INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY ENVIRONMENTAL HEALTH CRITERIA 131 DIETHYLHEXYL PHTHALATE 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. Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization First draft prepared by Dr P. Lundberg, Dr. J. Hogberg, and Dr P. Garberg, National Institute of Occupational Health, Sweden, Dr I. Lundberg, Karolinska Hospital, Sweden, and Dr S. Dobson and Mr. P. Howe, Institute of Terrestrial Ecology, United Kingdom World Health Orgnization Geneva, 1992 The International Programme on Chemical Safety (IPCS) is a joint venture of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization. The main objective of the IPCS is to carry out and disseminate evaluations of the effects of chemicals on human health and the quality of the environment. Supporting activities include the development of epidemiological, experimental laboratory, and risk-assessment methods that could produce internationally comparable results, and the development of manpower in the field of toxicology. Other activities carried out by the IPCS include the development of know-how for coping with chemical accidents, coordination of laboratory testing and epidemiological studies, and promotion of research on the mechanisms of the biological action of chemicals. WHO Library Cataloguing in Publication Data Diethylhexyl phthalate. (Environmental health criteria ; 131) 1.Diethylhexyl phthalate - adverse effects 2.Diethylhexyl phthalate - toxicity 3.Environmental exposure I.Series ISBN 92 4 157131 4 (NLM Classification: QV 612) ISSN 0250-863X The World Health Organization welcomes requests for permission to reproduce or translate its publications, in part or in full. Applications and enquiries should be addressed to the Office of Publications, World Health Organization, Geneva, Switzerland, which will be glad to provide the latest information on any changes made to the text, plans for new editions, and reprints and translations already available. (c) World Health Organization 1992 Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. All rights reserved. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. CONTENTS ENVIRONMENTAL HEALTH CRITERIA FOR DIETHYLHEXYL PHTHALATE 1. SUMMARY 1.1 Identity, physical and chemical properties, and analytical methods 1.2 Sources of human and environmental exposure 1.3 Environmental transport, distribution, and transformation 1.4 Environmental levels and human exposure 1.5 Kinetics and metabolism 1.6 Effects on laboratory mammals and in vitro test systems 1.7 Effects on humans 1.8 Effects on other organisms in the laboratory and field 1.9 Evaluation 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS 2.1 Identity 2.2 Physical and chemical properties 2.3 Conversion factors 2.4 Analytical methods 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1 Natural occurrence 3.2 Anthropogenic sources 3.2.1 Production levels 3.2.2 Uses 3.2.3 Disposal of plasticized products 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION 4.1 Environmental transport and distribution 4.1.1 Transport in air 4.1.2 Transport in soil and sediment 4.1.3 Transport in water 4.1.4 Transport between media 4.2 Biotransformation 4.2.1 Abiotic degradation 4.2.2 Biodegradation 188.8.131.52 Aerobic degradation 184.108.40.206 Anaerobic degradation 4.2.3 Bioaccumulation 220.127.116.11 Model ecosystems 18.104.22.168 Aquatic invertebrates 22.214.171.124 Fish 126.96.36.199 Amphibians 188.8.131.52 Plants 184.108.40.206 Birds 5. ENVIRONMENTAL LEVELS AND EXPOSURE 5.1 Environmental levels 5.1.1 Air 5.1.2 Precipitation 5.1.3 Water 5.1.4 Sediment 5.1.5 Soil 5.1.6 Food 5.1.7 Aquatic organisms 5.1.8 Terrestrial organisms 5.2 General population exposure 5.3 Occupational exposure during manufacture, formulation or use 6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS 6.1 Absorption 6.1.1 Inhalation 6.1.2 Dermal 6.1.3 Oral 6.1.4 Intraperitoneal 6.2 Distribution 6.3 Metabolism 6.4 Elimination and excretion 6.5 Retention and turnover 6.5.1 Half-life and body burden 6.5.2 Indicator media 7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS 7.1 Single exposure 7.2 Short-term exposure 7.3 Long-term exposure 7.4 Skin and eye irritation; sensitization 7.5 Reproduction, embryotoxicity, and teratogenicity 7.5.1 Reproduction 7.5.2 Embryotoxicity and teratogenicity 7.6 Mutagenicity and related end-points 7.6.1 Mutation 220.127.116.11 Bacteria 18.104.22.168 Fungi 22.214.171.124 Mammalian cells 126.96.36.199 Drosophila 7.6.2 DNA damage 7.6.3 DNA binding 7.6.4 Chromosomal effects 7.6.5 Cell transformation 7.6.6 In vivo effects 7.7 Carcinogenicity 7.8 Special studies 7.9 Mechanisms of hepatotoxicity 8. EFFECTS ON HUMANS 8.1 General population exposure 8.2 Occupational exposure 9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD 9.1 Toxicity to microorganisms 9.2 Toxicity to aquatic organisms 9.2.1 Invertebrates 9.2.2 Fish 9.2.3 Amphibians 9.3 Toxicity to terrestrial organisms 9.3.1 Plants 9.3.2 Earthworms 9.3.3 Insects 9.3.4 Birds 10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT 10.1 Evaluation of human health risks 10.1.1 Exposure levels 10.1.2 Toxic effects 10.1.3 Conclusion 10.2 Evaluation of effects on the environment 10.2.1 Exposure levels 10.2.2 Toxic effects 10.2.3 Conclusion 11. RECOMMENDATIONS FOR PROTECTION OF HUMAN HEALTH AND THE ENVIRONMENT 12. FURTHER RESEARCH 13. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES REFERENCES RESUME RESUMEN WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR DIETHYLHEXYL PHTHALATE Members Dr D. Anderson, British Industrial Biological Research Association, Carshalton, Surrey, United Kingdom Dr R. Cattley, Department of Experimental Pathology and Toxicology, Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina, USA Dr U. Chantharaksri, Department of Pharmacology, Mahidol University, Bangkok, Thailand Dr S.D. Gangolli, British Industrial Biological Research Association, Carshalton, Surrey, United Kingdom Dr J. Högberg, Department of Toxicology, National Institute of Occupational Health, Solna, Sweden Mr P. Howe, Institute of Terrestrial Ecology, Monks Wood Experimental Station, Abbots Ripton, Huntingdon, United Kingdom Dr F. Matsumura, Toxic Substances Program, Department of Environmental Toxicology, University of California, Davis, California, USA (Chairman) Dr S. Oishi, Department of Toxicology, Metropolitan Research Laboratory of Public Health, Tokyo, Japan Dr C.-N. Ong, Department of Community, Occupational and Family Medicine, National University of Singapore, Singapore (Joint Rapporteur) Professor G. Pliss, Laboratory for Chemical Carcinogenic Agents, N.N. Petrov Research Institute of Oncology, Leningrad, USSR Professor Y.-L. Wang, Department of Occupational Health, School of Public Health, Shanghai Medical University, Shanghai, China Mr G. Welter, Federal Environmental Protection Agency, Berlin, Germany Representatives of other intergovernmental organizations Dr M. De Smedt, Commission of the European Communities, Luxembourg Representatives of non-governmental organizations Dr C. Elcombe, European Chemical Industry Ecology and Toxicology Centre, Brussels, Belgium Dr B. Lake, Conseil Européen des Fédérations de l'Industrie chimique (CEFIC), Brussels, Belgium Secretariat Dr B.-H. Chen, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland (Secretary) Dr P. Lundberg, Department of Toxicology, National Institute of Occupational Health, Solna, Sweden (Joint Rapporteur) Dr D. McGregor, International Agency for Research on Cancer, World Health Organization, Lyon, France 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 kindly requested to communicate any errors that may have occurred to the Manager 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, Palais des Nations, 1211 Geneva 10, Switzerland (Telephone No. 7988400 or 7985850). ENVIRONMENTAL HEALTH CRITERIA FOR DIETHYLHEXYL PHTHALATE A WHO Task Group on Environmental Health Criteria for Diethylhexyl Phthalate (DEHP) met at the British Industrial Biological Research Association (BIBRA), Carshalton, Surrey, United Kingdom, from 3 to 7 June 1991. Dr S.D. Gangolli opened the meeting on behalf of BIBRA. Dr B.-H. Chen, IPCS, welcomed the participants on behalf of the Manager, IPCS, and the three IPCS cooperating organizations (UNEP/ILO/WHO). The Task Group reviewed and revised the draft criteria monograph and made an evaluation of the risks for human health and the environment from exposure to DEHP. The first draft of this monograph was prepared by Dr P. Lundberg, Dr J. Högberg, and Dr P. Garberg of the National Institute of Occupational Health, Sweden, Dr I. Lundberg of Karolinska Hospital, Sweden, and Dr S. Dobson and Mr P. Howe of the Institute of Terrestrial Ecology, Monks Wood Experimental Station, United Kingdom. The second draft was prepared by Dr P. Lundberg incorporating comments received following the circulation of the first draft to the IPCS Contact Points for Environmental Health Criteria monographs. Particularly valuable comments on the draft were made by the European Chemical Industry Ecology and Toxicology Centre (ECETOC), the International Agency for Research on Cancer (IARC), the Toxicology Division, Exxon Biomedical Sciences, and the Conseil European des Federations de L'industrie Chimique (CEFIC). Dr B.-H. Chen and Dr P.G. Jenkins, both members of the IPCS Central Unit, were responsible for the overall scientific content and technical editing, respectively. The efforts of all who helped in the preparation and finalization of the document are gratefully acknowledged. * * * Financial support for this Task Group was provided by the United Kingdom Department of Health as part of its contributions to the IPCS. ABBREVIATIONS DEHP diethylhexyl phthalate DBP di- n-butyl phthalate DiBP di-iso-butyl phthalate ECETOC European Chemical Industry Ecology and Toxicology Centre ECMO extracorporeal membrane oxidation HPLC high-performance liquid chromatography MEHP monoethylhexyl phthalate NDMA N-dimethylnitrosamine NOEL no-observed-effect level PVC polyvinyl chloride SHE Syrian hamster embryo SPF specific pathogen free UDS unscheduled DNA synthesis US ATSDR US Agency for Toxic Substances and Disease Registry US FDA US Food and Drug Administration 1. SUMMARY 1.1 Identity, physical and chemical properties, and analytical methods Di(2-ethylhexyl)phthalate (DEHP) is a benzenedicarboxylic acid ester which at room temperature is a colourless to yellow oily liquid. Its solubility in water is low (0.3-0.4 mg/litre), and is even lower in salt water. It is miscible with most common organic solvents. The volatility of DEHP is relatively low (8.6 x 10-4 Pa). Many sampling and analytical methods have been developed for the determination of DEHP in different media. Sensitive methods, such as gas chromatography, high-performance liquid chromatography, and mass spectrometry are being used increasingly. Analysis of low concentrations of DEHP is complicated by contamination from plastic equipment during sampling and analysis. 1.2 Sources of human and environmental exposure Almost all the DEHP present in the environment arises from anthropogenic sources rather than from natural ones. The worldwide production of DEHP has been increasing during recent decades and at present amounts to about 1 x 106 tonnes per year. One third of the total production is in the USA and one third in Europe. DEHP is the most widely used plasticizer (comprising 50% of all phthalate ester plasticizers) that softens resins. It may account for 40% (w/w) or more of the plastic. DEHP is used for making the polyvinyl chloride (PVC) utilized in building, construction and packaging, and for medical device components. Smaller amounts are used in industrial paints and as a dielectric fluid in condensers. Discarded plasticized products may be disposed of either by incineration or via dumping in a landfill site. During incineration at a low temperature, a large percentage of the DEHP may be lost to the atmosphere. The environmental fate of DEHP in landfill sites has not been well studied and no definite conclusions can be reached. 1.3 Environmental transport, distribution, and transformation Transport in the air is the major route by which phthalates enter the environment. From the atmosphere DEHP either falls or is washed out via rainfall. DEHP has a high octanol-water partition coefficient, so the equilibrium between water and an organic-rich soil or sediment is in favour of the soil or sediment. It is readily adsorbed by organic soil particles. Although the solubility of DEHP in water is low, the amount present in surface water may be higher due to adsorption onto organic particles and interaction with dissolved organic matter. It is adsorbed particularly by small particles, and adsorption is enhanced in salt water. Atmospheric photodegradation of DEHP is rapid, but its chemical hydrolysis in the environment is practically non-existent. Aerobic degradation has been found to be carried out by several soil microorganisms. However, the microbial degradation of DEHP in the environment has been reported to be slow. The biodegradation pathway begins with hydrolysis to the mono-ester, which is then converted to phthalic acid. The ring-opening degradation to pyruvate and succinate and then to CO2 and H2O is similar to the metabolic pathway of benzoic acid. The aerobic degradation is temperature dependent. Below 10 °C little degradation takes place. At higher temperatures biodegradation proceeds in the upper layer of the soil, but it is virtually non-existent deeper down where conditions are anaerobic. Anaerobic degradation, if it exists, is very much slower than aerobic degradation. DEHP is highly lipophilic and moderately persistent. The degree of bioaccumulation depends on the capability of an organism to metabolize DEHP. It has been shown to accumulate to a high degree in a variety of aquatic invertebrates, fish, and amphibians. When DEHP was applied to plant leaves, there was little loss over a 15-day period. Uptake by plants from soil or sewage sludge was found to be low. 1.4 Environmental levels and human exposure DEHP exists widely in the environment and is found in most samples, including air, precipitation, water, sediment, soil, and biota. Levels are generally highest in industrialized areas. DEHP concentrations of up to 300 ng/m3 have been found in urban and polluted air. Levels of between 0.5 and 5 ng/m3 have been reported in the air of oceanic areas, and the rainfall in these areas contained up to about 200 ng/litre. Precipitation samples from an area close to a plasticizer production plant indicated that the rate of dry deposition was 0.7 to 4.7 µg/m2 per day. In rivers and lakes the concentration of DEHP has been found to be up to 4 µg/litre, highest levels being associated with industrial effluent discharge points. The concentration in the sea is less than 1 µg/litre, highest levels being in estuaries. Due to its hydrophobic character, DEHP is readily absorbed to soil, sediment, and particulate matter. River sediment levels of up to 70 mg/kg (dry weight) have been reported, and these have reached 1480 mg/kg (dry weight) near discharge points. The concentration of DEHP in biota varies from less than 1 to 7000 µg/kg. It has been found in various types of food, such as fish, shellfish, eggs, and cheese. The estimated average exposure was around 300 µg/person per day in the USA in 1974 and 20 µg/person per day in the United Kingdom in 1986. Blood transfusions and other medical treatment using plastic devices may lead to involuntary human exposure to DEHP. Levels from 13.4 to 91.5 mg/kg (dry weight) in lung tissue have been detected in patients. The few data available indicate that workplace concentrations of DEHP are usually below 1 mg/m3. 