SJ & Woolhouse HW (1977) Some physiological aspects of
    copper and zinc tolerance in  Agrostis tenuis Sibth.: Cell
    elongation and membrane damage. J Exp Bot, 28: 1029-1036.

    Waldrop GL & Ettinger MJ (1990) The relationship of excess copper
    accumulation by fibroblasts from the brindled mouse model of Menkes
    disease to the primary defect. Biochem J, 267: 417-422.

    Walker-Smith J & Blomfield J (1973) Wilson's disease or chronic
    copper poisoning? Arch Dis Child, 48: 476-479.

    Walsh LH, Erhardt WH, & Seibel HD (1972) Copper toxicity in
    snapbeans  (Phaseolus vulgaris L.). J Environ Qual, 1: 197-200.

    Walsh FM, Crosson FJ, Bayley M, McReynolds J, & Pearson BJ (1977)
    Acute copper intoxication: Pathophysiology and therapy with a case
    report. Am J Dis Child, 131: 149-151.

    Walshe JM (1995) Copper: Not too little, not too much, but just
    right. J R Coll Phys (Lond), 29: 280-287

    Wapnir RA & Balkman C (1992) Intestinal absorption of copper:
    influence of carbohydrates. Biochem Med Metab Biol, 47: 47-53.

    Wapnir RA & Lee SY (1993) Dietary regulation of copper absorption
    and storage in rats: Effects of sodium, zinc and histidine-zinc. J
    Am Coll Nutr, 12: 714-719.

    Ward GM & Nagy JG (1976) Molybdenum and copper in Colorado forages,
    molybdenum toxicity in deer, and copper supplementation in cattle.
    In: Chappel WR & Petersen KK ed. Molybdenum in the environment:
    Volume 1. The biology of molybdenum. New York, Marcel Dekker, Inc.,
    pp 97-113.

    Watton AJ & Hawkes HA (1984) The acute toxicity of ammonia and
    copper to the gastropod  Potamopyrgus jenkinsi (Smith). Environ
    Pollut, A36: 7-29.

    Weant GE (1985) Sources of copper air emissions. Research Triangle
    Park, North Carolina, US Environmental Protection Agency, Air and
    Energy Engineering Research Laboratory (EPA 600/2-85-046).

    Weeks JM (1992a) The Talitrid amphipod (Crustacea)
     Platorchestia platensis as a biomonitor of trace metals (Cu and
    Zn) in Danish waters. In: Bjornestad E, Hagerman L, & Jensen K ed.
    Proceedings of the 12th Baltic Marine Biologists Symposium,
    Helsingor, Denmark, 25-30 August 1991. Fredensborg, Olsen & Olsen,
    pp 173-178.

    Weeks JM (1992b) Copper-rich granules in the ventral caeca of
    Talitrid amphipods (Crustacea; Amphipoda: Talitridae). Ophelia, 36:

    Weeks JM (1993) Effects of dietary copper and zinc concentrations
    on feeding rates of two species of Talitrid amphipods (Crustacea).
    Bull Environ Contam Toxicol, 50: 883-890.

    Weeks JM & Rainbow PS (1991) The uptake and accumulation of zinc
    and copper from solution by two species of Talitrid amphipods
    (Crustacea). J Mar Biol Assoc (UK), 71: 811-826.

    Weeks JM & Rainbow PS (1993) The relative importance of food and
    seawater as sources of copper and zinc to Talitrid amphipods
    (crustacea; Amphipoda; Talitridae. J Appl Ecol, 30: 722-735.

    Weeks JM, Rainbow PS, & Moore PG (1992) The loss, uptake and tissue
    distribution of copper and zinc during the moult cycle in an
    ecological series of Talitrid amphipods (Crustacea: Amphipoda).
    Hydrobiologia, 245: 15-25.

    Weeks JM, Jensen FB, & Depledge MH (1993) Acid-base status,
    haemolymph composition and tissue copper accumulation in the shore
    crab  Carcinus maenas exposed to combined copper and salinity
    stress. Mar Ecol Prog Ser, 97: 91-98.

    Weiss M, Müller-Höcker J, Wiebecke B, & Belohradsky BH (1989) First
    description of Indian childhood cirrhosis in a non-Indian infant in
    Europe. Acta Paediatr Scand, 79: 152-156.

    Welsh PG, Skidmore JF, Spry DJ, Dixon DG, Hutchinson NJ, & Hickie
    BE (1993) Effect of pH and dissolved organic carbon on the toxicity
    of copper to larval fathead minnow  (Pimephales promelas) in
    natural lake waters of law alkalinity. Can J Fish Aquat Sci, 50:

    Wharton DC & Rader M (1970) Rapid spectrophotometric method for
    determination of micro amounts of copper in proteins. Anal Biochem,
    33(2): 226-229.

    White SL & Rainbow PS (1985) On the metabolic requirements for
    copper and zinc in molluscs and crustaceans. Mar Environ Res, 16:

    WHO (1982) Evaluation of certain food additives and contaminants.
    Twenty-sixth report of the Joint FAO/WHO Expert Committee on Food
    Additives. Geneva, World Health Organization, pp 31-32 (WHO
    Technical Report Series, No. 683).

    WHO (1990) In: Akre J ed. Infant feeding -- the physiological
    basis. Supplement to Volume 67 of WHO Bulletin, 1989. Geneva, World
    Health Organization, pp 68-84.

    WHO (1993) Guidelines for drinking-water quality, 2nd ed. Volume 1:
    Recommendations. Geneva, World Health Organization, p 46.

    WHO (1996) Copper. In: Trace elements in human nutrition and
    health. Geneva, World Health Organization, chapter 7, pp 123-143.

    Widdowson EM & Dickerson JWT (1964) Chemical composition of the
    body. In: Comar CL & Bronner F ed. Mineral metabolism. New York,
    Academic Press, vol 2, chapter 17, p 1247.

    Widdowson EM, Dauncey J, & Shaw JCL (1974) Trace elements in fetal
    and early postnatal development. Proc Nutr Soc, 33: 275-284.

    Wieser W, Busch G, & Büchel L (1976) Isopods as indicators of the
    copper content of soil and litter. Oecologia, 23: 107-114.

    Williams DM (1982) Clinical significance of copper deficiency and
    toxicity in the world population. In: Prasad AS ed. A clinical,
    biochemical and nutritional aspects of trace elements. New York,
    Alan R. Lyss, pp 277-299.

    Williams DM (1983) Copper deficiency in humans. Semin Hematol,
    20: 118-128.

    Wilson SAK (1912) Progressive lenticular degeneration: A familial
    nervous disease associated with cirrhosis of the liver. Brain,
    34: 295-509.

    Wilson P, Cooke M, Cawley J, Giles L, & West M (1995) Comparison of
    the determination of copper, nickel and zinc in contaminated soils
    by X-ray fluorescence spectrometry and inductively coupled plasma
    spectrometry. X-Ray Spectrom, 24: 103-108.

    Windom HL, Byrd JT, Smith RG Jr, & Huan F (1991) Inadequacy of
    NASQAN data for assessing metal trends in the nation's river.
    Environ Sci Technol, 25(6): 1137-1142.

    Winge DR & Mehra RK (1990) Host defenses against copper toxicity.
    Int Rev Exp Pathol, 31: 47-83.

    Winner RA (1984) The toxicity and bioaccumulation of cadmium and
    copper as affected by humic acid. Aquat Toxicol, 5: 267-274.

    Winner RW (1985) Bioaccumulation and toxicity of copper as affected
    by interactions between humic acid and water hardness. Water Res,
    19(4): 449-455.

    Winner RW & Farrell MP (1976) Acute and chronic toxicity of copper
    to four species of  Daphnia. J Fish Res Board Can, 33: 1685-1691.

    Winner RW & Owen HA (1991a) Seasonal variability in the sensitivity
    of freshwater phytoplankton communities to a chronic copper stress.
    Aquat Toxicol, 19: 73-88.

    Winner RW & Owen HA (1991b) Toxicity of copper to
     Chlamydomonas reinhardtii (Chlorophyceae) and
     Ceriodaphnia dubia (Crustacea) in relation to changes in water
    chemistry of a freshwater pond. Aquat Toxicol, 21: 157-170.

    Winner RW, Owen HA, & Moore MV (1990) Seasonal variability in the
    sensitivity of freshwater lentic communities to a chronic copper
    stress. Aquat Toxicol, 17: 75-92.

    Wirth PL & Linder MC (1985) Distribution of copper among multiple
    components of human serum. J Natl Cancer Inst, 75: 277-284.

    Wong PK (1988) Mutagenicity of heavy metals. Bull Environ Contam
    Toxicol, 40: 597-603.

    Wong CK (1992) Effects of chromium, copper, nickel, and zinc on
    survival and feeding of the cladoceran  Moina macrocopa. Bull
    Environ Contam Toxicol, 49: 593-599.

    Wong MH & Bradshaw AD (1982) A comparison of the toxicity of heavy
    metals, using root elongation of rye grass, Lolium perenne. New
    Phytol, 91: 255-261.

    Wong PK & Chang L (1991) Effects of copper, chromium and nickel on
    growth, photosynthesis and chlorophyll a synthesis of
     Chlorella pyrenoidosa 251. Environ Pollut, 72: 127-139.

