Copper (EHC 200, 1998)

INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

ENVIRONMENTAL HEALTH CRITERIA 200

COPPER

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

First draft prepared by 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
Chemicals.

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
objectives of the IPCS are to establish the scientific basis for
assessment of the risk to human health and the environment from
exposure to chemicals, through international peer review processes,
as a prerequisite for the promotion of chemical safety, and to
provide technical assistance in strengthening national capacities
for the sound management of chemicals.

The Inter-Organization Programme for the Sound Management of
Chemicals (IOMC) was established in 1995 by UNEP, ILO, the Food and
Agriculture Organization of the United Nations, WHO, the United
Nations Industrial Development Organization, the United Nations
Institute for Training and Research, and the Organisation for
Economic Co-operation and Development (Participating
Organizations), following recommendations made by the 1992 UN
Conference on Environment and Development to strengthen cooperation
and increase coordination in the field of chemical safety. The
purpose of the IOMC is to promote coordination of the policies and
activities pursued by the Participating Organizations, jointly or
separately, to achieve the sound management of chemicals in
relation to human health and the environment.

WHO Library Cataloguing in Publication Data

Copper.

(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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letters.

CONTENTS

ENVIRONMENTAL HEALTH CRITERIA FOR COPPER

1. SUMMARY AND CONCLUSIONS

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. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES AND ANALYTICAL
METHODS

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

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

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

4. ENVIRONMENTAL TRANSPORT AND DISTRIBUTION

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. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

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

6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS

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,
selenium)

7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS

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
7.2.2.1 Copper(II) sulfate
7.2.2.2 Copper chloride
7.3. Repeated exposure: subchronic toxicity
7.3.1. Oral
7.3.1.1 Copper(II) sulfate
7.3.1.2 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
7.6.1.1 In vitro
7.6.1.2 In vivo
7.6.2. Other copper compounds
7.6.2.1 In vitro
7.7. Other studies
7.7.1. Neurotoxicity
7.7.1.1 Copper sulfate
7.7.1.2 Copper chloride
7.7.2. Immunotoxicity
7.7.2.1 Copper(II) sulfate
7.8. Biochemical mechanisms of toxicity

8. EFFECTS ON HUMANS

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
8.3.2.1 Gastrointestinal and hepatic effects
8.3.2.2 Reproduction and development
8.3.2.3 Cancer
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. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD

9.1. Bioavailability
9.1.1. Bioavailability in water
9.1.1.1 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
9.2.2.1 Aquatic plants
9.2.2.2 Terrestrial plants
9.3. Toxic effects: laboratory experiments
9.3.1. Microorganisms
9.3.1.1 Water
9.3.1.2 Soil
9.3.2. Aquatic organisms
9.3.2.1 Plants
9.3.2.2 Invertebrates
9.3.2.3 Vertebrates
9.3.2.4 Model ecosystems and community
effects
9.3.3. Terrestrial organisms
9.3.3.1 Plants
9.3.3.2 Invertebrates
9.3.3.3 Vertebrates
9.4. Field observations
9.4.1. Microorganisms
9.4.2. Aquatic organisms
9.4.3. Terrestrial organisms
9.4.3.1 Tolerance
9.4.3.2 Copper fungicides and fertilizers

10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT

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
10.3.2.1 General population
10.3.2.2 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
10.5.1.1 Overview of exposure data
10.5.1.2 Overview of toxicity data
10.5.2. Terrestrial biota
10.5.2.1 Overview of exposure data
10.5.2.2 Plant foliar levels
10.5.2.3 Assessment of toxicity of copper in
soil

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

11.1. Human health
11.2. Environmental protection

12. FURTHER RESEARCH

12.1. Health protection
12.2. Environmental protection

13. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

REFERENCES

RESUME ET CONCLUSIONS

RESUMEN Y CONCLUCIONES

NOTE TO READERS OF THE CRITERIA MONOGRAPHS

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

* * *

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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WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR COPPER

Members

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

Observers

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

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)

Secretariat

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)

WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR COPPER

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
moriograph.

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

ABBREVIATIONS

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

CT^MAX critical thermal maxima

DT-OCEE deficiency toxicity optimum concentration for essential
elements

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. SUMMARY AND CONCLUSIONS

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
essential.

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
voltametry.

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
epithelium.

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
processes).