1.5 Kinetics and metabolism Available data on oral administration indicate that DEHP is hydrolysed in the gut by pancreatic lipase. The metabolites formed, i.e. mono(2-ethylhexyl)phthalate (MEHP) and 2-ethyl-hexanol, are rapidly absorbed. When 14C-labelled DEHP (2.9 mg/kg) was given orally to rats, more than 50% was recovered in the urine or bile. The bioavailability of an oral dose of DEHP seems to be higher in young rats than in older ones. When administered orally, DEHP is extensively hydrolysed in the gut in certain animals, e.g., rats, and is mainly distributed as monoethylhexyl phthalate (MEHP). However, hydrolysis occurs to a much lesser extent in primates and humans. MEHP binds to plasma proteins. The liver seems to be the major organ for the metabolism of MEHP and 2-ethylhexanol. Several further metabolites have been identified, omega- and omega-1-oxidation being the major metabolic pathways. One or several of the products of omega-oxidation may be further metabolized by ß-oxidation. Non-linear kinetics have been observed for the omega-oxidation. DEHP metabolism shows considerable species differences; e.g., the omega-oxidation pathway is less extensive in humans than in rats. Almost 100% of an oral dose of DEHP (2.9 mg/kg) was recovered in rat faeces and urine after a week. Bile and urine are the major excretory pathways. In a human study, 15-25% of an oral dose (0.45 mg/kg) of DEHP was excreted as MEHP, and oxidized metabolites constituted a major portion of the excretion products. 1.6 Effects on laboratory mammals and in vitro test systems The oral LD50 for DEHP is about 25-34 g/kg, depending on the species, but the value for MEHP is lower. In feeding studies on rats and mice, DEHP dosages greater than 3 g/kg per day caused deaths within 90 days, and a level of 0.4 g/kg per day reduced weight gain within a few days. In other studies, 6.3-12.5 g/kg diet caused a body weight reduction. Hepatomegaly and increased relative kidney weights have been observed in treated animals in long-term studies. In one study, there were also hypertrophic cells in the anterior pituitary. Several studies have shown testicular atrophy, evident within a few days, related to DEHP administration (dietary levels of 10-20 g DEHP/kg). Younger rats seem to be more susceptible than older ones, and rats and mice seem to be more sensitive than marmosets and hamsters. Reversibility of the atrophy has been observed. MEHP has toxic effects on Sertoli cells in vitro. DEHP, as well as MEHP, shows teratogenic properties. Malformations were observed at dietary levels of 0.5-2 g/kg in mice, and embryotoxic effects were observed at dietary levels greater than 10 g/kg. Tests for mutagenicity and related end-points have been negative in most studies. DEHP may induce cellular transformation, and it has been shown to be carcinogenic at doses of 6 and 12 g DEHP/kg diet in rats and 3 and 6 g/kg diet in mice. There was a dose-related increase in hepatocellular tumours in both sexes of both species. The induction of hepatic peroxisome proliferation and cell replication is strongly associated with the liver carci-nogenic effect of certain non- genotoxic carcinogens including DEHP. However, marked differences have been observed among animal species with respect to DEHP-induced peroxisome proliferation. In contrast to rat hepatocytes, DEHP metabolites do not produce peroxisome proliferation in cultured human hepatocytes. 1.7 Effects on humans Only very limited information is available on the effects of DEHP on humans. Mild gastric disturbances, but no other deleterious effects, were reported for two subjects given 5 or 10 g DEHP. 1.8 Effects on other organisms in the laboratory and field Most studies have yielded nominal LC50 values in acute toxicity tests that are in excess of 10 mg/litre, values which give a low toxicity rating for DEHP. However, these levels exceed the DEHP water solubility (0.3-0.4 mg/litre). One study suggested greater sensitivity of the water flea Daphnia pulex, with a nominal 48-h LC50 of 0.133 mg/litre. The only acute test with measured DEHP concentrations was on the fathead minnow and revealed a 96-h LC50 of > 0.33 mg/litre. In prolonged studies, the no-observed-effect level (NOEL) for Daphnia magna was 72 µg/litre. For adult fish a NOEL of > 62 µg/litre was determined. An exposure of 14 µg/litre, from 12 days prior to hatching, caused a significant increase in trout fry mortality. DEHP concentrations of between 3.7 and 11 µg/litre led to a reduction in the vertebral collagen of fish. The survival of zebra fish fry is adversely affected by DEHP concentrations of 50 mg/kg food. Sediment concentrations of 25 mg/kg (w/w) significantly reduced microbial activity and the number of tadpoles hatching. The acute toxicity of DEHP to algae, plants, earthworms, and birds is low. 1.9 Evaluation DEHP causes reproductive and hepatocarcinogenic effects in rats and mice. Testicular atrophy is the main reproductive effect in rats and mice, and young animals are more susceptible than older ones to this effect. The induction of hepatic peroxisome proliferation and cell replication are strongly associated with the liver carcinogenic effect of certain non-genotoxic carcinogens including DEHP. However, marked differences have been observed among animal species with respect to DEHP-induced peroxisome proliferation. Currently there is not sufficient evidence to suggest that DEHP is a potential human carcinogen. There is no documented information that DEHP presents any hazard, based on acute exposure to fish and daphnids. However, a reduction of microbial activity in sediment at environmental levels of DEHP was reported. A comparison between environmental levels and the concentrations that produce effects in prolonged studies, especially early life-stage tests on fish and amphibians, indicates that a hazard for the environment, particularly via water and sediment, cannot be excluded. Adverse effects on organisms are likely in areas with highly contaminated water and sediments which are near to point emission sources. Although few relevant studies have been reported, the acute toxicity of DEHP to algae, plants, earthworms, and birds appears to be low. 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS 2.1 Identity Common name: di(2-ethylhexyl) phthalate Structural formula: Empirical formula: C24H38O4 Abbreviation: DEHP Relative molecular mass: 390.57 Common synonyms: 1,2-benzenedicarboxylic acid bis(2-ethylhexyl) ester (CAS name); phthalic acid bis(2-ethylhexyl) ester (IUPAC name); BEHP; 1,2-benzenedicarboxylic acid bis(ethylhexyl) ester; bis(2-ethylhexyl) 1,2-benzenedicarboxylate; bis(2-ethyl-hexyl) ester of phthalic acid; bis(2-ethylhexyl) phthalate; di(2-ethylhexyl) ortho-phthalate; di(ethylhexyl) phthalate; dioctyl phthalate; DOP; ethylhexyl phthalate; 2-ethylhexyl phthalate; octyl phthalate; di- sec-octyl phthalate; phthalic acid dioctyl ester Common trade names: Bisoflex 81; Bisoflex DOP; Compound 889; DAF 68; Ergoplast FDO; Eviplast 80; Eviplast 81; Fleximel; Flexol DOP; Goodrite GP 264; Hatcol DOP; Kodaflex DOP; Mollan O; Nuoplaz DOP; Octoil; Palatinol AH; Platinol DOP; Pittsburgh; PX-138; Reomol DOP; Reomol D 79P; Sicol 150; Staflex DOP; Truflex DOP; Vestinol AH; Vinicizer 80; Witcizer 312 (IARC, 1982; NIOSH, 1985b) CAS registry number: 117-81-7 RTECS number: TI 035000 Di(2-ethylhexyl) phthalate (DEHP) is available in a variety of technical grades. In the USA typical product specifications are: minimal ester content, 99.0-99.6%; maximal moisture content, 0.1%; acidity (as acetic acid or phthalic acid), 0.007-0.01%; specific gravity, 0.980-0.985 (25 °C/25 °C); refractive index, 1.4850-1.4870 (23 °C); and minimal flash-point, 216 °C (IARC, 1982). In western Europe, DEHP is available with the following specifications: maximal acid value, 0.06; maximal weight loss on heating at 140 °C for 3 h, 1%; and saponification value, 284-290 mg KOH/g (IARC, 1982). In Japan, DEHP must fulfill the following specifications: maximal acid value, 0.05; maximal weight loss on heating at 125 °C for 3 h, 0.1%; and specific gravity, 0.983-0.989 (20 °C/20 °C) (IARC, 1982). 2.2 Physical and chemical properties DEHP is a colourless to yellow, oily liquid at room temperature and normal atmospheric pressure. The melting point is -46 °C (pour point) and the boiling point is 370 °C (at atmospheric pressure, 101.3 kPa; 236 °C at 1.33 kPa, and 231 °C at 0.67 kPa). The flash point is 425 °C (open cup) (Clayton & Clayton, 1981; IARC, 1982; SAX, 1984). At 20 °C, the density is 0.98 g/ml (Fishbein & Albro, 1972) and the vapour pressure 8.6 x 10-4 Pa (Howard et. al., 1985). The log n- octanol-water partition coefficient is 3-5. The solubility of uncolloidal DEHP in water is low (45 µg/litre at 20 °C) (Leyder & Boulanger, 1983). However, DEHP may form colloidal dispersions which lead to higher values for solubility (Klöpfer et al., 1982). Values of 285 µg/litre (Hollifield, 1979), 340 µg/litre (Howard et al., 1985), and 360 µg/litre (Defoe et al., 1990) have been determined at 20-25 °C. These higher values are probably more realistic in the environment. Howard et al. (1985) determined a value of 160 µg/litre at 25 °C in salt water. DEHP is miscible with most common organic solvents and is more soluble in blood than water. It is lipophilic and the distribution ratio in dichloromethane-Krebs bicarbonate buffer has been measured to be 1130 (Krauskopf, 1973; Clayton & Clayton, 1981; IARC, 1982; Sax, 1984; Weast et al., 1984; Sittig, 1985). 2.3 Conversion factors 1 ppm = 15.87 mg/m3 1 mg/m3 = 0.063 ppm 2.4 Analytical methods Methods used for the analysis of di(2-ethylhexyl) phthalate in many types of samples are summarized in Table 1. Analysis of samples with low concentrations of DEHP is complicated by the risk of contamination from plastic equipment. Table 1. Methods for the analysis of di(2-ethylhexyl) phthalate Sample Sample Assay Limit of matrix preparation procedure detection Reference Air collect on cellulose GC/FID range: NIOSH (1977) ester membrane filter; 2.03-10.9 NIOSH (1985a) extract disulfide) mg/m3 for a 32-litre sample at 23 °C Air collect with impinger GC/ECD not given Thomas (1973) (ethylene glycol); GC/MS extract (hexane) Marine air trap on glass-fibre filters GC/ECD 0.5 ng/m3 Giam et al. with foam plugs; Soxhlet (1980) extract (petroleum ether); concentrate extracts; clean-up on deactivated Florisil columns Air-borne Soxhlet extract (methanol); GC/MS not given Karasek et al. particulate concentrate; centrifuge (1978) matter River water extract (hexane); filter HPLC/UV 2 ng at Mori (1976) (normal and 224 nm reversed-phase adsorption chromatography and gel chromatography Table 1 (contd). Sample Sample Assay Limit of matrix preparation procedure detection Reference River water extract (chloroform); thin-layer 50 µg/litre Kataeva (1988) concentrate extract; dry by chromatography sodium sulfate treatment; evaporate; dissolve residue (chloroform) Industrial add hydrochloride acid; GC/MC (EI not given Sheldon & and municipal extract (dichloromethane); and CI modes); Hites (1979) waste water clean-up by liquid with SIM chromatographic fractionation River freeze-dry; homogenize; HPLC/UV 10 ng Schwartz et al. sediment extract (hexane; acetone; (233 nm) (1979) methanol); evaporate; dissolve (hexane); filter Human serum centrifuge; extract (chloroform: GC/FID 50 µg/litre Lewis et al. methanol); evaporate; dissolve GC/MS (1977) (ethyl acetate); treat with alumina; decant; rinse; filter; evaporate; dissolve residue (hexane-containing butyl benzyl phthalate as an internal standard) Human plasma separation on Celite 545; GC/FID 50 ng Piechocki & extract (diethyl ether); Purdy (1973) evaporate; dissolve (carbon disulfide) Table 1 (contd). Sample Sample Assay Limit of matrix preparation procedure detection Reference Stored blood; lyophilize; suspend; filter; GC/FID not given Contreras et whole blood wash residue; mix with distilled al. (1974) water; centrifuge; add silicic acid to chloroform phase; mix; centrifuge; decant; evaporate; dissolve; centrifuge Human and grind wet tissue samples GC/FID 0.3 µg/g Chen et al. animal in saline; extract homogenate (wet tissue) (1979b) tissue and or urine; dilute with 15 ng/ml Chen et al. urine chloroform:methanol (urine) (1979a) Intravenous add hydrochloric acid; GC/ECD 4 µg/litre Arbin & solutions extract (dichloromethane); Östelius redissolve (1980) Organic evaporate; dissolve GC/FID not given Ishida et al. solvents (diethyl ether) (1980) Solid immerse in chloroform: GC/FID not given Ishida et al. reagents methanol; filter; rinse; (1980) evaporate Aluminium cut into small pieces; GC/FID not given Ishida et al. foil; rubber immerse in chloroform: (1980) tubing, etc. methanol; extract Abbreviations: GC = gas chromatography; FID = flame-ionization detection; ECD = electron capture detection; MS = mass spectrometry; HPLC = high- performance liquid chromatography; UV = ultraviolet spectroscopy; EI = electron impact; CI = chemical ionization; SIM = selected ion monitoring. 