    Wong PK, Lam KC, & So CM (1993a) Removal and recovery of Cu(II)
    from industrial effluent by immobilized cells of Pseudomonas putida
    II-11. Appl Microbiol Biotechnol, 39: 127-131.

    Wong CK, Chu KH, Tang KW, Tan TW, & Wong LJ (1993b) Effects of
    chromium, copper and nickel on survival and feeding behaviour of
     Metapenaeus ensis larvae and postlarvae (Decapoda: Penaeidae).
    Mar Environ Res, 36: 63-78.

    Wong YS, Lam EKH, & Tam NFY (1994) Physiological effects of copper
    treatment and its uptake pattern in  Festuca rubra cv. Merlin.
    Resour Conserv Recycl, 11(1-4): 311-319

    Wood AM (1983) Available copper ligands and the apparent
    bioavailability of copper to natural phytoplankton assemblages. Sci
    Total Environ, 28: 51-64.

    Wood CM (1992) Flux measurements as indices of H+ and metal
    effects on freshwater fish. Aquat Toxicol, 22: 239-264

    Woolhouse HW (1983) Toxicity and tolerance in the responses of
    plants to metals Chapter 7. In: Lange OC, Nobel PS, Osmond CB, &
    Ziegler H ed. Encyclopedia of plant physiology. New York, Basel,
    Springer-Verlag, chapter 7, pp 245-300.

    Wu L & Bradshaw AD (1972) Aerial pollution and the rapid evolution
    of copper tolerance. Nature (Lond), 238: 167-169.

    Wu L & Kruckeberg AL (1985) Copper tolerance in two legume species
    from a copper mine habitat. New Phytol, 99: 565-570.

    Wu L & Lin S-L (1990) Copper tolerance and copper uptake of Lotus
    purshianus (Benth.) Clem. & Clem. and its symbiotic  Rhizobium loti
     derived from a copper mine waste population. New Phytol, 116:

    Wu ZY, Han M, Lin ZC, & Ondov JM (1994) Chesapeake Bay atmospheric
    deposition study, year 1: sources and dry deposition of selected
    elements in aerosol particles. Atmos Environ, 28(8): 1471-1486.

    Wurtsbaugh WA & Horne AJ (1982) Effects of copper on nitrogen
    fixation and growth of blue-green algae in natural plankton
    associations. Can J Fish Aquat Sci, 39: 1636-1641.

    Wyllie J (1957) Copper poisoning at a cocktail party. Am J Public
    Health, 47: 617.

    Yang FM, Friedrichs WE, Cupples RL, Bonifacio MJ, Sanford JA,
    Horton WA, & Bowman BH (1990) Human ceruloplasmin. Tissue-specific

    expression of transcripts produced by alternative splicing. J Biol
    Chem, 265: 10780-10785.

    Yeowell HN, Marshall MK, Walker LC, Ha V, & Pinnell SR (1994)
    Regulation of lysyl oxidase mRNA in dermal fibroblasts from normal
    donors and patients with inherited connective tissue disorders.
    Arch Biochem Biophys, 308: 299-305.

    Yip R, Reeves JD, Lönnerdal B, Keen CL, & Dallman PR (1985) Does
    iron supplementation compromise zinc nutrition in healthy infants?
    Am J Clin Nutr, 42: 683-687.

    Zabowski D & Zasoski RJ (1987) Cadmium, copper, and zinc adsorption
    by a forest soil in the presence of sludge leachate. Water Air Soil
    Pollut, 36: 103-113.

    Zak B (1958) Simple procedure for the single sample determination
    of serum copper and iron. Clin Chim Acta, 3: 328-334.

    Zhang M & Florence TM (1987) A novel adsorbent for the
    determination of the toxic fraction of copper in natural waters.
    Anal Chim Acta, 197: 137-148.

    Zhou P & Theil DJ (1991) Isolation of a metal-activated
    transcription factor gene from Candida glabrata by complementation
    in Saccharomyces cervisiae. Proc Natl Acad Sci (USA), 88:

    Zia S & Alikhan MA (1989) Copper uptake and regulation in a
    copper-tolerant decapod  Cambarus bartoni (Fabricius) (Decapoda,
    Crustacea). Bull Environ Contam Toxicol, 42: 103-110.

    Zidar BL, Shadduck RK, Zeigler Z, & Winkelstein A (1977)
    Observation of the anemia and neutropenia of human copper
    deficiency. Am J Hematol, 3: 177-185.

    Zitko V & Carson WG (1976) A mechanism of the effects of water
    hardness on the lethality of heavy metals to fish. Chemosphere, 5:

    Zoller WH, Gladney ES, & Duce RA (1974) Atmospheric concentrations
    and sources of trace metals at the South Pole. Science, 183:

    Zou E & Bu S (1994) Acute toxicity of copper, cadmium, and zinc to
    the water flea,  Moina irrasa (Cladocera). Bull Environ Contam
    Toxicol, 52: 742-748.

    Zucker SD & Gollan JL (1996) Wilson's disease and hepatic copper
    toxicosis. In: Zakin D & Boyer T ed. Hepatology: A textbook of
    liver disease. Philadelphia, Pennsylvania, Saunders.

    Zwozdziak J, Zwozdziak A, Matyniak Z, & Lisowski A (1985)
    Atmospheric sulphate formation in the vicinity of a copper smelter.
    Sci Total Environ, 46: 95-106.


    1.  Résumé

    1.1  Identité, propriétés physiques et chimiques

         Le cuivre est un métal ductile et malléable, de couleur brun
    rougeâtre. Il appartient au groupe IB de la Classification
    périodique. Il est généralement présent dans l'environnement au
    degré d'oxydation +2 mais peut également exister au degré 0,
    c'est-ŕ-dire ŕ l'état métallique, ainsi qu'aux degrés +1 et +3.A
    l'état naturel, il se présente sous la forme de sels minéraux et de
    composés organiques trčs divers ou encore sous forme métallique. Le
    métal est ŕ peine soluble dans l'eau et les solutions salines ou
    légčrement acides mais il est dissous par l'acide nitrique et
    l'acide sulfurique ainsi que par les solutions basiques d'hydroxyde
    ou de carbonate d'ammonium.

         Le cuivre présente une forte conductivité électrique et
    thermique et il est résistant ŕ la corrosion.

    1.2  Méthodes d'analyse

         La grande diversité des dérivés du cuivre, qu'ils soient
    minéraux ou organiques, a conduit ŕ la mise au point de tout un
    arsenal de techniques d'échantillonnage, de préparation et
    d'analyse en vue du dosage de cet élément dans les échantillons
    biologiques ou ceux qui proviennent de l'environnement. Il est
    essentiel de mettre en oeuvre des techniques "propres" car la
    contamination des échantillons par du cuivre provenant de l'air, de
    la poussičre, des récipients ou des réactifs est une source
    importante d'erreurs d'analyse.

         Le dosage colorimétrique ou gravimétrique du cuivre est bon
    marché et d'exécution simple. Son intéręt se limite cependant aux
    cas oů il n'est pas essentiel d'avoir une sensibilité extręmement
    élevée. Pour doser de faibles concentrations de cuivre dans des
    matrices diverses, on fait le plus souvent appel ŕ la
    spectrophotométrie d'absorption atomique. En opérant par la męme
    méthode mais avec un four ŕ électrodes de graphite, le gain de
    sensibilité est considérable par rapport ŕ la spectrophotométrie de
    flamme. En fonction du traitement préalable subi par l'échantillon
    ainsi que des techniques de séparation et de concentration
    utilisées, la limite de détection dans l'eau atteint environ 1
    µg/litre par absorption atomique avec électrodes de graphite
    (GF-AAS) et 20 µg/litre par absorption atomique classique; on a pu
    aller jusqu'ŕ 0,05-0,2 µg/g de tissu biologique par GF-AAS. On peut
    parvenir ŕ une sensibilité encore meilleure en ayant recours ŕ des
    techniques d'émission comme par exemple le plasma d'argon ŕ
    couplage inductif associé ŕ la spectroscopie d'émission atomique ou
    ŕ la spectrométrie de masse. Il existe encore d'autres méthodes
    comme la fluorescence X, l'utilisation d'électrodes ŕ membrane
    sélective ou la voltampérométrie avec redissolution anodique ou

    1.3  Sources d'exposition humaine et environnementale

         Parmi les sources d'exposition au cuivre on peut citer la
    poussičre soulevée par le vent, les volcans, les végétaux en
    décomposition, les feux de foręt et les embruns marins. Parmi les
    sources d'émission anthropogéniques, on compte les fours de fusion,
    les fonderies de fonte, les centrales thermiques ainsi que les
    sources de combustion telles que les installations municipales
    d'incinération. Les rejets de cuivre dans le sol proviennent
    essentiellement des résidus et terres de recouvrement des
    exploitations miničres et des boues d'égouts. Les produits
    agricoles ŕ base de cuivre représentent 2% des rejets de cuivre
    dans le sol.