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. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES AND ANALYTICAL METHODS

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^-,
CO₃^2-, SO₄^2-, 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.

2.3.1.1 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 salts^a

Copper Copper(II) Cuprous(I) Copper(II) Copper(II) Oxine-copper^b
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.

2.3.1.2 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.

2.3.1.3 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).

2.3.1.4 "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
limits
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

2.3.2.1 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
samples.

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.

2.3.2.2 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.

2.3.2.3 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 Method^a 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)
HNO₃

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
water

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).

2.4.1.1 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.,
1994).

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. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

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
organisms.

Table 3. Copper consumption in 1988^a (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. ENVIRONMENTAL TRANSPORT AND DISTRIBUTION

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.,
1990).

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)
area

Copper Zinc Lead Cadmium

EU^a 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
Norway^a 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
concentrations.

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
salinity.

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
hepatopancreas.

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
faeces.

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
exposures.

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,
respectively).

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. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

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/m³with 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.,
1985).

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.,
1989).

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,
1989).

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,
respectively.

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. Håkansson 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
activity.

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 æ 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

5.1.4.1 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
mineralogy.

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.

5.1.4.2 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,
respectively.

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
sites.

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

5.2.3.1 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.

5.2.3.2 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

Meat
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

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

Kidney
beef 3.7 2.8 4.2 6
pork 6.1 2.9 15 75

Fruit
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

Vegetables
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

Fish
cod 0.19 0.12 0.28 5
tuna 0.64 0.48 0.80 9

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

Milk
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
sampling^a (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.
(1990)

Finland TD^b 2.00 Kumpulainen
et al. (1987)

Germany DD 0.95 Anke (1991)

The Netherlands^c MB 1.5 Slooff et al.
(1989)

Norway DD 1.0 Pettersson &
Sandström
(1995)

Sweden MB 1.20 Becker &
Kumpulainen
(1991)

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.
(1995)

USA^c 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.,
1992).

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.

6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS

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 proteins^a

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 oxidase^a 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

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
postnatally.

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
increases.

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
cornstarch

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
analysis.

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
animals.

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).

7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS

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
exposure.

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 710^a 226 Smyth et al.
(1969)
mouse 1600^a 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 940^b 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 960^c 244 Smyth et al.
(1969)
mouse 50 (LD₁₀₀) 20 Venugopal &
Luckey (1978)
rabbit 125 50 Eden & Green
(1939)

^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
hamsters.

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.,
1993).

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

7.2.2.1 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).

7.2.2.2 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

7.3.1.1 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,
1985).

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)

7.3.1.2 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
microscopically

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
microscopically

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

7.6.1.1 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).

7.6.1.2 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

7.6.2.1 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.,
1980).

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.

7.7.1.1 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).

7.7.1.2 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.

7.7.2.1 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
studies.

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. EFFECTS ON HUMANS

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
diet.

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
abnormalities.

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

8.3.2.1 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
subjects.

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).

8.3.2.2 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.

8.3.2.3 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)
diagnosis)

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
determined

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
expression.

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
state.

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
disorder.

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,
nephrolithiasis

Cardiovascular cardiomyopathy, arrhythmias, conduction disturbances,
autonomic dysfunction

Musculoskeletal osteomalacia, osteoporosis, degenerative joint disease

Gastrointestinal cholelithiasis, pancreatitis, spontaneous bacterial
peritonitis

Endocrine amenorrhoea, spontaneous abortion, delayed puberty,
gynaecomastia

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
deficit.

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.,
1993).

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
ICC.

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
cirrhosis

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
uncertain.

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,
1980).

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,
1988).

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.,
1991).

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
conjuncture.

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
below.

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.,
1981).

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. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD

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.

9.1.1.1 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
following:

* it is mechanistically based
* for the first time in aquatic toxicology it allows estimation of
metal dose at the receptor surface directly associated with
toxicity
* 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
sediments.

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
anaemia.

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
requirements.

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
levels.

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

9.2.2.1 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).

9.2.2.2 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
plants.

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

9.3.1.1 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,
respectively.

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,
respectively.

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.

9.3.1.2 Soil

Toxicity of copper to soil microorganisms is summarized in Table
17.

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)
reduction)

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)
cucculus)

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.

Frostegård 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

9.3.2.1 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
Chlamydomonas.

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,
respectively.

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 Conditions^a 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.
(1993)
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.