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE It seems likely that by far the major part of the phthalate esters present in the environment arises from human activity and not from natural sources. Some dialkyl esters may be found in coals, crude oil, and shales while others may have plant origins, but most originate either directly or indirectly from industrial processes. Phthalic acid has been reported to be formed during the bacterial metabolism of phenanthrene. 3.1 Natural occurrence Phthalates have been reported in a wide variety of substances (oil, soil, plants, and animals) and over a wide geographical area. Most occurrences have anthropogenic origins but some could be of natural origin. The nature of the origin is further complicated by the fact that sampling techniques often lead to contamination of samples via contamination from plastic bags or bottles. Mathur (1974a) critically reviewed this question and concluded that the possibility of the phthalic acid esters found in biological and geochemical samples being of biosynthetic origin cannot be ruled out. Both Mathur (1974a) and Peakall (1975) cite reports where phthalates were detected yet no anthropogenic source could be found. Studies by Manandhar et al. (1979) and Pare et al. (1981) also revealed residues of phthalates in biological samples where the source seemed to be natural. Peterson & Freeman (1984) suggested that some of the phthalates found in older samples (from the 1920s and 1930s) of sediment cores from Chesapeake bay, USA, may have been of natural origin. An ECETOC (European Chemical Industry Ecology and Toxicology Centre) task force concluded that, although knowledge of naturally produced phthalates is limited or uncertain, it is unlikely that this contribution is of significance except, possibly, in very localized areas (ECETOC, 1985). 3.2 Anthropogenic sources 3.2.1 Production levels About 2.7 x 106 tonnes of total phthalates are produced annually, of which the non-plasticizer (dimethyl and diethyl) phthalates represent a very small fraction. Of the plasticizer phthalates, DEHP accounts for well over 50% of the tonnage, the contribution of the remaining compounds ranging from about 1% to 10% each (ECETOC, 1985). The production of DEHP has been increasing since it was first used commercially in 1949. During the period 1950-1954, the production in the USA was 106 x 103 tonnes, and by the period 1965-1969 the level had risen to 650 x 103 tonnes (Peakall, 1975). The estimated world consumption of DEHP in 1984 was 1.09 x 106 tonnes (SRI, 1985). 3.2.2 Uses Phthalate acid esters are the most widely used plasticizers for the production of polyvinyl chloride (PVC) products (with DEHP as the plasticizer). Phthalates are used for the insulation of wires and cables, in floor tiles, weatherstripping, upholstery, garden hose, swimming pool liners, footwear and clothing. They are also used in food wrapping and containers, although in some countries this use is prohibited by law. They also have non-plasticizer uses, e.g., as pesticide carriers. DEHP has been widely used since 1949. An important property is that it softens resins without reacting with them chemically. This has led to about 95% of DEHP production being directed towards plasticizer use, particularly in PVC products such as tubing and medical device components. It is also used as a plasticizer in cellulose ester plastics and synthetic elastomers. The DEHP content of these products generally ranges from 20 to 40%, but for some uses it is up to 55%. The most important non-plasticizer use of DEHP is as a dielectric fluid in capacitors. 3.2.3 Disposal of plasticized products Most discarded plasticized products are disposed of either by incineration or via dumping in a tip/landfill site. When incinerated at high temperature the combustion of phthalates is nearly complete. However, if combustion is uncontrolled and occurs at a low temperature, a large percentage of the phthalates may be lost to the atmosphere. After dumping in a landfill site, phthalates may leach into the aquatic environment, but because of their high affinity for organic soil particles and their low water solubility this is not likely to be a major route into the environment. Indiscriminate dumping is more likely to lead to volatilization of phthalates to the atmosphere rather than leaching to the aquatic environment (ECETOC, 1985). 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION 4.1 Environmental transport and distribution The release of phthalates to the environment may occur as follows: a) during production and distribution; b) during the manufacture of plasticized products; c) during the use of plasticized product; d) after disposal. It was concluded by ECETOC (1985) that most of the phthalates entering the environment are likely to do so by volatilization to the atmosphere, only a minor part (perhaps 10%) entering the aquatic environment by leaching. The estimated worldwide emissions of DEHP are given in Table 2. However, as reported by ECETOC (1985), the loss of DEHP from modern production plants is negligible. 4.1.1 Transport in air ECETOC (1985) suggested that transport in air is the major route by which phthalates enter the environment. DEHP is volatilised to the atmosphere and then either falls as dry deposition or is "washed out" via rainfall. DEHP has been measured in air samples from remote sites at Enewetak Atoll in the North Pacific Ocean (Atlas & Giam, 1981), the North Atlantic (Giam et al., 1978), the Gulf of Mexico (Giam et al., 1978, 1980), and Sweden (Thurén & Larsson, 1990). 4.1.2 Transport in soil and sediment DEHP has a high n-octanol-water partition coefficient, so the equilibrium between water and an organic-rich soil or sediment will be very much in favour of the soil or sediment. Absorbance of DEHP by soil or sediment is also enhanced by van de Waals type bonding with natural soil minerals, promoted by the presence of benzene rings and carbonyl groups, and also by the low solubility of DEHP (ECETOC, 1985). From results with other organic substances, Wams (1987) estimated that 90% of DEHP is readily adsorbed by organic soil particles. As can be seen from section 5.1.4, the sediment or hydrosoil tends to act as a sink for DEHP. Table 2. Estimated worldwide emission of DEHP, based on an estimated total annual production of 4 x 106 tonnesa Phase Emission (tonnes/year) Route Production up to 40 000 waste water Distribution 2000 sewage systems Production of PVC 32 000 air and water During use of plastics 14 000 air 6000 water After disposal: to landfill sites up to 200 000 percolating water to waste incinerators ? air uncontrolled burning ? air a Adapted from Wams (1987). The values are higher than those from other sources (ECETOC, 1985) 4.1.3 Transport in water The solubility of DEHP in water is low (0.3 mg/litre at 25 °C). However, the amount present in surface water may be higher than the actual solubility as a result of adsorption onto organic particles (Taylor et al., 1981) and interactions with dissolved organic matter of high relative molecular mass, such as humic and fulvic acid (Matsuda & Schnitzer, 1971). DEHP has been found to adsorb to suspended particulate matter fairly rapidly, in less than 2 to 3 h, especially to small particles (Al-Omran & Preston, 1987). This adsorption was more rapid in salt water than in fresh water. Taylor et al. (1981) reported that between one-half and two-thirds of the DEHP in Mississippi River water is associated with particulate matter. By extrapolating laboratory data on the volatilization of DEHP from water under defined conditions, Klopffer et al. (1982) obtained a half-life in water of 146 days, although on purely theoretical grounds a value of only 25 days was calculated. Using the Exposure Analysis Modeling System (EXAMS), Wolfe et al. (1980a) calculated that at equilibrium the loss of DEHP via volatilization from a model river, a pond, an eutrophic lake, and an oligotrophic lake would be 0%, 2.8%, 2.2% and 2.3%, respectively. 4.1.4 Transport between media Eisenreich et al. (1981) estimated that the total annual deposition of DEHP from air into the Great Lakes, North America, varied from 3.7 tonnes (Lake Ontario) to 16 tonnes (Lake Superior). DEHP has a high n-octanol/water partition coefficient. This means that biota living in phthalate-containing water would be expected to have a higher phthalate level than the water itself (see section 4.2.3). However, many organisms are able to metabolize DEHP, and the concentrations found may be lower than those expected on the sole basis of partition coefficient. During the 33-day period of a model ecosystem study, the concentration of 14C in the aquatic phase reached a peak of 31 µg/litre at the fifth day after treatment and had declined to 7.7 µg/litre by the end of the experiment. This decline was stated to be the result of the uptake of DEHP and its degradation products by the organisms in the model ecosystem (Metcalf et al., 1973). Lokke & Bro-Rasmussen (1981) treated the leaves of Sinapsis alba with a mixture of di-iso-butyl phthalate (DiBP), di- n-butyl phthalate (DBP), and DEHP at a rate of 2.5 µg/cm2. Only very small amounts of DEHP evaporated from the leaves during the 15-day experiment, compared with DiBP (71%) and DBP (43%). 4.2 Biotransformation 4.2.1 Abiotic degradation As a result of atmospheric photodegradation, the atmospheric half-life of DEHP is less than one day (ECETOC, 1985). Chemical hydrolysis of DEHP is practically non-existent, the half-life being > 100 years in water at pH 8 and 30 °C (Wolfe et al., 1980b). 4.2.2 Biodegradation Aerobic degradation has been found from several micro-organisms in soil, sludge, sediment, and water. Anaerobic degradation is very much slower, or possibly even non-existent. The first step in the metabolic pathway for the biodegradation of DEHP is the hydrolysis of the diester to the monoester by esterases with low substrate specificity (Kurane et al., 1980; Taylor et al., 1981). The monoester is then converted into phthalic acid (Engelhardt et al., 1975). The ring-opening degradation to pyruvate and succinate and then to CO2 and H2O is similar to the metabolism of benzoic acid. According to Kurane et al. (1984), this is probably why the biodegradation of phthalic esters is so widespread. It appears that mixed populations of microorganisms are the most successful at completely degrading DEHP (Engelhardt et al., 1975; Kurane et al., 1979). When pure cultures of bacteria, selectively isolated in the laboratory, are used for the biodegra-dation of phthalates, accumulation of the breakdown products tends to occur (Keyser et al., 1976). 188.8.131.52 Aerobic degradation Aerobic degradation of DEHP has been found with several microorganisms, including bacteria and fungi. Overall, it appears that phthalates with short alkyl chains undergo rapid degradation, whereas those with longer chains, such as DEHP, are only 40-90% degraded after 10-35 days (ECETOC, 1985). Graham (1973) reported that laboratory-scale activated sludge processes degraded 91% of introduced DEHP within 38 h. Saeger & Tucker (1973) demonstrated that all phthalates tested underwent complete aerobic degradation in activated sludge and river water. Aerobic degradation of DEHP depends on temperature. Mathur (1974b) incubated a loam soil with DEHP at 4, 10, 22-25, and 32 °C, and soil respiration rates were measured after 14 weeks. Increased rates of respiration, showing that microbial degradation was taking place, were found at all temperatures. However, at 4 and 10 °C results indicated that only marginal degradation was taking place. Johnson & Lulves (1975) incubated freshwater hydrosoil containing 14C-DEHP (1 mg/litre) under aerobic conditions, and after 14 days, 50% of the DEHP had been degraded. This was a much slower rate of degradation than that found with DBP, where 98% was degraded within 5 days. Johnson et al. (1984) studied the biodegradation of phthalic acid esters in freshwater sediment and found that the length and configuration of the alkyl phthalate diester significantly affected the primary biodegradation rate. After a 14-day incubation in aerobic sediment at 22 °C, less than 2% of the branched-chain alkyl phthalate DEHP had been degraded whereas over the same period the linear alkyl DBP showed 85% degradation. DEHP degradation was significantly greater at very high concentrations (10 mg/litre) and at temperatures above 22 °C. Neither inorganic nitrogen nor phosphorus influenced the degradation of DEHP. Engelhardt et al. (1977) found that the fungus Penicillium lilacinum degraded approximately half of the initial amount of DEHP within 30 days, yielding the corresponding monoester, a second metabolite, which is hydroxylated in the alcohol moiety, and at least four minor metabolites. The bacterium Pseudomonas acidovorans completely degraded DEHP at a medium concentration of 5000 mg/kg within 72 h (Kurane et al., 1977). Saeger & Tucker (1976) found that 60% of the DEHP had undergone primary biodegradation within 5 weeks in Mississippi River water. Rapid primary degradation was found when DEHP was added to activated sludge at the rate of 5 mg/24 h. Depending on the source of the sludge, between 70% and 78% was degraded. To monitor whether complete biodegradation was being achieved, the authors measured CO2 evolution. Within the 14 days of incubation DEHP had essentially been completely degraded to CO2 