         L'extraction, la fusion et le raffinage des minerais de cuivre
    débouchent sur la fabrication d'un grand nombre de produits
    industriels et commerciaux. Le cuivre est trčs utilisé pour la
    fabrication d'ustensiles de cuisine, dans les réseaux de
    distribution d'eau, ainsi que sous la forme d'engrais, de
    bactéricides, d'algicides et de peintures antisalissures. Il sert
    également ŕ la préparation d'additifs pour l'alimentation des
    bestiaux et de produits favorisant la croissance, et il entre dans
    la composition de substances permettant de lutter contre les
    maladies du bétail et de la volaille. Dans l'industrie, on
    l'utilise comme activateur pour la flottation des minerais
    sulfurés, pour la production d'agents de protection du bois, en
    galvanoplastie, dans la fabrication des colorants azoďques, comme
    mordant pour les colorants des tissus, dans le raffinage du pétrole
    et enfin pour la préparation de composés divers.

    1.4  Transport, distribution et transformation dans l'environnement

         Le cuivre est libéré dans l'atmosphčre en association avec des
    particules de matičre. Il s'élimine par gravité, dépôt ŕ sec,
    lessivage et entraînement par les précipitations. La vitesse
    d'élimination et la distance parcourue depuis la source dépendent
    des caractéristiques de cette derničre, de la granulométrie des
    particules et de la vitesse du vent.

         Le lessivage naturel du sol par les intempéries et les
    décharges provenant de l'industrie et des stations d'épuration sont
    ŕ l'origine du cuivre présent dans l'eau. Il est également possible
    que des composés cupriques soient volontairement introduits dans
    l'eau pour détruire les algues. Le devenir du cuivre dans le milieu
    aquatique est tributaire d'un certain nombre de processus. Il
    s'agit notamment de la formation de complexes, de la sorption par
    des oxydes métalliques hydratés, des argiles et des substances
    organiques ou encore de la bioaccumulation. La connaissance de la
    forme physicochimique sous laquelle se trouve le cuivre (l'espčce
    chimique en cause) apporte plus de renseignements que la
    concentration totale de l'élément. Une grande partie du cuivre
    rejeté dans l'eau se trouve sous forme particulaire et il a
    tendance ŕ se déposer, ŕ précipiter ou ŕ s'adsorber ŕ des matičres
    organiques, ŕ des oxydes de fer et de mangančse hydratés ou aux
    argiles présents dans les sédiments ou dans la couche aqueuse.

    Dans l'environnement aquatique, la concentration du cuivre dépend
    de facteurs tels que la dureté et l'alcalinité de l'eau, sa force
    ionique, son pH et son potentiel redox, la présence de substances
    complexantes, de matičres en suspension et de carbone et enfin, des
    interactions entre l'eau et les sédiments.

         C'est dans le sol que le cuivre est rejeté en majeure partie;
    les sources principales en sont les exploitations miničres,
    l'agriculture et les déchets solides ou les boues provenant des
    stations d'épuration. La plus grande partie du cuivre déposé dans
    le sol est fortement adsorbée et demeure dans les premiers
    centimčtres de la couche supérieure. Le cuivre s'adsorbe aux
    matičres organiques, aux carbonates, aux argiles ainsi qu'aux
    oxydes hydratés de fer et de mangančse. C'est dans les sols sableux
    acides que le lessivage est le plus important. Dans l'environnement
    terrestre, un certain nombre de facteurs importants conditionnent
    le devenir du cuivre. Il s'agit notamment de la nature du sol
    lui-męme, de la présence d'oxydes, du potentiel redox, des surfaces
    porteuses de charges électriques, des matičres organiques et des
    échanges de cations.

         Il peut y avoir bioaccumulation du cuivre présent dans
    l'environnement s'il est biologiquement disponible. La valeur du
    facteur de bioaccumulation varie beaucoup d'un organisme ŕ l'autre,
    mais il a tendance ŕ ętre plus élevé en cas d'exposition ŕ de
    faibles concentrations. Par accumulation, il peut arriver que
    certains animaux (par exemple, les bivalves) ou certaines plantes
    terrestres (comme celles qui poussent sur des sols contaminés) se
    chargent d'une quantité exceptionnellement élevée de cuivre.
    Néanmoins, de nombreux organismes sont capables de réguler leur
    concentration totale de cuivre.

    1.5  Concentrations dans l'environnement et exposition humaine

         La concentration du cuivre dans l'air d'un site est liée ŕ la
    proximité de sources polluantes importantes comme les fours, les
    centrales électriques et les installations d'incinération. Le
    cuivre étant un élément naturel, il est largement disséminé dans
    l'eau. Il faut cependant ętre prudent lorsqu'on cherche ŕ
    interpréter la teneur en cuivre d'un environnement aquatique donné.
    En effet, dans un systčme aquatique, la quantité de cuivre qui est
    mesurée correspond généralement soit au cuivre total, soit au
    cuivre dissous, ce dernier étant plus représentatif de la
    biodisponibilité du métal.

         En milieu rural, la concentration moyenne de fond dans l'air
    va de 5 ŕ 50 ng/m3. Dans les zones non contaminées, la
    concentration est de 0,15 µg/litre dans l'eau de mer et de 1 ŕ 20
    µg/litre dans l'eau douce. Les sédiments constituent un important
    réservoir et milieu récepteur pour le cuivre. La concentration de
    fond dans les sédiments naturels en eau douce va de 16 ŕ 5000 mg/kg
    de poids sec. Dans les sédiments marins, la teneur en cuivre va de
    2 ŕ 740 mg/kg de poids sec. En milieu dépourvu d'oxygčne, le cuivre
    présent dans les sédiments est fortement lié sous la forme de
    sulfure et il n'est donc

    pas biodisponible. Dans des sols non contaminés, on a relevé une
    concentration médiane en cuivre de 30 mg/kg (limites: 2-250 mg/kg).
    Le cuivre s'accumule dans les végétaux, les invertébrés et les
    poissons. La teneur de divers organismes en cuivre est plus élevée
    dans les zones contaminées que dans celles qui ne le sont pas.

         Chez les personnes en bonne santé qui ne sont pas soumis ŕ une
    exposition professionnelle, la principale voie d'exposition est la
    voie buccale. L'apport journalier moyen par l'alimentation est de
    0,9 ŕ 2,2 mg pour un adulte. La plupart des études montrent que
    dans la majorité des cas, cet apport est voisin de l'extrémité
    inférieure de la fourchette. Les variations que l'on peut constater
    traduisent la diversité des habitudes alimentaires et des pratiques
    agricoles ou culinaires de par le monde. Parfois, l'eau de boisson
    peut contribuer de façon substantielle ŕ l'apport journalier total
    de cuivre, en particulier dans les habitations dont la tuyauterie
    est au contact d'une eau corrosive. Dans les habitations dont la
    tuyauterie n'est pas en cuivre ou qui ne sont pas alimentées par
    une eau corrosive, l'apport de cuivre par l'eau de boisson ne
    dépasse que rarement 0,1 mg/jour, alors que si l'eau distribuée est
    corrosive, cet apport peut excéder plusieurs mg par jour. En
    général, l'apport journalier total par la voie buccale
    (alimentation et eau de boisson) se situe entre 1 et 2 mg par jour,
    avec des pointes occasionnelles ŕ plus de 5 mg/jour. L'apport de
    cuivre par les autres voies (respiratoire ou percutanée) est
    négligeable par rapport ŕ la voie buccale. L'inhalation de
    poussičres et de fumée ajoute quelque 0,3-2,0 mg de Cu par jour.
    Les femmes qui portent des DIU en cuivre ne sont exposées de ce
    fait qu'ŕ un apport supplémentaire de 80 µg au maximum.

    1.6  Cinétique et métabolisme chez les animaux de
         laboratoire et l'Homme

         L'homéostase du cuivre est liée ŕ la dualité du cuivre,
    élément ŕ la fois essentiel et toxique. Son caractčre essentiel
    tient au fait qu'il intervient dans un grand nombre de protéines,
    tant comme élément structural que comme catalyseur. Les męmes
    processus cellulaires de fixation et d'incorporation dans les
    protéines ainsi que les sorties de cuivre se retrouvent chez tous
    les mammifčres et sont modulés par le métal lui-męme.

         Le cuivre est principalement absorbé dans les voies
    digestives. Le taux de résorption du cuivre alimentaire est de 20
    ŕ 60%, le reste étant excrété dans les matičres fécales. Aprčs ętre
    passé ŕ travers la membrane basolatérale, le cuivre se fixe ŕ
    l'albumine qui le transporte jusqu'au foie. Cet organe joue un rôle
    déterminant dans l'homéostase du cuivre. Le cuivre se répartit
    ensuite en deux fractions, l'une qui est excrétée par la bile, et
    l'autre qui est incorporée aux protéines intra- et
    extracellulaires. La bile constitue la principale voie d'excrétion.
    Le transport du cuivre vers les tissus périphériques est assuré par
    l'albumine plasmatique, la céruléoplasmine et des complexes de
    faible masse moléculaire.

         Parmi les méthodes utilisées pour étudier l'homéostase du
    cuivre chez les mammifčres figurent les analyses de rations
    alimentaires et les études de bilan. Il est essentiel de recourir
    ŕ des méthodes isotopiques et ŕ des analyses biochimiques
    standardisées pour bien établir l'existence de carences ou d'excčs
    de cuivre.