and water under the conditions of this test. Taylor et al. (1981) showed the presence of significant populations of taxonomically distinct bacteria that grew on a range of phthalic acid esters, including DEHP, in the water and sediments of the Mississippi River region. Sugatt et al. (1984) used an acclimated shake-flask CO2-evolution test to study the biodegradation of DEHP and reported an initial breakdown of the parent compound of > 99% within the 28 days. The authors calculated a half-life of 5.25 days for the primary biodegradation of DEHP. In surface waters, DEHP is strongly adsorbed to organic particles (Taylor et al., 1981), which tends to reduce degradation (Baughman et al., 1980). In the upper layer of soil, biodegradation of phthalates proceeds as in surface water, but deeper down, where conditions are anaerobic, it is virtually nonexistent (Engelhardt & Wallnofer, 1978). Shanker et al. (1985) incubated garden soil containing DEHP at a concentration of 500 mg/kg. Within 20 days, 75% of the DEHP had been degraded and, after 30 days, more than 90%. Again the rate of degradation was much slower than that found for either di- n-methyl or di- n-butyl phthalate. No degradation was detectable when sterilized soil was used. 184.108.40.206 Anaerobic degradation Johnson & Lulves (1975) found DEHP to be completely resistant to microbial attack under anaerobic conditions. After 30 days, there was no significant loss of 14C-DEHP activity in freshwater hydrosoils overlaid with nitrogen. Shanker et al. (1985) reported that degradation of DEHP was much slower in anaerobic soil, flooded with sterile water to reduce the oxygen tension. After a 30-day incubation, 33% of the DEHP had been degraded, compared with more than 90% in the case of aerobic soil. O'Connor et al. (1989) studied the biodegradation of DEHP under anaerobic conditions in a medium containing municipal digester sludge over a period of 140 days. DEHP, which was the only carbon source, was added at a rate of 20, 100, and 200 mg/litre, and 100%, 69%, and 54% of the DEHP was degraded at the three respective concentrations. However, complete biodegradation to carbon dioxide and methane was minimal. Ziogou et al. (1989) studied the behaviour of DEHP (0.5, 1, and 10 mg/litre) during batch anaerobic digestion of sludge over a 32-day period. No degradation of DEHP was observed during this period. 4.2.3 Bioaccumulation DEHP is highly lipophilic, the log n-octanol-water partition coefficient being 3 to 5, and it is moderately persistent. The accumulation of DEHP is also influenced by the capability of an organism to metabolize it. Melancon (1979) reviewed the metabolism of phthalates in aquatic organisms. Bioconcentration factors for DEHP in a variety of aquatic organisms are given in Table 3. Table 3. Bioaccumulation of DEHP in aquatic organisms Organism Stat/ Exposure Exposure Bioconcentration Reference flowa period concentration factorc (µg/litre) Freshwater organisms Canadian pondweed stat 24 h 10 274.8d Metcalf et al. (1973) (Elodea canadensis) stat 12 h 10 000 133.8d Metcalf et al. (1973) Snail stat 48 h 10 857.5d Metcalf et al. (1973) ( Physa sp) stat 6 h 10 000e 402d Metcalf et al. (1973) Scud flowb 7 day 0.1 13 600 Sanders et al. (1973) (Gammarus pseudolimnaeus) flow 7 day 0.1 3900 Mayer & Sanders (1973) Water flea flowb 7 day 0.3 5200d Sanders et al. (1973) (Daphnia magna) flow 7 day 0.3 420 Mayer & Sanders (1973) stat 1 h 10 421d Metcalf et al. (1973) stat 12 h 10 000e 133.8d Metcalf et al. (1973) Sowbug flowb 21 day 62.3 250d Sanders et al. (1973) (Asellus brevicaudus) Mosquito (larvae) stat 12 h 10 1320.2d Metcalf et al. (1973) (Culex pipiens stat 24 h 10 000e 1187.3d Metcalf et al. (1973) quinquefasciatus) stat 24 h 10 20.3d Metcalf et al. (1973) stat 48 h 10 000e 434.6d Metcalf et al. (1973) Midge larvae (3rd instar) flowb 7 day 0.2 408 Streufert et al. (1980) (Chironomus plumosus) flowb 7 day 0.3 3100d Sanders et al. (1973) flow 7 day 0.3 350d Mayer & Sanders (1973) Mayfly flowb 7 day 0.1 2300d Sanders et al. (1973) (Hexagenia bilineata) flow 7 day 0.1 575d Mayer & Sanders (1973) Table 3 (contd). Organism Stat/ Exposure Exposure Bioconcentration Reference flowa period concentration factorc (µg/litre) Mosquito fish stat 48 h 100 265.3d Metcalf et al. (1973) (Gambusia affinis) stat 12 h 10 000e 129.4d Metcalf et al. (1973) Fathead minnow flow 14 day 1.9 458d Mayer & Sanders (1973) (Pimephales promelas) flow 56 day 1.9 886d Mehrle & Mayer (1976) Marine organisms Eastern oyster (muscle) stat 24 h 100 11.2 Wofford et al. (1981) (Crassostrea virginica) stat 24 h 500 6.9 Wofford et al. (1981) Brown shrimp stat 24 h 100 10.2 Wofford et al. (1981) (Penaeus aztecus) stat 24 h 500 16.6 Wofford et al. (1981) Sheepshead minnow stat 24 h 100 10.7 Wofford et al. (1981) (Cyprinodon variegatus) stat 24 h 500 13.5 Wofford et al. (1981) a Stat = static conditions (water unchanged for the duration of the test); flow = flow-through conditions (DEHP concentration in water continuously maintained, unless stated otherwise) b Intermittent flow-through conditions c Bioconcentration factor = concentration of DEHP in organism divided by concentration of DEHP in water d Bioconcentration factor calculated using a radioactive isotope (values represent parent compound plus radiolabelled products) e DEHP applied directly to water 220.127.116.11 Model ecosystems Metcalf et al. (1973) studied the uptake of 14C-labelled DEHP from water by aquatic organisms in a model ecosystem containing algae (Oedogonium), snails ( Physa sp.), mosquito larvae (Culex pipiens quinquefasciatus), and fish ( Gambusia sp). The mosquito larvae showed the highest concentration factor and the fish the lowest. Labelled DEHP was added to Sorghum plants and at the end of the 33- day experiment the water contained 0.34 µg DEHP per litre, the algae 18.32 mg/kg (53 890 x), the snails 7.3 mg/kg (21 480 x), the mosquito larvae 36.61 mg/kg (10 7670 x), and the fish 0.044 mg/kg (130 x). Sodergren (1982) exposed fish, aquatic invertebrates, and plants to 14C-labelled DEHP at a concentration of 1.4 mg/litre for 27 days under static conditions. After 5 days, 1/50 of the added amount of DEHP was still present in the water, and at the end of the experiment 62% was recovered from the various surfaces (glass walls, sediment and surface microlayer). All organisms accumulated DEHP. The amphipod Gammarus pulex, larvae of trichopterans, and the snail Planorbis corneus accumulated the DEHP to the highest degree, the concentration factors ranging from 17 000 to 24 000. The submerged plants, Mentha aquatica and Chara chara, also showed uptake and storage of large amounts (concentration factor of 18 000). However, the fish (stickleback, Pungitius pungitius, and minnow, Phoxinus phoxinus) did not accumulate 14C-DEHP to any great extent (concentration factors of 300 or less). Large accumulations of DEHP occurred in organisms living and/or feeding at interfaces. 18.104.22.168 Aquatic invertebrates Brown & Thompson (1982a) exposed Daphnia magna to nominal 14C-labelled DEHP concentrations of 3.2, 10, 32, and 100 µg/litre for 21 days and obtained bioconcentration factors of 166, 140, 261, and 268 at the four respective concentrations. When Brown & Thompson (1982b) exposed mussels (Mytilus edulis) to labelled DEHP at concentrations of 4.1 and 42.1 µg per litre, in both cases equilibrium was reached within 14 days with a concentration factor of 2500. Exposure ceased on day 28 but the mussels were monitored for a further 14 days. The half-life for loss of DEHP over this period was calculated to be 3.5 days. Laughlin et al. (1978) exposed grass shrimp, during larval development, to DEHP concentrations of up to 1 mg/litre for 28 days. DEHP was not detectable in shrimp tissues at or above a level of 2 mg/kg. When Streufert et al. (1980) exposed midge larvae (Chironomus plumosus) to a radioactively labelled DEHP concentration of 0.2 µg/litre, the larvae accumulated DEHP to 292 times the concentration in water within 2 days. DEHP levels in the midge larvae reached a plateau after 7 days at a bioconcentration factor of 408. Some of the larvae were transferred to clean water after 4 days, by which time they had accumulated 56 µg DEHP/kg, and the half-life for loss was calculated to be 3.4 days. After 9 weeks of exposure to sediment containing approximately 600 mg/kg, dragonfly larvae had taken up 14.7 mg DEHP per kg. This was significantly more than control larvae, which contained 2.9 mg/kg (Woin & Larsson 1987). Hobson et al. (1984) fed penaeid shrimps on a diet containing between 40 and 50 000 mg DEHP/kg for 14 days at a rate of 40 g/kg body weight per day. Whole body residues ranged from 0.249 to 18.3 mg/kg in a dose-related manner. 22.214.171.124 Fish Macek et al. (1979) exposed bluegill sunfish (Lepomis macrochirus) to 14C-DEHP, both via food at a concentration of 2.8 mg/kg and via water at 5.6 µg/litre, for up to 35 days. The steady- state body burden of 14C-DEHP after exposure via food and water was 0.73 mg/kg and via water alone was 0.64 mg/kg. The authors concluded that the uptake of DEHP via the aquatic food chain was statistically indistinguishable from that due to aqueous exposure. The time required for the fish to eliminate 50% of the residue burden during depuration in uncontaminated water was < 3 days. In a study by Karara & Hayton (1989), sheepshead minnows (Cyprinodon variegatus) were exposed to a 14C-DEHP concentrations of 60 ng/litre at temperatures ranging from 10 °C to 35 °C for a period of between 72 h and 160 h. The amount of DEHP accumulated after 72 h was 6 times greater at 35 °C than at 10 °C, and the bioconcentration factors increased exponentially with temperature from 45 at 10 °C to 6510 at 35 °C. Metabolic clearance also increased as a function of temperature, the maximum value being reached at a temperature of between 29 °C and 35 °C. Tarr et al. (1990) exposed three sizes (2.9 g, 61 g, and 440 g) of rainbow trout (Oncorhynchus mykiss) to 14C-DEHP at 20 ng/ml under static conditions for up to 96 h at 12 °C. The body-weight- associated changes in the pharmacokinetic parameters caused the bioconcentration factor to decline from 51.5 to 1.6 as body weight increased. When Mehrle & Mayer (1976) exposed rainbow trout (Salmo gairdneri) eggs (12 days prior to hatching to 24 days post-hatching) to 14C-labelled DEHP at concentrations of 5, 14, and 54 µg/litre, the bioconcentration factors were 78, 113, and 42, respectively. Mayer (1976) exposed fathead minnows (Pimephales promelas) to DEHP concentrations ranging from 1.9 to 62 µg/litre for 56 days under flow-through conditions. As the exposure concentration increased, concentration factors, measured after 56 days, decreased from 569 to 91. Equilibrium was attained after 28 days at the lowest dose and after 56 days at the highest dose. After exposure the fish were placed in uncontaminated water for 28 days, and the half-lives for loss ranged from 6.2 days (at 2.5 µg/litre) to 18.3 days (at 62 µg/litre). 126.96.36.199 Amphibians Larsson & Thuren (1987) exposed moorfrog eggs to sediment DEHP concentrations ranging from 10 to 800 mg/kg (fresh weight of sediment). The eggs hatched after about 3 weeks and the tadpoles were analysed after 60 days. The DEHP was released from the sediment to the overlying water, and the losses to the water increased linearly with increasing levels in the sediment (from 0.89 to 187.4 µg/litre). DEHP accumulated in the tadpoles at concentrations ranging from 0.28 to 246.8 mg/kg wet weight, and the accumulation increased with increasing DEHP concentration, both in sediment and water. 188.8.131.52 Plants Lokke & Bro-Rasmussen (1981) applied DEHP as a mixture that also contained DiBP and DBP at a concentration of 2.5 µg/cm2 to the leaves of Sinapis alba. The residue level of DEHP on the leaves immediately after application was 2.7 µg/cm2. After 15 days, DEHP levels had decreased to 0.8 µg/cm2, but when the growth of the plant was taken into account, no significant loss of DEHP over this period was found. Lokke & Rasmussen (1983) also found little loss of DEHP over a 15-day period when they applied it (as a mixture with DBP) to Achillea at a concentration of 3.5 µg/cm2 or to Sinapis at 3.1 µg/cm2. Residues ranged from 120 to 155 µg/plant, and approximately 80% of the DEHP accumulated was on the surface of the leaf. When Schmitzer et al. (1988) grew barley from seed in soil containing 1 or 3.33 mg 14C-DEHP/kg dry soil for 7 days, only 0.61% and 1.25%, respectively, of the applied 14C was taken up by the plants. Aranda et al. (1989) also found low accumulation of DEHP by plants grown in sewage sludge containing DEHP. Lettuce (Lactuca sativa), carrot (Daucus carota), chile pepper (Capiscum annuum), and tall fescue (Festuca arundinaca) were all grown in sludge containing 14C-DEHP levels of between 2.57 and 14.07 mg/kg. Bioconcentration factors ranged from 0.06 to 0.53 (based on initial soil concentration and plant dry weight). 184.108.40.206 Birds Belisle et al. (1975) fed mallard ducks (Anas platyrhynchos) on a diet containing 10 mg DEHP/kg for a period of 5 months. No DEHP was detected in fat tissue, but 0.1 and 0.15 mg/kg (wet weight) were found in breast muscle and lung tissue, respectively. O'Shea & Stafford (1980) exposed starlings (Sturnus vulgaris) to a dietary DEHP concentration of 25 or 250 mg/kg for 30 days. One of eight birds fed 25 mg/kg contained detectable residues (1.6 mg/kg) after 30 days exposure, and five of eight birds fed 250 mg/kg contained an average of 1.8 mg/kg. The same proportion of birds fed the higher dose still had detectable residues (an average of 1.3 mg/kg) 14 days after dosing had finished. When Ishida et al. (1982) fed hens on a diet containing 5 or 10 g/kg for up to 230 days, DEHP was detected in all tissue monitored except the brain. Residues ranged from non-detectable to 42.5 mg/kg for most tissues. However, adipose tissue (192.7 to 899.6 mg/kg) and feathers (513.1 to 1165.2 mg/kg) accumulated the highest concentrations. A similar pattern of uptake was observed in hens fed 2 g/kg for 25 days, although the amount of DEHP accumulated was much lower. At this dose level no accumulation had occurred in tissues other than liver and feather within 5 days. 5. ENVIRONMENTAL LEVELS AND EXPOSURE 5.1 Environmental levels DEHP exists widely in the environment and is found in most samples, including air, precipitation, water, sediment, soil, and biota. Residues have also been detected in food and in humans. In many cases it is not clear whether the phthalate measured in samples is naturally occurring or is exogenous. However, there seem to be clear indications that high levels of DEHP are anthropogenic in origin. 5.1.1 Air The levels of DEHP in air have been monitored in the North Atlantic, the Gulf of Mexico, and on Enewetak Atoll in the North Pacific and found to range from not detectable to 4.1 ng/m3 (Giam et al., 1978; Giam et al., 1980; Atlas & Giam 1981). Similar levels (between 0.5 and 5 ng/m3) have been found in the Great Lakes ecosystem (Eisenreich et al., 1981) and in the Swedish atmosphere (Thurén & Larsson, 1990). The DEHP content of these samples was at least an order of magnitude lower than those found in urban areas such as New York city, where levels of up to 28.6 ng/m3 have been found (Bove et al., 1978). Based on the analysis of snow samples, Lokke & Rasmussen (1983) calculated DEHP concentrations in air of 22 ng/m3 at Lyngby, Denmark. Levels of 29-132 ng/m3 have been found in Antwerp, Belgium (Cautreels et al., 1977), 126 ng/m3 in polluted air in Belgium (Cautreels & Van Cauwenberghe, 1978), 300 ng/m3 in polluted air in Canada (Thomas, 1973), and 38-790 ng/m3 in Japan in 1985 (Environment Agency of Japan, 1989). 5.1.2 Precipitation Atlas & Giam (1981) measured levels of DEHP in rainfall at Enewetak Atoll, North Pacific, of 5.3-213 ng/litre (mean, 55 ng/litre). Eisenreich et al. (1981) reported between 4 and 10 ng/litre in precipitation falling on the Great Lakes ecosystem and Thurén & Larsson (1990) a level of 55 ng/litre in Sweden. Goto (1979) found a range of mean rainwater concentrations of 0.65 to 3.16 µg/litre in various Japanese cities. Lokke & Rasmussen (1983) analysed snow sampled near a plasticizer production plant 14 days after a snowfall. Levels of DEHP ranged from 0.7 to 4.7 µg/m2 per day over this period, the highest levels being within 150 m of the plant and the lowest levels at least 600 m away. 5.1.3 Water Levels of DEHP found in water are summarized in Table 4. Table 4. Concentrations of DEHP in water Location Country Year Concentration Reference µg/litrea Marine Northern Atlantic 0.0001-0.006 Giam et al. (1978) Gulf of Mexico 0.006-0.316 Giam et al. (1978) Estuaries Germany ND-0.3 Weber & Ernst (1983) Nueces Estuary, Texas USA 1980 0.2-0.77 Ray et al. (1983b) Estuaries United Kingdom 1981 0.058-0.078 Waldock (1983) Freshwater Various rivers Japan ND-3.1 Kodama et al. (1975) Various cities Japan 1974 0.1-2.19 Goto (1979) River Meuse Netherlands 1983 < 0.1-3.5 Wams (1987) River Rhine Netherlands 1983 ND-1.2 Wams (1987) River Rhine Netherlands 1982 ND-4.0 Wams (1987) a ND = not detectable Thuren (1986) analysed water samples from the Rivers Ronnebyan and Svartan, Sweden, and found DEHP concentrations ranging from 0.32 to 3.1 µg/litre and from 0.39 to 1.98 µg/litre, respectively. The highest concentrations were associated with industrial effluent discharge points. Few samples of ground water have been analysed for DEHP. Wams (1987) reported that contaminated ground water in the Netherlands contained between 20 and 45 µg/litre, while Rao et al. (1985) found DEHP levels of up to 170 µg/litre in New York state ground water. In a non-industrialised estuary in the United Kingdom, Waldock (1983) measured DEHP levels of 58 to 78 ng/litre. Ray et al. (1983b) found levels of DEHP ranging from 210 to 770 ng/litre in a Nueces estuary in Texas, USA, while Weber & Ernst (1983) found DEHP levels of up to 300 ng/litre in German estuaries. DEHP levels of up to 316 ng/litre have been found in the Gulf of Mexico, but levels in the North Atlantic were much lower (Giam et al., 1978). In Japan, river and marine levels in 1982 ranged from 0.1 to 0.8 µg/litre (Environment Agency of Japan, 1989). Ritsema et al. (1989) analysed samples from Lake Yssel, Netherlands, and found DEHP levels of < 0.1 to 0.3 µg/litre in water and levels of 12-25 mg/kg in suspended particulate matter. The authors concluded that the probable source of DEHP was the River Yssel. Preston & Al-Omran (1986) sampled water and suspended particulates from the Mersey estuary, United Kingdom, in 1985, and reported DEHP concentrations ranging from 83 to 335 ng/litre in water and from 182 to 1700 µg/kg in particulate matter. However, in 1986, levels were 125-693 ng/litre in water and 280-640 µg/kg in particulate matter (Preston & Al-Omran, 1989). 5.1.4 Sediment DEHP levels in sediment are summarized in Table 5. Being lipophilic DEHP tends to be adsorbed onto sediment, which acts as a sink. Sediment samples from various Dutch rivers have been found to contain between 1 and 70 mg/kg (Schwartz et al., 1979; Wams, 1987). Taylor et al. (1981) analysed sediment samples from the Mississippi River and found similar levels. Sediment levels of DEHP in the Chester River Maryland, USA, were found to be less than 45 µg/kg dry weight, but, in a tributary of this river, sediment levels were up to 4.8 mg/kg about 2 km downstream from a phthalate ester plant outfall. The Chester River flows into Chesapeake Bay, which contained sediment DEHP levels of 110 µg/kg (Peterson & Freeman 1984). In Sweden, Thuren (1986) found sediment DEHP levels ranging from 1.2 to 628 mg/kg (dry weight) in the River Ronnebyan and 0.15 to 1480 mg/kg in the River Svarten. As was found with water samples (see section 5.1.3), in both rivers the highest residues of DEHP were near to industrial effluent discharge points. Giam et al. (1978) analysed sediment from the Mississippi delta and reported mean DEHP levels of 69 µg/kg. Sediment samples from Nueces Estuary, Texas, USA, unlike the water samples, reflected local inputs of pollutants. The highest levels (up to 16 mg/kg) were associated with industrial areas, whereas other areas of the estuary contained levels ranging from 40 to 330 µg/kg. Much lower levels of DEHP were found on the Gulf of Mexico coast and in the open sea (mean levels of 6.6 and 2 µg/kg, respectively) (Giam et al., 1978). Table 5. Concentrations of DEHP in sediment Location Country Year Concentration Reference (µg/kg)a Gulf of Mexico < 0.1-248 ns Giam et al. (1978) Nueces Estuary, Texas USA 1980 40-16 000 dw Ray et al. (1983b) Portland, Maine USA 1980 60-7800 dw Ray et al. (1983a) Chester River, Maryland USA 1978 20-4800 dw Peterson & Freeman (1984) River Mississippi USA 1981 140 dw Taylor et al. (1981) River Meuse Netherlands 1977 1000-17 000 dw Schwartz et al. (1979) River Ijssel Netherlands 1977 2500-52 500 dw Schwartz et al. (1979) River Rhine Netherlands 1978 4000-36 000 ns Wams (1987) River Rhine Netherlands 1977 6500-70 500 dw Schwartz et al. (1979) Various cities Japan 1974 80-1360 dw Goto (1979) Crouch Estuary, Essex United Kingdom 1981 11.2-26.2 ww Waldock (1983) River Usk United Kingdom 1974 30 000 dw Eglinton et al. (1975) a dw = dry weight; ww = wet weight; ns = not stated whether concentrations refer to dry or wet weight In Japan, the levels of sediments in rivers and seas in 1982 ranged from 9 to 35 000 µg/kg dry weight (Environment Agency of Japan, 1989). 5.1.5 Soil Contaminated soil analysed in the Netherlands was found to contain up to 1.5 mg DEHP/kg (Wams 1987), while residues of DEHP in soil collected in the vicinity of a DEHP manufacturing plant contained up to 0.5 mg/kg (Persson et al., 1978). Fatoki & Vernon (1990) reported a DEHP concentration of 1.9 µg/litre in treated sewage effluent from the Manchester area, United Kingdom, and stated that such a level was consistent with the industrial activities of the city. 5.1.6 Food DEHP has been found in many samples of fish and shellfish (see Table 6). It has also been detected in milk (Cerbulis & Ard, 1967), bovine pineal gland (Taborsky, 1967), bovine heart muscle (Nazir et al., 1971), and chicken eggs (Ishida et al., 1982). Perkins (1967) isolated a substance similar to DEHP from corn oil. The US ATSDR (1988) quoted an US FDA survey of various foods in 1974 which showed that DEHP levels in most foods were less than 1 mg/kg; the foods surveyed included margarine, cheese, meat, cereal, eggs, milk, white bread, canned corn, corn meal, and baked beans. Ishida et al. (1981) collected and analysed chicken eggs available in Japanese markets. DEHP levels in egg white ranged from 0.05 to 0.4 mg/kg, but no DEHP was detected in the egg yolk. In a study by Antonyuk (1975), DEHP migration from PVC materials into foodstuffs was noted following 7 days of contact. Levels of 4-16 mg DEHP/kg were detected in cheese, sausage, meat, flour, and rice while after 30 days levels of 30-150 mg/kg were found in sunflower oil. The permissible level of DEHP migration into foodstuffs was considered to be 2.0 mg/kg. Zitko (1972) detected DEHP concentrations in hatchery-reared juvenile Atlantic salmon of 13 to 16 mg/kg lipid; fish food contained 8 to 9 mg/kg lipid. When Williams (1973) analysed fish available to the Canadian consumer, only 6 out of 21 samples contained measurable amounts of DEHP. Levels of 0.1 and up to 0.16 mg/kg were found in unprocessed eels and in processed canned tuna fish, respectively. Table 6. Concentrations of DEHP in biota Organisms Location Country Concentration Reference (µg/kg)a Mollusc (digestive gland) Crouch Estuary, Essex United Kingdom 9.2-214 ww Waldock (1983) Dragonfly naiads Iowa (industrial) USA 200 Mayer et al. (1972) Commercial fish food North America 2000-7000 Mayer et al. (1972) Channel catfish Mississippi & Arkansas USA 3200 Mayer et al. (1972) (industrial) Channel catfish Iowa (industrial) USA 400 Mayer et al. (1972) Various fish species Japan 70-450 Kodama & Takai (1974) Various fish species Japan < 50-1800 ww Kamata et al. (1978) Various fish species various cities Japan 50-720 Goto (1979) Mainly fish Gulf of Mexico < 1-135 Giam et al. (1978) Various fish species (liver) Tees Bay United Kingdom 43-85.9 ww Waldock (1983) Various fish species (muscle) Tees Bay United Kingdom 13-51.3 ww Waldock (1983) Walleye Lake Superior, Ontario Canada 800 Mayer et al. (1972) Tadpoles Iowa (industrial) USA 300 Mayer et al. (1972) Common seal pup (blubber) 10 600 lw Zitko (1972) a ww = wet weight; lw = lipid weight 5.1.7 Aquatic organisms Ray et al. (1983a) found DEHP levels of up to 490 µg/kg in sandworms (Neanthes virens) and up to 170 µg/kg in clams collected from Portland, Maine, USA, but these levels did not seem to reflect the sediment levels and local pollutant sources. Musial & Uthe (1980) collected fish of various species from the Gulf of St Lawrence, Canada, and analysed them for DEHP in lipid extracts. They reported levels of up to 6.5 mg/kg on a wet weight basis (51.3 mg/kg on fat weight basis) in mackerel muscle and up to 7.2 mg/kg (47.1 mg/kg fat weight) in herring muscle. Lower levels of 0.37 mg/kg (wet weight) were found in eels, and in both plaice and redfish concentrations were less than 0.001 mg/kg. Persson et al. (1978) collected aquatic organisms from the vicinity of a DEHP factory in Finland. Invertebrates contained up to 0.1 mg DEHP/kg, and levels of 1.1 mg/kg in roach muscle and 2.3 mg/kg in pike liver were measured. Thuren (1986) analysed biota near to an industrial discharge point and reported levels of up to 5.3 mg/kg (fresh weight) in Odonata sp and up to 14.4 mg/kg in Asellus aquaticus. In Japan, DEHP levels in various species of fish in rivers and seas ranged from 0.01 to 19 mg/kg wet weight in 1974 (Environment Agency of Japan, 1989). 5.1.8 Terrestrial organisms Persson et al. (1978) analysed soil arthropods collected near a DEHP factory in Finland and found residues of 2.8 mg/kg. 5.2 General population exposure Few data are available on general population exposure. Based on an analysis of DEHP levels in various foods, the average exposure in the USA has been estimated to be around 0.3 mg/person per day and the maximum 2 mg/person per day (see section 5.1.6). In a survey of plasticizer levels in food-contact material and food, the United Kingdom Ministry of Agriculture, Fisheries and Food (MAFF, 1987) stated that DEHP has very limited use in food-contact material, and the maximum daily intake from food sources has been estimated to be less than 20 µg/person per day. DEHP found in human tissues may be derived from medical devices, since it was recognised by Trimble et al. (1966) and by Guess & Haberman (1968) that DEHP is leached from certain medical devices. Samples of soft PVC fluid bags containing normal saline and glucose (50 mg/ml) were shaken for 24 h and analysed for plastic additives (Smistad et al., 1989). The PVC plastic materials contained DEHP, epoxidized vegetable oils, and stearates as the main additives. The same components were found in the solutions. MEHP was also detected, but only in the solutions. Marcel & Noel (1970) reported the presence of phthalate esters in human plasma that had been stored in plastic blood bags. Jaeger & Rubin (1972) found that DEHP was extracted from PVC plastic blood bags by human blood at the rate of 2.5 mg/litre per day at 4 °C. The DEHP was found in both lipid-containing and lipid-free fractions of plasma, whereas the red cells contained only minor amounts. Seven out of twelve samples of lung tissue, taken at autopsy from patients who had received transfusions of stored blood, contained DEHP at concentrations of 13.4-91.5 mg/kg (dry weight). Rubin & Nair (1972) reported that DEHP had also been found in the tissues and urine of patients who had not received blood transfusions, but no details were given. Mes et al. (1974) analysed human adipose tissue in Canada and found the levels of DEHP in most samples to range from 0.3 to 1.0 mg/kg. Schneider et al. (1989) estimated that the highest exposure to DEHP associated with medical devices resulted from extracorporeal membrane oxygenation (ECMO), which could result in an exposure of 14 mg/kg per day. In an infant receiving ECMO for 14 and 24 days, the DEHP serum levels were 26.8 and 33.5 mg/litre, respectively. In another infant, ECMO for 6 days resulted in liver, heart, and testicular concentrations of 3.5, 1.0, and 0.4 mg/kg, respectively. Newborn infants given exchange transfusions may have plasma levels of about 10 mg/litre (Sjöberg et al., 1985b). 