         La toxicité biochimique du cuivre, lorsque la régulation
    homéostatique devient inopérante, résulte de l'effet que cet
    élément exerce sur la structure et la fonction des biomolécules
    comme l'ADN, les membranes et les protéines, soit directement, soit
    par l'intermédiaire de mécanismes faisant intervenir des radicaux

    1.7  Effets sur les animaux de laboratoire et les systčmes
         d'épreuve in vitro

         La toxicité d'une dose unique de cuivre varie largement selon
    l'espčce en cause (DL50 comprise entre 15 et 1664 mg Cu/kg de poids
    corporel). Parmi les sels de cuivre, ceux qui présentent une bonne
    solubilité (sulfate de Cu(II), chlorure de Cu (II)), sont
    généralement plus toxiques que par exemple l'hydroxyde de Cu(II) ou
    l'oxyde de Cu (II), moins solubles. Des symptômes tels
    qu'hémorragie gastrique, tachycardie, hypotension,crise
    hémolytique, convulsions et paralysie précčdent l'issue fatale.
    Pour la DL50 en exposition percutanée, on a fait état de valeurs
    > 1124 et >2058 mg Cu/kg p.c. respectivement chez le rat et chez
    le lapin. La CL50 pour une exposition par inhalation (durée non
    précisée) a été trouvée > 1303 mg Cu/kg p.c. chez des lapins et on
    a constaté une détérioration de la fonction respiratoire chez des
    cobayes exposés ŕ une dose de 1,3 mg Cu/m3 pendant 1 h.

         Des rats qui avaient reçu quotidiennement pendant 15 jours 305
    mg Cu/kg dans leur nourriture, sous la forme de sulfate de Cu (II),
    on présenté des modifications de leurs paramčtres biochimiques
    sanguins accompagnées d'autres anomalies hématologiques (anémie en
    particulier) et l'on a également observé des effets nocifs au
    niveau du foie, des reins et des poumons. Ces effets étaient de
    męme nature que ceux observés chez d'autres espčces avec d'autres
    dérivés du cuivre. La dose sans effet observable (NOEL) a été
    évaluée dans cette étude ŕ 23 mg Cu/kg p.c. par jour. On a
    cependant relevé que les moutons étaient particuličrement sensibles
    et des doses de Cu (II) réitérées correspondant ŕ 1,5-7,5 mg Cu/kg
    p.c. administrées chaque jour sous la forme de sulfate ou d'acétate
    ont entraîné des lésions hépatiques progressives, une crise
    hémolytique et la mort.

         Exposés pendant une longue période, des rats et des souris
    n'ont pas présenté de signes manifestes de toxicité autres qu'une
    réduction de croissance liée ŕ la dose, aprčs ingestion de doses
    quotidienne équivalant ŕ 138 mg Cu/kg p.c. (rats) et 1000 mg Cu/kg
    p.c. (souris). La dose sans effet nocif observable (NOAEL) a été
    évaluée ŕ 17 mg Cu/kg p.c. par jour pour les rats et ŕ 44 et 126 mg

    Cu/kg p.c. par jour, respectivement pour les souris mâles et les
    souris femelles. Les effets observés consistaient notamment en une
    inflammation du foie et en une dégénérescence de l'épithélium
    tubulaire rénal.

         Les études consacrées aux effets toxiques sur la reproduction
    et le développement sont limitées. On a constaté une certaine
    dégénérescence testiculaire et une réduction du poids du corps et
    des organes chez des rats nouveau-nés ŕ des doses dépassant 30 mg
    Cu/kg p.c. par jour et administrées sur de longues périodes. On a
    également observé des malformations foetales et autres effets
    foetotoxiques ŕ dose élevée (> 80 mg Cu/kg p.c. par jour).

         Le sulfate de Cu (II) ne s'est pas révélé mutagčne dans les
    épreuves sur bactéries. Toutefois, on a observé une synthčse non
    programmée de l'ADN qui augmentait en fonction de la dose dans des
    hépatocytes de rat. Lors du test des micronoyaux sur la souris, on
    a observé- dans une étude tout du moins- une augmentation
    significative des cassures chromosomiques ŕ la dose I.V. la plus
    élevée (1,7 mg Cu/kg p.c.), mais aucun effet n'a été constaté lors
    d'une autre étude ŕ des doses allant jusqu'ŕ 5,1 mg Cu/kg p.c.

         Les études de neurotoxicité n'ont révélé aucun effet sur le
    comportement mais des modifications neurochimiques ont été
    signalées aprčs administration par voie buccale de doses
    correspondant ŕ 20-40 mg Cu/kg p.c. par jour. D'aprčs un nombre
    limité d'études d'immunotoxicité, il y a eu une détérioration de la
    fonction immunitaire humorale et ŕ médiation cellulaire aprčs
    ingestion, avec l'eau de boisson, de doses équivalant ŕ environ 10
    mg Cu /kg p.c. par jour.

    1.8  Effets sur l'Homme

         Le cuivre est un élément essentiel et les effets indésirables
    qui lui sont imputables peuvent provenir d'une carence comme d'un
    excčs. La carence en cuivre est ŕ l'origine d'anémies, de
    neutropénies et d'anomalies osseuses mais il est rare qu'elle se
    manifeste cliniquement chez l'Homme. On peut faire un bilan
    cuprique pour essayer de prévoir certains effets cliniques ou
    encore procéder ŕ un dosage du cuivre et de la céruléoplasmine
    sériques pour évaluer une carence modérée ŕ forte, dosage qui
    n'offre toutefois pas autant de sensibilité dans le cas d'une
    carence limite.

         Si l'on excepte les cas d'intoxication aiguë, on n'observe
    gučre d'effets dans les populations normales. L'absorption d'une
    dose unique d'un dérivé du cuivre soit accidentellement, soit dans
    un but de suicide, donne lieu aux symptômes suivants: goűt
    métallique, douleurs épigastriques, céphalées, nausées,
    étourdissements, vomissements et diarrhée, tachycardie, difficultés
    respiratoires, anémie hémolytique, hématurie, hémorragie
    gastrointestinale massive, insuffisance hépatique et rénale

    aboutissant finalement ŕ la mort. On a observé des effets
    gastrointestinaux aprčs ingestion unique ou répétée d'eau ŕ forte
    teneur en cuivre et on a fait état d'insuffisance hépatique
    consécutive ŕ l'absorption de cuivre pendant une longue période. Il
    ne semble pas qu'une exposition cutanée puisse entraîner une
    intoxication générale, mais le cuivre peut provoquer des réactions
    allergiques chez certains individus. On a mentionné des cas de fičvre
    des fondeurs consécutifs ŕ l'inhalation, sur le lieu de travail,
    d'air fortement chargé en cuivre mais, bien que d'autres effets
    respiratoires aient été attribués ŕ l'inhalation de mélanges
    contenant du cuivre (par ex. bouillie bordelaise, travail ŕ la
    mine, travail auprčs des fours), la responsabilité du cuivre n'a
    pas été démontrée. Des ouvriers apparemment exposés ŕ des
    concentrations atmosphériques correspondant ŕ l'absorption d'une
    dose de 200 mg Cu/jour, ont présenté des signes évocateurs d'une
    intoxication cuprique (par ex. élévation du Cu sérique,
    hépatomégalie). Les données dont on dispose au sujet de la
    cancérogénicité et des effets toxiques du cuivre sur la
    reproduction sont insuffisantes pour permettre une évaluation du

         On a décrit un certain nombre de groupes que des troubles de
    l'homéostase cuprique semblent rendre plus sensibles que le reste
    de la population ŕ une carence ou ŕ un excčs de cuivre. Certains
    troubles ont une origine génétique précise. Il s'agit notamment de
    la maladie de Menkes, une carence cuprique généralement mortelle,
    de la maladie de Wilson (dégénérescence hépatolenticulaire), une
    pathologie qui conduit ŕ une accumulation progressive de cuivre et
    de l'acéruléoplasminémie héréditaire, qui s'accompagne des
    manifestations cliniques d'une surcharge martiale. La cirrhose
    infantile indienne et la cuprotoxicose idiopathique sont des
    affections liées ŕ un excčs de cuivre et peut-ętre associées ŕ une
    sensibilité au cuivre d'origine génétique, encore que cette
    hypothčse n'ait pas été indiscutablement prouvée. Il s'agit lŕ
    d'affections mortelles de la petite enfance dans lesquelles le
    cuivre s'accumule dans le foie. On a pu mettre ces maladies en
    parallčle avec une forte consommation de cuivre, tout du moins dans
    certains cas.

         Parmi les autres groupes potentiellement sensibles ŕ l'excčs
    de cuivre on peut citer les personnes en hémodialyse et les malades
    atteints d'une affection hépatique chronique. Parmi les groupes
    exposés au risque de carence en cuivre figurent les nourrissons
    (notamment les enfants de faible poids de naissance et les
    prématurés, les enfants qui se remettent d'une malnutrition et les
    enfants nourris exclusivement au lait de vache), les sujets
    souffrant d'un syndrome de malabsorption (maladie coeliaque, sprue,
    mucoviscidose) et les malades nourris exclusivement par voie
    parentérale. On a également incriminé une carence en cuivre dans la
    pathogénčse de certaines maladies cardiovasculaires.