5.3 Occupational exposure during manufacture, formulation or use Few data on occupational exposure to DEHP have been reported. In a phthalate manufacturing plant in the USA producing DEHP from phthalic anhydride and alcohols, Liss et al. (1985) measured, in the case of six heavily exposed workers, 8-h TWA workplace air concentrations of DEHP ranging from 0.02 to 4.1 mg/m3. The exposure level for 44 other workers in the same plant was below the detection limit (10 µg/sample). In an Italian factory producing n-butyl phthalate, isobutyl phthalate, and DEHP, Gilioli et al. (1978) measured total phthalate exposure concentrations of between 1 and 60 mg/m3, the average being 5 mg/m3. Nielsen et al. (1985) measured total phthalic acid esters in air in a PVC-processing plant in Sweden where diisodecyl phthalate, DEHP, and some butylbenzylphthalate were used. Concentrations of between 0.01 and 2.0 mg/m3 were recorded in 96 2-h personal samples from 54 workers. Total phthalates concentrations in air of between 1.7 and 66 mg/m3 were recorded in a PVC-processing plant in the USSR using mainly dibutyl phthalate and higher alkyl phthalates but also some DEHP and other phthalates (Milkov et al., 1973). Stankevich & Zarembo (1978) measured DEHP levels of 1.5 to 40 mg/litre in the blood of workers manufacturing PVC in the USSR. DEHP concentrations in air of between 0.09 and 0.16 mg/m3 were recorded in a German factory for phthalate production (Thiess et al., 1978a). In 9 PVC-processing plants in Finland, the mean concentration of DEHP ranged from less than 0.02 mg/m3 to 0.5 mg/m3 and the highest single value was 1.1 mg/m3 (Vainiotalo & Pfäffli, 1990). 6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS 6.1 Absorption 6.1.1 Inhalation There is no quantitative information available on the pulmonary route of absorption, although aerosols of DEHP are readily formed (Albro & Lavenhar, 1989). 6.1.2 Dermal After a single application of 14C-labelled DEHP (61.5 mg/kg) to the back of F-344 rats, clipped one hour before treatment, urine and faeces were collected every 24 h for 7 days. The amount of 14C excreted was taken as an index of the percutaneous absorption, and about 5% of the dose given was excreted (Elsisi et al., 1989). 6.1.3 Oral Studies with 14C-labelled DEHP indicated that at least 50% of the radioactivity of a single dose (2.9 mg/kg) was absorbed in the rat intestine since 42% and 14% were excreted in urine and bile, respectively, after 7 days (Daniel & Bratt, 1974). The same authors also found that DEHP was rapidly hydrolysed by pancreatic lipase, suggesting that DEHP is hydrolysed in the gut before absorption. This was supported by the fact that no unmetabolized DEHP was found in liver after the administration of low oral doses (< 0.4 g/kg), although at higher doses (> 0.5 g/kg) DEHP was detected (Albro et al., 1982). At an oral dose level of 2 g/kg, the bioavailability of DEHP in rats, as measured in blood by HPLC, was 14%, whereas at an intraperitoneal dose level of 4 g/kg only 5% was recovered, again indicating a role for hydrolysis of DEHP in the gut (Pollack et al., 1985b). Using an inhibitor of mucosal esterases ( S,S,S-tributyl phosphorothionate), White et al. (1980) observed a marked inhibition in the uptake of DEHP by the gut. Studies involving oral administration of MEHP indicated that this metabolite is well absorbed. After radiolabelled MEHP or DEHP was given to rats, the radioactivity recovered in plasma from MEHP was 16 times more than that from DEHP (Teirlynck & Belpaire, 1985). Oral administration of DEHP (1 g/kg) to young rats leads to a larger area under the plasma concentration-time curve (measured with gas chromatography) for MEHP (twice that of DEHP) than in older rats (Sjöberg et al., 1985a). This indicates either a more rapid hydrolysis of DEHP or a more efficient absorption of MEHP in young rats. Cynomolgus monkeys hydrolyse DEHP in the gut less efficiently than rats or mice (Astill, 1989). Rhodes et al. (1986) also noted that there is less absorption from the gastrointestinal tract in marmosets than in rodents. 6.1.4 Intraperitoneal The systemic availability of DEHP was only 5% when a dose of 4 g/kg was given intraperitoneally to rats. Relatively small amounts of MEHP were recovered in the blood in this study (Pollack et al., 1985b). 6.2 Distribution Intravenously administered DEHP is rapidly eliminated from blood. This was demonstrated in experiments where radioactive DEHP was injected into male CFN rats and blood levels were determined by thin- layer chromatography (Schulz & Rubin, 1973). At a low dose level (0.1 mg/kg), there was an initial phase with a half-time of 4.5 min and a second phase with a half-time of 22 min. At a higher dose level (200 mg/kg) the initial phase had a half-time of 9 min. This indicated that DEHP was taken up in a tissue compartment by a saturable process (Schulz & Rubin, 1973). Radioactivity from DEHP was rapidly distributed to the liver, lungs, and spleen when administered intravenously (Schulz & Rubin, 1973; Daniel & Bratt, 1974). Orally administered DEHP is mainly distributed as MEHP in rats (Pollack et al., 1985b; Teirlynck & Belpaire, 1985). Unmetabolized DEHP was recovered in the liver only after large oral doses (> 0.5 g/kg) were given, indicating a threshold phenomenon in the absorption and distribution (Albro et al., 1982; Agarwal, 1986). The distribution kinetics of MEHP have been analysed by Pollack et al. (1985b) and by Teirlynck & Belpaire (1985). Pollack et al. (1985b) found that the peak concentration of MEHP in blood was reached 15 min after oral or intraperitoneal administration of MEHP. The half-time of MEHP in blood or plasma in the rat is shorter than that of DEHP (Pollack et al., 1985b; Teirlynck & Belpaire, 1985). The in vitro plasma protein binding of MEHP in the rat reaches approximately 98% (Sjöberg et al., 1985a). A phenomenon known as "shock-lung" has been reported to occur following intravenous administration of DEHP to rats and other species. Two hours after an emulsion of DEHP was given, between 13% and 48.6% of DEHP-radiolabelled material was found in the lungs of rats, as compared to 26.3-38.2% in the liver (Daniel & Bratt, 1974). This phenomenon may be relevant to human exposure via intravenous administration from bags and tubing containing DEHP. The DEHP plasma level in newborn infants given exchange transfusions may reach about 10 mg/litre (Sjöberg et al., 1985b). This level is about twice as high as those found in leukaemia patients receiving platelet concentrations and about five times as high as levels found in haemodialysed patients. After treatment, this level falls rapidly to about 3 mg/litre within 2 h, and then there is a further drop with a half-time of about 10-12 h (Sjöberg et al., 1985b). 6.3 Metabolism DEHP is hydrolysed in vitro by pancreatic lipase to MEHP (Daniel & Bratt, 1974), indicating that this metabolism would occur mainly in the gut lumen. In rats about 80% of an oral dose of DEHP undergoes mono-deesterification (Pollack et al., 1985b), while intra- arterially administered DEHP is only slowly converted to MEHP (Pollack et al., 1985b). Studies on the hydrolysis of DEHP in homogenates from different organs (Table 7) indicate a very high activity in pancreatic juice and a comparatively low activity in liver (Albro & Thomas, 1973; Daniel & Bratt, 1974). At relatively high doses of DEHP (2 g/kg body weight per day), administered by gavage for up to 14 days, approximately 25-40% of the dose in rats and 50-75% of the dose in marmosets was excreted in the faeces. This implies that incomplete intestinal hydrolysis and absorption may occur (Rhodes et al., 1986). MEHP may in turn be metabolized in the gut wall (Pollack et al., 1985b) or in other organs. Rat liver cell cultures have been shown to convert MEHP to several metabolites, as occurs in the intact rat (Albro et al., 1973; Lhuguenot et al., 1985). Cultures of testicular cells were, however, apparently not able to metabolize MEHP beyond slight hydrolysis to phthalic acid within 18-24 h (Albro et al., 1989). The metabolic pathways for MEHP are shown in Fig. 1. The omega- and omega-1-carbon oxidation products constitute more than 85% of the metabolites (Albro et al., 1973; Mitchell et al., 1985a; Lhuguenot et al., 1985). The ethyl side chain may also be oxidized (Lhuguenot et al., 1985). It has been suggested that omega-oxidation leads to a metabolite that is further degraded by ß-oxidation in the peroxisomes (Albro et al., 1973; Lhuguenot et al., 1985). Non-linear dose- dependency has been reported for these pathways in the rat; the predominance of omega-oxidation over omega-1-oxidation was increased by high doses of MEHP (Lhuguenot et al., 1985). The administration of 2-ethyl-(1-14C)-hexyl-labelled DEHP led to a low level of radioactivity being recovered from purified rat liver DNA (Albro et al., 1982). In a more recent study (Lutz, 1986), the administration of 14C-carboxylate-labelled DEHP resulted in no measurable radioactivity in DNA, whereas radioactivity was clearly measurable after the administration of DEHP that was 14C- or 3H-labelled in the alcohol moiety. Table 7. DEHP hydrolase activity of various tissue lipasesa DEHP hydrolase activity Enzyme preparation units/mg protein units/g tissue Liver acid lipase, homogenate 0.34 101 Liver alkaline lipase, homogenate 0.14 43 Liver "lysosomal" concentrate (acacia)b 8.80 - Liver microsome + supernatant fraction, pH 8.2 1.22 - Kidney acid lipase, homogenate 0.15 45 Lung homogenate (cholate)b 0.072 15 Lung homogenate (acacia)b 0.10 21 Mucosal homogenate, pH 7.4 0.86 83 Muscosal homogenate, pH 9.0 0.43 41 Pancreas homogenate 54.9 34 400 Adipose "monoglyceride lipase" 0.021 0.53 Adipose "hormone-sensitive lipase" 0.14 0.82 Purified cholesteryl esterase 0 - a From: Albro & Thomas (1973) b 14C-labelled DEHP supplied as dispersion in either sodium cholate or gum acacia There are marked species differences in the metabolism of DEHP. Thus omega-oxidation seems to play a dominante role in the rat and guinea-pig (Albro et al., 1982; Lhuguenot & Elcombe, 1984; Lhuguenot et al., 1985), but to be a minor pathway in the mouse, hamster, green monkey, cynomolgus monkey, and marmoset (Albro et al., 1982; Lhguenot & Elcombe, 1984). In guinea-pigs there are few omega-1 metabolites of MEHP (Albro et al., 1982). This may have toxicological significance because certain omega-1-oxidation metabolites have been identified as active agents in peroxisomal proliferation in rat hepatocytes (Mitchell et al., 1985a). No conjugated metabolites were detected in the urine of DEHP-treated rats, but a minor portion was conjugated in the urine of hamsters (Albro et al., 1982; Lhuguenot & Elcombe, 1984). A major portion of glucuronide conjugates was found in the urine of the marmoset, mouse, guinea-pig, and green monkey, and in human urine (Albro et al., 1982). Albro (1986) reported that glucuronidation of DEHP metabolites was insignificant in rats. Studies in primates, including the African green monkey (Albro et al., 1981), marmoset (Rhodes et al., 1986), cynomolgus monkey (Short et al., 1987; Astill, 1989), and man (Schmid & Schlatter, 1985), demonstrated that conjugation of DEHP can occur at the carboxylic acid moiety following a single ester hydrolysis. The level of unmetabolized MEHP excreted in urine also varies considerably between species; it is low in the rat and hamster, but high in the mouse, guinea-pig, green monkey, and man (Albro et al., 1982). Repeated oral administration of DEHP or MEHP at high doses (500 mg/kg) to rats leads to a change in the metabolic profile; there is an increase in omega-oxidized metabolites and a decrease in omega-1-oxidized metabolites (Lhuguenot et al., 1985). In rats given 2% DEHP in the diet for one week, a 4-fold increase in peroxisomal ß- oxidation was found. ß-Oxidation of fatty acids induced by DEHP appears to occur via mitochondrial and peroxisomal pathways that are similar to normal pathways (Ganning et al., 1989). Drug-metabolizing enzyme activities have been studied after DEHP administration, and in some cases changes were observed (Walseth et al., 1982; Agarwal et al., 1982; Gollamudi et al., 1985; Pollack et al., 1989). The same metabolites as those found in rat urine can be detected in human urine. One study on intravenously injected DEHP (Albro et al., 1982) and one on orally administered DEHP (Schmid & Schlatter, 1985) indicated that humans metabolize DEHP by omega- and omega-1-oxidation as well as by oxidation of the ethyl side chain. However, the omega-oxidation-pathway seems to be a minor pathway in man (Albro et al., 1982; Schmid & Schlatter, 1985). More than half of the metabolites recovered in human urine are conjugated metabolites (Albro et al., 1982; Schmid & Schlatter, 1985). Time-averaged concentrations of DEHP, MEHP, and phthalic acid in the blood of patients undergoing maintenance haemodialysis were 1.9, 1.3, and 5.2 mg/litre, respectively (Pollack et al., 1985a). Such patients are considered to be at risk of potential DEHP toxicity through prolonged contact with medical plastic products that contain DEHP. The relatively high circulating level of phthalic acid may indicate an altered metabolism of DEHP in uraemic patients (Pollack et al., 1985a). The levels of DEHP and MEHP in plasma have been studied in newborn infants given blood exchange transfusions. In one case the MEHP half-life was the same as for DEHP (about 12 h), indicating that the hydrolysis of DEHP was the rate-limiting metabolic step. However, in other children the half-time of MEHP was longer than that of DEHP (Sjöberg et al., 1985b). 