    1.9  Effets sur les autres ętres vivants au laboratoire et dans
         leur milieu naturel

         Il faut mettre en balance les effets indésirables du cuivre et
    son caractčre essentiel. Cet élément est en effet essentiel pour
    tout les ętres vivants et il faut veiller ŕ ce que ces organismes
    reçoivent la quantité de cuivre qui correspond ŕ leur besoins. Il
    y a au moins 12 protéines importantes dont le cuivre fait partie
    intégrante de la structure. Il joue un rôle essentiel dans
    l'utilisation du fer pour la formation de l'hémoglobine et la
    plupart des crustacés et des mollusques possčdent une protéine,
    l'hémocyanine, qui contient du cuivre et représente leur principal
    transporteur d'oxygčne. Chez les végétaux, le cuivre entre dans la
    composition de plusieurs enzymes qui interviennent dans le
    métabolisme des sucres, de l'azote et de la paroi cellulaire.

         Dans l'évaluation du risque imputable au cuivre, la
    biodisponibilité de cet élément joue un rôle déterminant.
    L'adsorption du cuivre ŕ des particules de matičre ou sa
    complexation par des substances organiques peuvent en limiter
    fortement l'accumulation et par voie de conséquence, les effets.
    Les autres cations ainsi que le pH peuvent également avoir une
    influence importante sur la biodisponibilité.

         On a montré que le cuivre exerçait des effets nocifs sur la
    reproduction, les paramčtres biochimiques, les fonctions
    physiologiques et le comportement chez divers organismes
    aquatiques. Ainsi, des effets toxiques se manifestent chez ces
    organismes ŕ des concentrations ne dépassant pas 1-2 µg/litre. Il
    est vrai cependant qu'il faut prendre en considération les
    importantes variations de sensibilité et de biodisponibilité
    interspécifiques lorsque l'on se propose d'interpréter et
    d'appliquer ces données.

         Dans des communautés naturelles de phytoplancton, on a
    constaté que la chlorophylle  a et la fixation de l'azote étaient
    sensiblement réduites ŕ des concentrations de cuivre > 20
    µg/litre et que la fixation du carbone était aussi notablement
    réduite ŕ une concentration > 10 µg/litre. Pour les algues, on
    a obtenue une CE50 basée sur l'inhibition de la croissance qui
    allait de 47 ŕ 120 µg Cu/litre.

         Chez les invertébrés dulçaquicoles, la valeur de la CL ou de
    la CE50 ŕ 48 h varie de 5 µg Cu/litre pour une espčce de daphnie
    ŕ 5300 µg Cu/litre pour un ostracode. Dans le cas des invertébrés
    marins, on a obtenu une CL50 ŕ 96 h de 29 µg Cu/litre pour une
    coquille saint-jacques et de 9400 µg Cu/litre pour les crabes du
    genre  Uca.  La toxicité aiguë du cuivre pour les poissons d'eau
    douce et les poissons de mer est trčs variable. Pour les poissons
    d'eau douce, la valeur de la CL50 ŕ 96 h va de 3 µg Cu/litre (ombre
    arctique  Thymallus signifer) ŕ 7340 µg Cu/litre

     (Lepomis machrochirus).  Dans le cas des espčces marines, la Cl50 ŕ
    96 h va de 60 µg Cu/litre pour un saumon,  Onchorhynchus tschawtscha, ŕ
    1400 µg Cu/litre pour le mulet.

         Le cuivre joue le rôle d'oligoélément pour les plantes mais un
    sol trop riche en cuivre peut se révéler extręmement toxique. En
    général, les signes d'une toxicité d'origine métallique consistent
    dans l'apparition de petites feuilles chlorotiques qui tombent
    prématurément. Il y a rabougrissement de la plante dont les racines
    démarrent mal et ne forment pas de départs latéraux. La réduction
    du développement des racines peut conduire ŕ une moindre fixation
    d'eau et de nutriments par la plante avec perturbation du
    métabolisme et de la croissance. Au niveau cellulaire, le cuivre
    inhibe un grand nombre d'enzymes et perturbe plusieurs processus
    biochimiques (notamment la photosynthčse, la synthčse des pigments
    et l'intégrité des membranes) ou physiologiques (notamment le
    métabolisme des acides gras et des protéines avec également un effet
    inhibiteur sur la respiration et les processus de fixation de

         Des effets toxiques ont également été observés au laboratoire
    chez des lombrics placés dans une terre riche en cuivre; le
    paramčtre le plus sensible qui ait été mesuré était la formation de
    cocons et des effets nocifs ont été notés ŕ des concentrations de
    50-60 mg Cu/kg.

         Certains effets délétčres observés chez des microorganismes
    terricoles ont pu ętre mis en corrélation avec la présence de
    fortes concentrations de cuivre dues ŕ l'épandage d'engrais ŕ base
    de cuivre ou ŕ l'implantation de fonderies de zinc dans le
    voisinage. Dans des plantations d'agrumes traitées par des
    fongicides ŕ base de cuivre, on a constaté une chlorose foliaire en
    corrélation significative avec la teneur du sol en cuivre.

         On a montré que dans le milieu naturel, le phytoplancton, les
    invertébrés aquatiques et terrestres, de męme que les poissons et
    les plantes terrestres, faisaient preuve d'une certaine tolérance
    au cuivre. Parmi les mécanismes invoqués pour expliquer cette
    tolérance chez les plantes, on peut citer la fixation du métal ŕ
    certains composants de la paroi cellulaire, la présence d'enzymes
    métallo-tolérantes, la formation de complexes avec des acides
    organiques suivie d'une élimination dans la vacuole et enfin, la
    combinaison avec des protéines spécialisées riches en thiols ou
    avec des phytochélatines.

    2. Conclusions

    2.1  Santé humaine

         La limite inférieure de l'intervalle de dose acceptable par
    ingestion (AROI) est égale ŕ 20 µg Cu/kg de poids corporel par
    jour. Pour obtenir cette valeur, on est parti de l'apport minimal
    requis pour un adulte en tenant compte des variations du taux
    d'absorption, de rétention et d'accumulation du cuivre (OMS, 1996).
    Pour les enfants en bas âge, ce chiffre est égal ŕ 50 µg Cu/kg p.c.
    par jour.

         La limite supérieure de l'intervalle précité n'est pas connue
    avec certitude chez l'adulte mais il est trčs probable qu'elle est
    de l'ordre de quelques mg par jour et pas davantage (par  quelques
    on entend plus de 2 ŕ 3 mg/jour). Cette évaluation ne repose que
    sur l'étude des effets gastrointestinaux d'une consommation d'eau
    contaminée par du cuivre. Il n'a pas été possible de donner une
    limite supérieure plus spécifique pour un groupe quelconque de
    population. Nous ne disposons que de données limitées sur la
    quantité de cuivre d'origine alimentaire qui serait susceptible de
    nuire ŕ la santé.

         On a estimé que les données toxicologiques obtenues sur
    l'animal n'étaient d'aucun secours pour l'établissement de la
    limite supérieure de l'intervalle de dose acceptable par ingestion
    chez l'Homme, du fait de l'incertitude quant ŕ l'applicabilité ŕ
    l'Homme des modčles utilisés. En outre, les méthodes auxquelles on
    a habituellement recours pour évaluer l'innocuité d'une substance,
    méthodes qui impliquent l'application d'un coefficient de sécurité
    aux données obtenues sur l'animal, ne sauraient convenir dčs lors
    que l'on doit prendre en considération des caractéristiques
    particuličres qui sont celles d'éléments essentiels comme le

         A la lumičre des données dont on dispose sur l'exposition
    humaine au cuivre dans l'ensemble du monde, mais plus spécialement
    en Europe et dans les Amériques, il semble que les dangers d'une
    carence en cuivre sont plus grands que ceux d'un excčs de cet

    2.2  Effets sur l'environnement

         Pour assurer la protection des organismes aquatiques dans les
    eaux oů la biodisponibilité est forte, il faut que le cuivre total
    en solution reste en dessous de 10 µg/litre environ, la valeur la
    plus appropriée étant fonction des espčces présentes et des
    conditions d'exposition du site en cause; elle devra ętre fixée
    aprčs étude approfondie de tous les paramčtres ŕ prendre en

         En de nombreux endroits, l'existence de facteurs
    physico-chimiques limitant la biodisponibilité permettra de relever
    les limites de concentration. La réglementation devra prendre en
    considération les espčces chimiques en présence si les auteurs de
    rejets sont ŕ męme de prouver que la biodisponibilité du cuivre
    dans les eaux réceptrices peut ętre mesurée avec une fiabilité

         Lors des prélčvements et des analyses effectués dans
    l'environnement en vue de la recherche et du dosage du cuivre, il
    est essentiel d'utiliser des techniques "propres".

         Etant donné que le cuivre est un élément essentiel, il faut,
    lorsqu'on cherche ŕ éviter l'absorption de quantités toxiques de
    cuivre, se garder d'introduire des coefficients de sécurité qui
    aboutissent finalement ŕ des concentrations recommandées
    inférieures aux teneurs naturelles.


    1.  Resumen

    1.1  Identidad, propiedades físicas y químicas

         El cobre es un metal de color pardo rojizo, dúctil y maleable.
    Pertenece al grupo IB de la Tabla periódica. Se suele encontrar en el
    medio ambiente formando compuestos con valencia 2, pero pueden existir
    estados metálicos de valencia +1 y +3. Está presente en la naturaleza
    en una gran variedad de sales minerales y compuestos orgánicos, y en
    forma metálica. El metal es muy poco soluble en soluciones acuosas,
    salinas o ligeramente ácidas, pero se puede disolver en los ácidos
    nítrico y sulfúrico, así como en soluciones básicas de hidróxido o
    carbonato de amonio.

         El cobre posee una elevada conductividad eléctrica y térmica y es
    resistente a la corrosión.

    1.2  Métodos analíticos

         La gran variedad de especies de cobre, inorgánicas y orgánicas,
    ha dado lugar a una serie de técnicas de muestreo, preparación y
    métodos analíticos para cuantificar el elemento en muestras del medio
    ambiente y biológicas. La contaminación de las muestras por cobre
    procedente del aire, el polvo, los recipientes o los reactivos durante
    la preparación y el muestreo es una fuente importante de errores
    analíticos, por lo que es fundamental el uso de técnicas "limpias".

         Los métodos colorimétricos y gravimétricos para la medición del
    cobre son fáciles de usar y económicos; sin embargo, su utilidad se
    limita a las situaciones en las cuales no es indispensable una
    sensibilidad máxima. Para la medición de concentraciones bajas de
    cobre en diversas matrices, los métodos más utilizados son los de
    espectrofotometría de absorción atómica. La sensibilidad aumenta
    enormemente con la utilización de la espectrofotometría de absorción
    atómica en electrohorno de grafito, en lugar de la de llama. En
    función de los procedimientos de tratamiento previo, separación y
    concentración de la muestra, se han notificado límites de detección de
    alrededor 1 µg/litro en agua mediante espectrofotometría en
    electrohorno de grafito y 20 µg/litro por la de llama y niveles de
    0,05-0,2 µg/g de tejido con la primera. Se puede conseguir una
    sensibilidad mayor mediante el uso técnicas de emisión, como las
    técnicas de plasma de argon con acoplamiento inductivo de alta
    temperatura, seguidas de espectroscopia de emisión atómica o
    espectrometría de masas. Existen otras metodologías más sensibles y
    especializadas, como la fluorescencia por rayos X, los métodos de
    electrodos selectivos de iones y potenciométricos y la voltametría de
    descascarillado anódico y de descascarillado catódico.

    1.3  Fuentes de exposición humana y ambiental

         Las fuentes naturales de exposición al cobre son el polvo
    arrastrado por el viento, los volcanes, la vegetación en
    descomposición, los incendios forestales y la dispersión marina. Entre
    las emisiones antropogénicas cabe mencionar los hornos de fusión, las
    fundiciones de hierro, las centrales eléctricas y fuentes de
    combustión como los incineradores municipales. El desplazamiento
    principal del cobre a la tierra se produce a partir de las escorias y
    el manto de las minas de cobre y los fangos cloacales. El uso agrícola
    de productos de cobre representa el 2% de la liberación de cobre al

         Los minerales de cobre se extraen, funden y refinan para la
    fabricación de numerosos productos industriales y comerciales. Se
    utiliza ampliamente en utensilios de cocina y sistemas de
    abastecimiento de agua, así como en fertilizantes, bactericidas,
    fungicidas, alguicidas y pinturas antiincrustantes. Se emplea asimismo
    en aditivos de piensos y estimulantes del crecimiento, así como en la
    lucha contra determinadas enfermedades del ganado vacuno y de las
    aves. El cobre se utiliza en la industria como activador en la
    flotación por espuma de los minerales sulfurosos, la producción de
    conservantes de la madera, la galvanoplastia, la fabricación de
    colorantes nitrogenados, como mordiente para tintes de tejidos, en el
    refinado del petróleo y en la fabricación de los compuestos de cobre.

    1.4  Transporte, distribución y transformación en el medio ambiente

         El cobre se libera en la atmósfera asociado con materia
    particulada. Se elimina mediante sedimentación gravitatoria,
    deposición seca, arrastre y lavado por la lluvia. La velocidad de
    eliminación y la distancia recorrida desde la fuente dependen de las
    características de ésta, del tamańo de las partículas y de la
    velocidad del viento.

         El cobre se libera en el agua como consecuencia de la exposición
    natural a la intemperie del suelo y los vertidos de industrias y
    plantas de depuración de aguas residuales. Se pueden aplicar
    compuestos de cobre de manera intencionada al agua para destruir las
    algas. Hay varios procesos que influyen en el destino del cobre en el
    medio acuático. Son la formación de complejos, la sorción para formar
    óxidos metálicos hidratados, arcillas y materiales orgánicos y la
    bioacumulación. Los datos sobre las formas fisicoquímicas del cobre
    (especiación) son más informativos que las concentraciones totales de
    cobre. Gran parte del cobre vertido en el agua está en forma
    particulada y tiende a sedimentarse, precipitar o adsorberse en
    materia orgánica, hierro hidratado, óxidos de manganeso y arcilla en
    el sedimento o la columna de agua. En el medio acuático, la
    concentración de cobre y su biodisponibilidad dependen de factores
    como la dureza y la alcalinidad del agua, la fuerza iónica, el pH y el
    potencial redox, así como de la formación de ligandos complejos, la
    materia particulada y el carbón suspendidos y la interacción entre los
    sedimentos y el agua.

         La liberación más importante de cobre se produce hacia la tierra;
    sus fuentes principales son las operaciones de extracción, la
    agricultura, los residuos sólidos y los fangos procedentes de las
    actividades de tratamiento. La mayor parte del cobre depositado en el
    suelo se adsorbe fuertemente y se mantiene en los centímetros más
    superficiales. El cobre se adsorbe en la materia orgánica, los
    minerales carbonados, los minerales arcillosos, el hierro hidratado y
    los óxidos de manganeso. La mayor parte de la lixiviación se produce a
    partir de suelos arenosos ácidos. En el medio ambiente terrestre hay
    varios factores importantes que influyen en el destino del cobre en el
    suelo. Son las características del propio suelo, el pH, la presencia
    de óxidos, el potencial redox, las superficies cargadas, la materia
    orgánica y el intercambio de iones.

         Se produce bioacumulación de cobre procedente del medio ambiente
    si el cobre está biológicamente disponible. Los factores de
    acumulación varían enormemente entre los distintos organismos, pero
    tienden a ser más elevados a concentraciones de exposición más bajas.
    La acumulación puede dar lugar a concentraciones corporales
    excepcionalmente altas en algunos animales (como por ejemplo los
    bivalvos) y en plantas terrestres (como las que crecen en suelos
    contaminados). Sin embargo, muchos organismos son capaces de regular
    su concentración interna de cobre.

    1.5  Niveles medioambientales y exposición humana

         La concentración de cobre en el aire depende de la proximidad del
    lugar a fuentes importantes, como hornos de fusión, centrales
    eléctricas e incineradores. El cobre esta ampliamente distribuido en
    el agua, porque se encuentra en ella de forma natural. Sin embargo,
    sus concentraciones en el medio acuático se deben interpretar con
    cautela. En sistemas acuáticos, los niveles ambientales de cobre se
    suelen medir como concentración total o disuelta, siendo esta última
    más representativa de la biodisponibilidad del metal.

         El promedio de las concentraciones básicas de cobre en el aire de
    las zonas rurales oscila entre 5 y 50 ng/m3. En las zonas no
    contaminadas, en el agua marina se encuentran concentraciones de 0,15
    µg/litro y en el agua dulce de 1-20 µg/litro. Los sedimentos son un
    depósito y una reserva importantes de este metal. Las concentraciones
    básicas de cobre en sedimentos de agua dulce naturales oscilan entre
    16 y 5000 mg/kg (peso seco). Las concentraciones en sedimentos marinos
    varían entre 2 y 740 mg/kg (peso seco). En sedimentos anóxicos, el
    cobre se une fuertemente mediante sulfuros, por lo que no está
    biodisponible. Se notificaron concentraciones medias de cobre de 30
    mg/kg en suelos no contaminados (intervalo de 2-250 mg/kg). Este metal
    se acumula en las plantas, los invertebrados y los peces. En
    organismos de lugares contaminados por cobre se han notificado
    concentraciones más elevadas que en los de zonas no contaminadas.