6.4 Elimination and excretion Radioactivity from intravenously injected 14C-labelled DEHP is mainly recovered in urine and faeces after 24 h (Schulz & Rubin, 1973), indicating that urine and bile are major excretory pathways. When a low dose level (0.1 mg/kg) was given to rats, 50-60% of injected radioactivity was recovered in urine and faeces after 24 h, whereas at a high dose level (200 mg/kg) less than 50% was recovered (Schulz & Rubin, 1973). Seven days after an oral dose (2.9 mg/kg) of DEHP was given to rats, 42% of the radioactivity was recovered in the urine and 57% in the faeces (Daniel & Bratt, 1974). Biliary excretion was also measured in these experiments, and it was found that 14% of the radioactivity was recovered in bile after 4 days (Daniel & Bratt, 1974). The almost 100% recovery reported by Daniel & Bratt (1974) has been confirmed by Teirlynck & Belpaire (1985). Oral administration of MEHP (50-500 mg/kg) gave a higher urinary recovery than orally administered DEHP (50-500 mg/kg) as measured after 24 h (Lhuguenot et al., 1985). After the oral administration of non-radioactive DEHP (0.45 mg/kg) to human volunteers, it was found that 15-25% was excreted in urine as MEHP or oxidized metabolites within 2-3 days (Schmid & Schlatter, 1985). In the rat no unmetabolized DEHP is excreted in the urine, but small amounts are found in mouse or green monkey urine (Albro et al., 1982). Major amounts of MEHP are excreted in mouse, guinea-pig, green monkey, and human urine (Albro et al., 1982). However, oxidized metabolites, either free or conjugated, constitute a major portion of excretion products in rat, mouse, hamster, green monkey, and human urine (Albro et al., 1982). Changes in excretion pathways have been observed after prolonged administration of DEHP. After oral dosing of rats without pre-treatment with DEHP, the faecal excretion pathway dominated, while in rats fed with DEHP for 7 days the urinary pathway dominated (Daniel & Bratt, 1974). 6.5 Retention and turnover 6.5.1 Half-life and body burden After the intravenous administration of radiolabelled DEHP, at least two elimination phases of radioactivity, with short half-lives (4.5-9 and 22 min, respectively), were observed in rat blood (Schulz & Rubin, 1973). After 7 weeks of oral administration, the elimination phase in the liver was considerably slower, the half-life being 3-5 days (Daniel & Bratt, 1974). No accumulation of DEHP or MEHP was observed when the dosage was 2.8 g/kg per day for 7 days (Teirlynck & Belpaire, 1985), nor was there any in a long-term (5-7 weeks) feeding study at a dose level of 1 or 5 g/kg diet (corresponding to a daily dose of about 50 and 250 mg/kg body weight) (Daniel & Bratt, 1974). 6.5.2 Indicator media Analysis of the total amount of urinary metabolites, measured as derivatized phthalic acid, indicate a weak positive correlation between occupational exposure to phthalate and the presence of metabolites in the urine (Nielsen et al., 1985; Liss et al., 1985). In the study by Nielsen et al. (1985), workers were exposed mainly to DEHP and diisodecyl phthalate, and the urinary level of phthalate ester metabolites rose from the background level (17 µmol/litre) to 23-25 µmol/litre. In the study by Liss et al. (1985), workers were exposed to DEHP and phthalic anhydride. Urinary phthalate concentrations in exposed workers more than doubled after a workshift and levels up to 44 µmol/litre were recorded. The authors concluded that phthalic anhydride influenced the urinary level more than DEHP. Phthalic acid is not a specific marker for DEHP exposure. 7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS 7.1 Single exposure Numerous LD50 values have been reported for DEHP. Oral LD50 values generally exceed 25 g/kg in rats and 30 g/kg in mice (Stankevich et al., 1984; NIOSH, 1985b; Woodward et al., 1986); in the rabbit it is 33.9 g/kg (Shaffer et al., 1945) and in the guinea-pig 26.3 g/kg (Krauskopf, 1973). Dermal LD50 values for guinea-pigs and rabbits of 10 g/kg and 25 g/kg, respectively, have been reported (NIOSH, 1985b). The LD50 values after intraperitoneal administration were 30.7 g/kg in rats (Shaffer et al., 1945) and 14-75 g/kg in mice (Lawrence et al., 1975; Woodward et al., 1986). LD50 values in the range of 200-250 mg/kg were reported for the rat after intravenous administration of DEHP solubilized with a nonionic detergent (Schmidt et al., 1975; Rubin & Chang, 1978). The main symptom of DEHP toxicity after single oral or intraperitoneal dosing is diarrhoea (Hodge, 1943). An intraperitoneal dose of 500 mg/kg in rats decreased spontaneous running activity, thereby indicating behavioural changes (Rubin & Jaeger, 1973). After intravenous dosing, lung lesions including oedema, haemorrhage, and infiltrations of polymorphonuclear leucocytes were observed in rats at doses as low as 50 mg/kg (Schulz et al., 1975). The etiology of the lung lesions is unknown. It has, however, been suggested that some changes could be due to the release of lysosomal enzymes from alveolar macrophages, which was found to occur in vitro in rabbit alveolar macrophages cultured with DEHP (Bally et al., 1980). Rabbits treated intravenously with 350 mg DEHP/kg showed a decrease in blood pressure and an increase in breathing rate. No deaths occurred after doses up to 650 mg/kg were administered (Calley et al., 1966). The monoester, MEHP, may be more toxic than the diester but data are very limited. In a short note by Villeneuve et al. (1978), the oral LD50 of MEHP was reported to be 1.34 g/kg in female rats and 1.8 g/kg in males. 7.2 Short-term exposure Doses of 3.4 g/kg body weight per day given by gavage (in olive oil) for periods of up to 90 days caused the death of 15 out 20 rats (Nikonorow et al., 1973). However, no deaths were reported among rats fed 3% DEHP in the diet (1.9 g/kg body weight) for 90 days (Shaffer et al., 1945) or in a rat study of the US National Toxicology Program after dietary dosing (< 50 g/kg) for 14 days (NTP, 1982). Oral administration of DEHP at a rate of > 0.4 g/kg body weight per day resulted in a weight gain decrease in rats within a few days (Nikonorow et al., 1973). In a 17-week feeding study where rats were given 2, 10 or 20 g DEHP/kg diet, a decreased body weight was observed (Gray et al., 1977). Reduction in body weight was also observed in rats given dietary levels of 12.5 or 25 g/kg for 13 weeks. Dosages of 1.6-6.3 g/kg resulted in either slight elevations of body weight or no effect (NTP, 1982). MEHP given at a level of 6.4 g/kg diet caused reduction in the body weight gain of rats (Chu et al., 1981). No effects on body weight occurred at dietary levels of 0.625 g/kg given for 3 months, but a significant decrease in blood glucose was observed. Reductions in haemoglobin, packed cell volume and erythrocyte numbers were observed in rats given 10 or 20 g DEHP/kg diet for 17 weeks, but not when they were given 2 g/kg for the same period (Gray et al., 1977). Cystic kidneys and centrilobular necrosis were noted in one strain of mice (ddY) fed 2.5 or 25 g DEHP/kg for 2 weeks, but not in another strain (B6C3F1), even with a higher exposure level and a longer exposure period (Woodward et al., 1986). DEHP administered intravenously at a rate of 25-500 mg/kg per day for 2-4 weeks to beagle dogs resulted in pulmonary haemorrhage and inflammatory response similar in appearance to the "shock-lung" effect (Woodward et al., 1986). In an inhalation study, Wistar rats were exposed in a head-nose inhalation system to DEHP aerosols of respirable particle size. Exposure duration was 6 h/day, 5 days/week for 4 weeks at target concentrations of 0, 0.01, 0.05, and 1.0 mg/litre. A statistically significant increase in relative lung weights was found in the males given the highest dosage, and this was accompanied by foam cell proliferation and thickening of the alveolar septa (Klimisch et al., 1991). Another inhalation study has been reported, but this is inadequate for assessment (Timofievskaja et al., 1980). A discussion of the effects of DEHP on the liver is given in section 7.9. When rats were treated with DEHP by intraperitoneal injection (Walseth et al., 1982) or by repeated oral dosing (Agarwal et al., 1982), an increase in cytochrome P-450 levels was observed. The increase in hepatic microsomal oxidation appears to be primarily due to the deesterification products of DEHP, i.e. MEHP and 2-ethoxyhexanol, in long-term exposure, whereas DEHP and its two metabolites may inhibit microsomal oxidation after acute exposure to DEHP (Pollack et al., 1989). However, in vitro rat liver microsomal cytochrome P-450 levels were not affected by DEHP (Gollamudi et al., 1985). Liver mitochondrial enzymes and mitochondrial morphology have been reported to be influenced by DEHP administration (Ohyama, 1977; Shindo et al., 1978). Recent results suggest that the in vitro effects of DEHP (> 20 µmol/litre) on mitochondrial functions are mainly related to the action on membrane lipids surrounding the adenine nucleotide translocator, which reduces the rate of adenine nucleotide exchange across the mitochondrial membrane (Kora et al., 1989). In male rats given DEHP in the diet, the urinary excretion of zinc was enhanced and the testicular level of zinc decreased (Gray et al., 1982; Oishi, 1985) (section 7.5). These changes in zinc homeostasis could be due to altered levels of metallothionein. In mice fed 6 or 12 g DEHP/kg diet for 24 weeks, hepatic levels of metallothionein were increased up to 11-fold (Waalkes & Ward, 1989). Several studies on rats have shown that DEHP given in the diet (5-20 g/kg) decreases plasma triglyceride and cholesterol levels (Yanagita et al., 1978; Sakurai et al., 1978; Bell et al., 1978a; Bell et al., 1978b; Bell et al., 1979; Yanagita et al., 1979; Curstedt & Sjövall, 1983). DEHP inhibits the biosynthesis of cholesterol, an effect which is accompanied by phospholipidosis, and the same effects have been observed with MEHP (Oishi & Hiraga, 1982). 7.3 Long-term exposure In a 24-month study by Harris et al. (1956), three groups of Wistar rats each comprising 43 males and 43 females were fed diets containing 0, 1, and 5 g DEHP/kg, interim kills being made at 3, 6, and 12 months. At the end of the study, only two control, four low-dose, and seven high-dose animals were alive. During the first year the body weights of the high-dose group were slightly reduced, but by the second year the body weights of all groups were similar. During the first 6 months an increase in relative liver and kidney weights was seen in DEHP-treated animals but later they were similar in control and treated animals. After 3 months of treatment, one out of eight rats in the low-dose group was found to have mild renal tubular atrophy. After 6, 12, and 24 months of treatment, no compound-related pathological changes were evident. Because of high mortality due to disease, this study is difficult to validate. In another 24-month study (Carpenter et al., 1953), groups of Sherman rats consisting of 32 males and 32 females were given diets containing 0, 0.4, 1.3 or 4 g DEHP/kg. Owing to reduced life expectancy due to disease and the small numbers of animals used, the study was inadequate for assessing the chronic toxicity of DEHP. In a 12-month study by Nikonorow et al. (1973), a group of 20 male and 20 female Wistar rats was given a diet containing 3.5 g DEHP/kg, and a control group received the diet without DEHP. The only gross or micropathological change noted in exposed animals at necropsy was hepatomegaly. During the study, however, about 30% of the animals died due to congestion of the small intestine and loss of the gastric and/or intestinal mucosa, which was complicated by purulent pneumonia and endometritis. Crocker et al. (1988) described the renal effects of DEHP given by gavage to young male rats at a dosage of 2.14 mg/kg body weight three times per week for up to 12 months. A 50% reduction in creatinine clearance and an increase in the severity of renal cyst formation was observed. This lesion was consistent with spontaneous nephropathy commonly observed in old rats; exposure may cause an onset in younger rats. Furthermore, DEHP fed at 6 and 12 g/kg diet for two years did not produce renal lesions in male and female F-344 rats (NTP, 1982). In a 2-year study (NTP, 1982; Kluwe et al., 1982), groups of F-344 rats were given dietary levels of 0, 6, and 12 g DEHP/kg. Decreased body weight in exposed groups was noted from week 30 until the cessation of exposure. In addition to neoplastic effects (section 7.7) and testicular atrophy (section 7.5), a compound-related hypertrophy of cells in the male anterior pituitary was noted in the high-dose group. In both exposed groups an increased incidence of clear changes in liver cells was observed. This study also investigated B6C3F1 mice exposed to 3 or 6 g DEHP/kg diet. A dose-related decrease of body weight was observed in female mice. There was no increased incidence of non-neoplastic lesions except for seminiferous tubular degeneration in the testes of male mice (section 7.5.1). In a study on groups of male and female guinea-pigs fed diets containing 0, 0.4 or 1.3 g DEHP/kg for 12 months (Carpenter et al., 1953), the body weight of the low-dose animals was statistically higher than that of controls, and liver weight relative to body weight of dosed females slightly increased. No other exposure-related lesions were found. A study on groups of male ferrets fed diets containing 0 and 10 g DEHP/kg for 14 months revealed a reduction in body weight and an increase in relative liver weight, but no evidence of peroxisome proliferation (Lake et al., 1976). 7.4 Skin and eye irritation; sensitization DEHP has been shown to be a weak irritant to mammalian skin when administered topically or intradermally (0.2 ml of an emulsion of 100 g/litre) (Calley et al., 1966; Woodward et al., 1986). In a study by Lawrence et al. (1975), no irritation occurred when undiluted DEHP was instilled into the eye of rabbits. No data are available on the potential for DEHP to induce skin sensitization in animals. 