         Para las personas sanas no expuestas al cobre en el puesto de
    trabajo la vía principal de exposición es la oral. La ingesta diaria
    media con los alimentos oscila en las personas adultas entre 0,9 y 2,2
    mg. En la mayoría de los estudios se ha encontrado que los valores de
    la ingesta se sitúan en el extremo inferior del intervalo. La
    variación refleja los diferentes hábitos alimentarios, así como las
    distintas prácticas agrícolas y de preparación de alimentos utilizadas
    en todo el mundo. En algunos casos, el agua potable puede contribuir a
    un aumento importante del valor total de la ingesta diaria de cobre,
    sobre todo en hogares con aguas corrosivas y tuberías de cobre. En los
    hogares sin tuberías de cobre o con aguas no corrosivas, la ingesta de
    cobre a partir del agua potable raramente supera el valor de 0,1
    mg/día, aunque puede haber valores superiores a unos pocos mg al día a
    causa de la distribución de agua corrosiva a través de tuberías de
    cobre. En general, la ingesta diaria total de cobre por vía oral
    (alimentos más agua potable) oscila entre 1 y 2 mg/día, aunque
    ocasionalmente puede alcanzar un valor superior a 5 mg/día. Todas las
    demás ingestas de cobre (inhalación y cutánea) son insignificantes en
    comparación con la vía oral. La inhalación ańade 0,3-2,0 µg/día
    procedente del polvo y el humo. Las mujeres que utilizan DIU están
    expuestas sólo a 80 µg o menos de cobre al día a partir de esta

    1.6  Cinética y metabolismo en animales de laboratorio y en
         el ser humano

         La homeóstasis del cobre se debe al doble carácter del elemento,
    esencial y tóxico. El carácter esencial se deriva de su incorporación
    específica a un gran número de proteínas con fines catalíticos y
    estructurales. En los mamíferos se conservan las rutas celulares de
    absorción, incorporación a las proteínas y exportación del cobre,
    reguladas por el propio metal.

         El cobre se absorbe fundamentalmente a través del tracto
    gastrointestinal. Se absorbe del 20% al 60% del cobre procedente de
    los alimentos, mientras que el resto se excreta a través de las heces.
    Una vez que el metal ha atravesado la membrana basolateral, es
    transportado hasta el hígado unido a la seroalbúmina. El hígado es el
    órgano fundamental para la homeóstasis del cobre. El metal se reparte
    entre la excreción a través de la bilis y la incorporación a proteínas
    intracelulares y extracelulares. La vía de eliminación más importante
    es la biliar. El transporte de cobre hasta los tejidos periféricos se
    realiza a través del plasma, unido a seroalbúmina, ceruloplasmina o
    complejos de bajo peso molecular.

         Los métodos utilizados para estudiar la homeóstasis del cobre en
    los mamíferos son el análisis de los alimentos y los estudios del
    balance. Para conocer la deficiencia y el exceso de cobre son
    imprescindibles los isótopos y los análisis bioquímicos normalizados
    de estos procesos.

         La toxicidad bioquímica del cobre, cuando supera el control
    homeostático, se debe a sus efectos en la estructura y la función de
    biomoléculas como el ADN, las membranas y las proteínas, directamente
    o mediante mecanismos con intervención de radicales de oxígeno.

    1.7  Efectos en los animales de laboratorio y en los sistemas
         de prueba in vitro

         La toxicidad de una dosis oral única de cobre varía enormemente
    entre las especies (DL50 de 15-1664 mg de Cu/kg de peso corporal).
    Las sales más solubles de cobre (sulfato de cobre (II) y cloruro de
    cobre (II)) son generalmente más tóxicas que las menos solubles
    (hidróxido de cobre (II), óxido de cobre (II)). La muerte se produce
    tras la aparición de hemorragia gástrica, taquicardia, hipotensión,
    crisis hemolítica, convulsiones y parálisis. Se notificaron valores de
    la DL50 para la exposición cutánea > 1124 y > 2058 mg de Cu/kg de
    peso corporal en ratas y conejos, respectivamente. La DL50 por
    inhalación (duración de la exposición no especificada) fue >1303 mg
    de Cu/kg de peso corporal en conejos, y en los cobayas expuestos a
    concentraciones de 1,3 mg de Cu/m3 durante una hora se observó
    insuficiencia respiratoria.

         Las ratas que recibieron 305 mg de Cu/kg al día por vía oral con
    los alimentos en forma de sulfato de cobre (II) durante 15 días
    mostraron alteraciones de la bioquímica sanguínea y los datos
    hematológicos (particularmente anemia) y efectos secundarios en el
    hígado, el rińón y los pulmones. Los efectos fueron cualitativamente
    semejantes a los de otros compuestos de cobre y en otras especies. La
    concentración sin efectos observados (NOEL) en este estudio fue de 23
    mg de Cu/kg de peso corporal al día. Sin embargo, las ovejas fueron
    particularmente sensibles, y dosis repetidas de 1,5-7,5 mg de Cu/kg de
    peso corporal al día en forma de sulfato de cobre (II) o acetato de
    cobre (II) produjeron lesiones hepáticas progresivas, crisis
    hemolítica y por último la muerte.

         La exposición prolongada de ratas y ratones no puso de manifiesto
    signos evidentes de toxicidad, salvo una reducción del crecimiento
    relacionada con la dosis tras la ingestión de 138 mg de Cu/kg de peso
    corporal al día (ratas) y 1000 mg de Cu/kg de peso corporal al día
    (ratones). La concentración sin efectos adversos observados (NOAEL)
    fue de 17 mg de Cu/kg de peso corporal al día en ratas y de 44 y 126
    mg de Cu/kg de peso corporal al día en ratones machos y hembras,
    respectivamente. Los efectos fueron la inflamación del hígado y la
    degeneración del epitelio tubular del rińón

         Los estudios de la toxicidad reproductiva y en el desarrollo
    fueron limitados. Se observó cierta degeneración testicular y una
    reducción del peso del cuerpo y de los órganos al nacer en ratas
    tratadas con dosis superiores a 30 mg de Cu/kg de peso corporal al día
    durante períodos prolongados de tiempo y efectos fetotóxicos y
    malformaciones con concentraciones altas (>80 mg de Cu/kg de peso
    corporal al día).

         El sulfato de cobre (II) no fue mutagénico en valoraciones
    realizadas con bacterias. Sin embargo, se observó un aumento
    relacionado con la dosis de la síntesis de ADN no programado en
    hepatocitos de rata. En el ensayo del micronúcleo, un estudio puso de
    manifiesto un aumento significativo de las fracturas cromosómicas con
    la dosis intravenosa más alta (1,7 mg de Cu/kg de peso corporal al
    día), pero en otro estudio realizado con dosis intravenosas de hasta
    5,1 mg de Cu/kg de peso corporal al día no se observó ningún efecto.

         Los estudios de neurotoxicidad no han puesto de manifiesto
    efectos en el comportamiento, pero se han notificado cambios
    neuroquímicos tras la administración oral de 20-40 mg de Cu/kg de peso
    corporal al día. En un número limitado de estudios de inmunotoxicidad
    se ha observado un trastorno de la función inmunitaria humoral y
    mediada por células en ratones después de la ingesta oral con agua de
    bebida de unos 10 mg de Cu/kg de peso corporal al día.

    1.8  Efectos en el ser humano

         El cobre es un elemento esencial y hay efectos perjudiciales para
    la salud relacionados tanto con su deficiencia como con su exceso. La
    deficiencia de cobre está asociada con anemia, neutropenia y anomalías
    óseas, pero la deficiencia clínicamente manifiesta es relativamente
    poco frecuente en el ser humano. Se pueden utilizar los datos del
    balance para prever los efectos clínicos, mientras que las
    concentraciones de cobre en el suero y en la ceruloplasmina son
    medidas útiles de la deficiencia entre moderada y grave, pero son
    medidas menos sensibles de la deficiencia marginal.

         Excepto en el caso de accidentes agudos ocasionales de
    intoxicación por cobre, se han observado pocos efectos en la población
    normal. Se han notificado efectos de una exposición única tras la
    ingestión oral con fines suicidas o accidental consistentes en sabor
    metálico, dolor epigástrico, dolor de cabeza, náuseas,
    desvanecimiento, vómitos y diarrea, taquicardia, dificultad
    respiratoria, anemia hemolítica, hematuria, hemorragia
    gastrointestinal masiva, insuficiencia hepática y renal y la muerte.
    También se han presentado efectos gastrointestinales por una ingestión
    única y repetida de agua de bebida con altas concentraciones de cobre
    y se ha notificado insuficiencia hepática tras la ingestión crónica de
    cobre. La exposición cutánea no se ha asociado con la toxicidad
    sistémica, pero el cobre puede inducir respuestas alérgicas en
    personas sensibles. Se han notificado casos de fiebre de los
    fundidores debidos a la inhalación de concentraciones elevadas en el
    aire en el puesto trabajo y, aunque se han atribuido otros efectos
    respiratorios a la exposición a mezclas que contenían cobre (por
    ejemplo, caldo bordelés, extracción y fundición), no se ha demostrado
    la función del cobre. Los trabajadores aparentemente expuestos a
    concentraciones elevadas en el aire que daban lugar a una ingesta
    estimada de 200 mg de Cu/día mostraron signos que parecían indicar una
    intoxicación por cobre (por ejemplo, concentraciones elevadas de cobre
    en el suero, hepatomegalia). Los datos disponibles sobre la toxicidad

    reproductiva y la carcinogenicidad son inadecuados para la evaluación
    del riesgo.

         Se describen varios grupos en los cuales los trastornos aparentes
    de la homeóstasis del cobre producen una sensibilidad mayor al déficit
    o el exceso de cobre que en la población general. Algunos trastornos
    tienen una base genética bien definida. Entre éstos figuran la
    enfermedad de Menkes, manifestación de la deficiencia de cobre
    generalmente fatal; la enfermedad de Wilson (degeneración
    hepatolenticular), enfermedad que lleva a una acumulación progresiva
    de cobre; y la aceruloplasminemia hereditaria, con síntomas clínicos
    de sobrecarga de hierro. La cirrosis infantil india y la toxicosis
    idiopática por cobre son enfermedades relacionadas con el exceso de
    cobre que pueden estar asociadas con una sensibilidad al cobre de base
    genética, aunque esto no se ha demostrado de manera inequívoca. Estas
    son enfermedades hepáticas fatales en la primera infancia, en las que
    el cobre se acumula en el hígado. Las incidencias de las enfermedades
    estaban relacionadas con un ingestión elevada de cobre, por lo menos
    en algunos casos.

         Otros grupos potencialmente sensibles al exceso de cobre son los
    pacientes sometidos a hemodiálisis y las personas con enfermedades
    hepáticas crónicas. Los grupos con riesgo de deficiencia de cobre
    incluyen los nińos pequeńos (en particular los recién nacidos de bajo
    peso al nacer/prematuros, los nińos que se están recuperando de una
    malnutrición, los nińos pequeńos alimentados exclusivamente con leche
    de vaca), las personas con síndrome de mala absorción (por ejemplo
    enfermedad celíaca, esprue, fibrosis cística) y los pacientes
    totalmente dependientes de una nutrición parenteral. Se ha relacionado
    la deficiencia de cobre con la patogénesis de las enfermedades

    1.9  Efectos en otros organismos en el laboratorio y en el
         medio ambiente

         Hay que buscar un equilibrio entre los efectos adversos del cobre
    y su carácter esencial. El cobre es un elemento esencial para toda la
    biota, y hay que tener cuidado para asegurar que queden cubiertas las
    necesidades nutricionales de cobre de los organismos. Este elemento
    forma parte integrante de la estructura de 12 proteínas importantes
    por lo menos. Es imprescindible para la utilización del hierro en la
    formación de la hemoglobina; en la mayor parte de los crustáceos y
    moluscos la principal proteína sanguínea transportadora de oxígeno es
    la hemocianina, en cuya estructura figura el cobre. En las plantas, el
    cobre forma parte de varias enzimas que intervienen en el metabolismo
    de los hidratos de carbono, del nitrógeno y de la pared celular.

         Un factor decisivo en la evaluación del peligro del cobre es su
    biodisponibilidad. La adsorción de cobre en las partículas y la
    formación de complejos con la materia orgánica puede limitar mucho el
    grado de acumulación del metal y sus efectos. Su biodisponibilidad
    puede verse afectada también en gran medida por la presencia de otros
    cationes y por el pH.

         Se ha demostrado que el cobre tiene efectos adversos en la
    reproducción, la bioquímica, la fisiología y el comportamiento de
    diversos organismos acuáticos. Se ha observado que concentraciones de
    cobre de apenas 1-2 µg/litro tienen efectos perjudiciales en
    organismos acuáticos; sin embargo, en la interpretación y aplicación
    de esta información se deben considerar grandes variaciones debidas a
    la sensibilidad y la biodisponibilidad de las especies.

         En las comunidades naturales de fitoplancton se observó una
    reducción significativa de la clorofila á y de la fijación del
    nitrógeno con concentraciones de cobre > 20 µg/litro, y la fijación
    del carbono disminuyó de manera considerable con concentraciones >
    10 µg/litro. La CE50 (72 horas) para las algas, basada en la
    inhibición del crecimiento, oscila entre 47 y 120 µg de Cu/litro.

         En los invertebrados de agua dulce, la C(E)L50 a las 48 horas
    oscila entre 5 µg de Cu/litro para una especie de dáfnidos y 5300 µg
    de Cu/litro para un ostrácodo. La CL50 a las 96 horas en los
    invertebrados marinos varía entre 29 µg de Cu/litro para el peine
    caletero y 9600 µg de Cu/litro para el cangrejo violinista. La
    toxicidad aguda del cobre para los peces de agua dulce y marinos es
    enormemente variable. En los primeros, la CL50 oscila entre 3 µg de
    Cu/litro (tímalo ártico) y 7340 µg de Cu/litro para
     Lepomis macrochirus. En los segundos, la CL50 a las 96 horas oscila
    entre 60 µg de Cu/litro para el salmón real y 1400 µg de Cu/litro para
    el mujol.

         Aunque las plantas necesitan cobre como elemento traza, la
    concentración elevada de este metal en el suelo puede ser muy tóxica.
    En general, los síntomas visibles de la toxicidad metálica son las
    hojas cloróticas pequeńas y la caída temprana de las hojas. También se
    produce un retraso del crecimiento y la iniciación de las raíces y el
    desarrollo de las laterales son escasos. El reducido crecimiento de
    las raíces puede dar lugar a una menor absorción de agua y de
    nutrientes, y esto provoca alteraciones en el metabolismo y retraso
    del crecimiento. A nivel celular, el cobre inhibe un gran número de
    enzimas e interfiere con varios aspectos de la bioquímica vegetal (por
    ejemplo la fotosíntesis, la síntesis de pigmentos y la integridad de
    la membrana) y la fisiología (en particular interfiere con los ácidos
    grasos y el metabolismo de las proteínas e inhibe la respiración y los
    procesos de fijación del nitrógeno).

         Se han observado efectos tóxicos en estudios de laboratorio
    realizados con lombrices de tierra expuestas a cobre en el suelo; la
    formación del cocón es el parámetro más sensible medido, con efectos
    adversos importantes en presencia de 50-60 µg de Cu/litro.

         En la naturaleza, los efectos adversos en los microorganismos del
    suelo se han relacionado con concentraciones más elevadas de cobre en
    zonas tratadas con fertilizantes que contenían este elemento y en
    lugares cercanos a fundiciones de cobre-zinc. En las zonas citrícolas
    en las cuales se han aplicado fungicidas con cobre se ha observado que

    la clorosis foliar está fuertemente relacionada con las
    concentraciones de cobre en el suelo.

         Se ha demostrado tolerancia al cobre en el medio ambiente para el
    fitoplancton, los invertebrados acuáticos y terrestres, los peces y
    las plantas terrestres. Los mecanismos de tolerancia propuestos en las
    plantas comprenden la unión del metal al material de la pared celular,
    la presencia de enzimas tolerantes al metal, la formación de complejos
    con ácidos orgánicos y la consiguiente eliminación en las vacuolas y
    la unión a proteínas específicas ricas en grupos tiol o a

    2.  Conclusiones

    2.1  Salud humana

         El límite inferior de la gama aceptable de ingesta oral (AROI) es
    de 20 µg/kg de peso corporal al día. Esta cifra se obtiene a partir de
    las necesidades basales de una persona adulta con un margen para tener
    en cuenta las variaciones en la absorción, retención y almacenamiento
    del cobre (OMS, 1996). En la infancia, esta cifra es de 50 µg/kg de
    peso corporal.

         El límite superior de la AROI en las personas adultas es
    incierto, pero muy probablemente es del orden de varios, pero no
    muchos, mg por día (por varios se entiende más de 2-3 mg/día). Esta
    evaluación se basa únicamente en los estudios de los efectos
    gastrointestinales del agua de bebida contaminada por cobre. No se
    pudo confirmar un valor más específico para el límite superior de la
    AROI con respecto a ningún sector de la población general. Es limitada
    la información disponible sobre el nivel de ingestión del cobre en los
    alimentos capaz de provocar efectos adversos para la salud.

         Los datos disponibles sobre la toxicidad en los animales no se
    consideraron de ayuda para establecer el límite superior de la AROI,
    debido a la incertidumbre acerca del modelo apropiado para el ser
    humano. Además, la metodología tradicional para la evaluación de la
    inocuidad, basada en la aplicación de factores de incertidumbre a los
    datos de los animales, no aborda de manera adecuada las
    características especiales de elementos esenciales como el cobre.

         De los datos disponibles sobre la exposición humana en todo el
    mundo, pero particularmente en Europa y en las Américas, se deduce que
    la deficiencia en la ingesta de cobre representa un riesgo de efectos
    en la salud mayor que el debido a un exceso.

    2.2  Efectos en el medio ambiente

         La protección de la vida acuática en las aguas con una elevada
    biodisponibilidad exigirá el mantenimiento de la concentración total
    de cobre disuelto en un valor inferior a 10 µg/litro; sin embargo, el
    límite adecuado de la concentración depende de la biota y de las
    condiciones de exposición en los lugares que despiertan preocupación y

    se debe establecer en función de una nueva evaluación de todos los
    datos pertinentes.

         En muchos lugares, los factores fisicoquímicos que limitan la
    biodisponibilidad permitirán valores de cobre más elevados. En los
    criterios reglamentarios se debe tener en cuenta la especiación del
    cobre si los autores de los vertidos pueden demostrar que se puede
    medir de forma fidedigna la biodisponibilidad del cobre en las aguas

         En el muestreo y el análisis del cobre en el medio ambiente es
    fundamental la utilización de técnicas "limpias".

         Habida cuenta de que el cobre es un elemento esencial, no se
    deben incorporar a los procedimientos para impedir niveles tóxicos de
    este metal factores de inocuidad que den lugar a concentraciones
    recomendadas inferiores a los niveles naturales.

    See Also:
       Toxicological Abbreviations
       Copper (ICSC)
       Copper (WHO Food Additives Series 17)
       COPPER (JECFA Evaluation)
       Copper (UKPID)