7.5 Reproduction, embryotoxicity, and teratogenicity 7.5.1 Reproduction The effects of DEHP on male reproductive organs have been studied extensively. The majority of the studies have been carried out on rats or mice given DEHP in the diet. Seminiferous tubular atrophy, comprising a loss of spermatids and spermatocytes, occurred when 4-week-old Wistar rats were given 2800 mg DEHP/kg by oral intubation for 10 days (Gray & Butterworth, 1980). In similarly treated 10-week-old rats, about 50% of the tubules were atrophic and the remainder unaffected. However, no testicular damage was detected in treated 15-week-old rats. When 20 g DEHP/kg was given in the diet (approximately 1200 mg DEHP/kg per day) to 4-week-old rats, the lesions produced were reversible whether treatment stopped before or continued until after the control rats had reached sexual maturity. In rats given 10 or 20 g DEHP/kg diet, the testis atrophy was dose dependent after approximately 2 weeks of feeding. This atrophy was accompanied by pituitary changes, i.e. enlargement and vacuolization of the basophils of the pars distalis, corresponding to the formation of the so-called castration cells seen after gonadectomy (Gray et al., 1977). In a subsequent study, there was a reduction in testicular and prostatic zinc levels concomitant with increased urinary excretion of zinc (Gray et al., 1982). In a rat study by Oishi & Hiraga (1980a), the serum testoster-one levels in rats fed 20 g DEHP/kg diet were reduced by approximately 50%. The total amount per testis decreased, but the concentration rose to 150% of the original value because of the testicular atrophy. Simultaneous administration of testosterone or zinc had no protective effect on the atrophy but did prevent the weight reduction of the sex organs such as the epididymis (Gray & Butterworth, 1980; Oishi & Hiraga, 1983). In a further study, it was found that a low-zinc diet aggravated the DEHP-induced testicular atrophy (Agarwal et al., 1986a). DEHP given to 7 young (5 weeks old) Wistar rats in the diet (20 g/kg) for one week decreased the testicular weight significantly (P < 0.05), compared to controls, and also the testicular zinc concentration (Oishi, 1984). In a study by Oishi (1985), groups of 20 male rats were given DEHP (2.0 g/kg per day) by gavage for 14 days. Ten rats were then killed and the remaining ten were kept for 45 days on a DEHP-free diet. The histopathological changes of the testes seen on day 15 were marked shrinkage of the seminiferous tubules, a germinal epithelium consisting only of Sertoli cells, and very few spermatogonia. After 45 days the percentage of spermatogenic tubules had increased from 0 (at day 15) to 12.8%, indicating a limited reversibility of the testicular atrophy. Similar degeneration of the seminiferous tubules was observed when 13-week-old Wistar rats were orally given 2 g DEHP/kg body weight for 7 consecutive days (Saxena et al., 1985) . In a dietary study, DEHP was administered to F-344 rats at 6 and 12 g/kg for 103 weeks (NTP, 1982). Degeneration of the seminiferous tubules was observed only at the higher dose. The testicular changes induced by oral DEHP administration appear to be age dependent. A daily dosage of 2.8 g DEHP/kg body weight given for 10 days caused seminiferous tubular atrophy in 4-week-old Wistar rats, but this effect was less severe in 10-week-old rats and absent in 15-week-old rats (Gray & Butterworth, 1980). Sjöberg et al. (1986) showed that DEHP fed at a level of 1.7 g/kg diet caused reductions in testis weight to 20%, 55%, and 92% of the control values in Sprague-Dawley rats at 25, 40, and 60 days of age, respectively. This was apparently not due to differences in pharmacokinetic parameters, since the plasma levels of the metabolite MEHP were identical in the 25- and 40-day-old rats (Sjöberg et al., 1982). Rats given DEHP intraperitoneally at a dosage of 100 mg/kg per day for 5 days did not develop testicular atrophy (Curto et al., 1982). However, this lack of response was probably a consequence of the dose used, rather than the dose route, and there was a 30% reduction in testicular zinc. Intraperitoneal administration of higher doses (1-25 g/kg per day for 5 days) to Wistar rats decreased serum testosterone levels (Oishi & Hiraga, 1979). Some degenerated primary spermatocytes and altered Sertoli cells were observed in Sprague-Dawley rats given 3-h intravenous infusions of an emulsion at a rate of 1 ml/h, which corresponded to a daily dose of 500 mg DEHP/kg (Sjöberg et al., 1985c). The infusions were given every other day on six occasions. The emulsion contained DEHP, fractionated egg yolk phosphatides, glycerol, and water. No effects were observed when emulsions corresponding to 0, 5 or 50 mg DEHP/kg were given. In a study by Curto & Thomas (1982), groups of sexually mature Swiss-Webster mice were given intraperitoneal injections of 50 or 100 mg DEHP/kg either daily for 5 days or alternate daily for 20 days (10 injections). The animals were killed 24 h after the last injection. No significant alterations in testicular weight or zinc levels occurred. Young rats were not more susceptible to testicular damage than older ones following intravenous infusion of DEHP. It has been suggested that the age-related difference observed in some studies may be due to the fact that the rate of gastrointestinal absorption of the DEHP-derived metabolite MEHP is higher in younger animals than in older ones (Sjöberg et al., 1986). In a NTP-sponsored study (Melnick et al., 1987), CD-1 mice given 3 g DEHP/kg diet showed significantly diminished testis and epididymis weights compared to controls. In addition, the sperm concentration in the cauda epididymis was reduced and the percentage of abnormal sperm in the cauda was significantly higher in the treated mice than in the controls. A high oral dose (4.2 g/kg) of DEHP gave minimal tubular atrophy in hamsters but did not produce any effects on urinary zinc excretion, testicular zinc levels or testicular weights (Gray et al., 1982). The effects of the monoester MEHP have not been as well studied as those of DEHP. An oral dosage of 1 g MEHP/kg per day for 5 days produced a significant decrease in rat testis weight and extensive testicular atrophy (Gray et al., 1982). On the other hand, rats given intraperitoneal doses of up to 100 mg MEHP/kg daily for 5 days showed no abnormal histology (Curto et al., 1982) and an intraperitoneal dose of 50 mg/kg given on alternate days for 20 days produced only a reduction in prostatic zinc levels (Curto & Thomas, 1982). Mice fed diets containing 20 g MEHP/kg for 1 week revealed markedly reduced testicular zinc and testosterone levels, but there were no reductions in testicular weight (Oishi & Hiraga, 1980b). Hamsters given MEHP (1 g/kg per day) for 9 days showed more severe testicular effects than those given DEHP at a level of 4.2 g/kg per day for the same period (Gray et al., 1982). More recent studies (Albro et al., 1989) indicate that testicular atrophy resulting from DEHP exposure is most probably due to the formation of MEHP. These studies indicate that the rat is the species most susceptible to DEHP-induced testicular atrophy. The mechanism of phthalate-induced testicular damage is not fully understood. Testicular zinc depletion has been suggested to be a primary event (Foster et al., 1982, 1983). However, Gray et al. (1982) showed that an effect on zinc level is not always associated with testicular atrophy in all species tested. Zinc is essential for normal testicular function and its depletion is known to lead to testicular atrophy (Barney et al., 1969). Inhibition of dehydrogenase enzymes, e.g., those controlling the biosynthesis of testosterone, leads to reduced testosterone levels. DEHP administration has been shown to reduce the level of serum testosterone in the rat (Oishi & Hiraga, 1979; Oishi & Hiraga, 1980a) as well as in the mouse (Gray et al., 1982), although in the mouse no testicular atrophy was observed. Administration of testosterone or zinc did not prevent the testicular damage induced by DEHP (Gray & Butterworth, 1980; Oishi & Hiraga, 1983). Co-administration of DEHP and testosterone, on the other hand, apparently enhanced the testicular damage caused by DEHP (Oishi, 1989a). In a similar study (Oishi, 1989b), luteinizing hormone-releasing hormone (LRH) significantly decreased testis weight, sulfhydryl content, and lactate dehydrogenase when given together with DEHP, whereas LRH or DEHP alone had no effects. Similar effects were observed with exogenously added follicle stimulating hormone (FSH) in an in vitro study. Primary testicular cell cultures pretreated with MEHP showed a dose-related reduction in FSH-stimulated cyclic adenosine monophosphate (cAMP) production (Lloyd & Foster, 1988). These results suggest that MEHP produces a perturbation at the level of the FSH membrane receptor, causing an inhibition of FSH action. In vitro studies have indicated that the Sertoli cell is the target cell. Mixed cultures of Sertoli and germ cells prepared from rat testes were exposed to DEHP or MEHP (10-7-10-4 mol/litre) (Gray & Beamand, 1984; Gray & Gangolli, 1986). DEHP had no effect, but MEHP caused a dose-dependent increase in the rate of germ cell detachment from Sertoli cells, accompanied by changes in Sertoli cell morphology. More recent data indicate that Sertoli cell mitochondria are a target for MEHP (Chapin et al., 1988). However, it has also been shown that testicular mitochondrial respiratory functions are decreased in rats given 2 g DEHP/kg body weight by gavage (Oishi, 1990). In a study by Melnick et al. (1987), CD-1 mice were given 0, 0.1, 1 or 3 g DEHP/kg diet during a 7-day pre-mating period and a subsequent 98-day cohabitation period. There was complete suppression of fertility in the 3-g/kg group and a significant reduction in fertility in the 1-g/kg group, compared to controls, but no effect on fertility at 0.1 g/kg. In a fertility study designed to investigate earlier significant results (Singh et al., 1972), groups of male ICR mice were dosed subcutaneously with undiluted DEHP at levels of 1, 2, 5, and 10 ml/kg (equivalent to 0.99, 1.97, 4.93, and 9.86 g/kg) on days 1, 5, and 10 of the study (Agarwal et al., 1985a). They were subsequently mated with virgin females on days 2, 6, 11, 16 and 21, and then at weekly intervals until week 8. There were reductions in the incidence of pregnancies in several groups, but these were dose related only at the first mating (i.e. on day 2 of the experiment). Examination of pregnant mice on day 13 of gestation showed that there were pre-implantation losses corresponding to a DEHP dose level of 10 ml/kg and a mating on day 6. There were consistent increases in early fetal deaths relating to matings at day 21, week 4, and week 5. In a similar study, male and female mice were treated subcutaneously with 1-100 ml DEHP/kg, and this led to a reduction in testicular but not ovarian weight. The ovaries exhibited histological injury at lower doses of DEHP than the testes. Unlike the situation in the testes, there was no significant dose-related increase in histopathological changes in the ovaries (Agarwal et al., 1989). In an investigation of the effects of phthalates on female reproductive organs, three doses of 4.93 g DEHP/kg were given intraperitoneally at 5-day intervals to female rats (Seth et al., 1976). No histopathological changes in the ovaries were seen 22 days after the first injection, but reductions in the activities of some enzymes were noted. The administration of DEHP (1000 mg/kg intraperitoneal or 2000 mg/kg oral) to marmosets daily for 14 days did not lead to testicular atrophy (Rhodes et al., 1986). Hence, it appears that rats and guinea-pigs are sensitive to DEHP-induced testicular atrophy, while mice are fairly resistant and hamsters and marmosets are highly resistant. The fact that at least some of the effects associated with this atrophy can be produced in vitro argues against a hormonally mediated indirect effect. The earliest effects are seen in Sertoli cells and are described as vacuolation (Albro, 1987). 7.5.2 Embryotoxicity and teratogenicity In a study by Nikonorow et al. (1973) on female Wistar rats given 0.34 or 1.7 g DEHP/kg by gavage during the first 21 days of gestation, the only untoward effect was a reduction in fetal body weight. When DEHP (0, 5, 10, 15 or 20 g/kg) was administered orally to Fischer-344 rats on gestational days 0 to 20, maternal toxicity and reduced fetal body weight per litter were observed at the three highest dose levels. The number of fetuses per litter was unaffected by the treatment (Tyl et al., 1988). Intraperitoneal injections of 4.93 or 9.86 g DEHP/kg on days 5, 10, and 15 of gestation resulted in an increase in the number of resorptions and reduced fetal weight in Sprague-Dawley rats (Singh et al., 1972). In the highest-dose group, gross abnormalities, such as twisted hind legs and anophthalmia, were noted but no skeletal defects were observed. Rat plasma soluble extracts of two PVC plastics containing DEHP were administered intravenously to groups of pregnant Sprague-Dawley rats daily from the 6th to the 15th day of gestation, and the animals were killed at day 20 (Lewandowski et al., 1980). The daily doses of DEHP were equivalent to 1.3 mg/kg and 5.2 mg/kg. No significant teratogenic or embryotoxic effects were noted. Groups of ICR mice were given DEHP in the diet at levels of 0.5 to 10 g/kg for the first 18 days of gestation and were then sacrificed (Shiota et al., 1980; Shiota & Nishimura, 1982). The food intake was an average of 7 g/day. At the 4 g/kg and 10 g/kg dose levels, no live fetuses were found. At 2 g/kg, 40% of the fetuses had malformations including exencephaly, spina bifida, and malformed tail. Delayed ossification was seen in about 15% of the fetuses at 1 g/kg and 2 g/kg. In a study by Tyl et al. (1988), DEHP was administered in the diet to CD-1 mice on the first 17 days of gestation at levels of 0, 0.25, 0.5, 1, and 1.5 g/kg. At the two highest dose levels maternal toxicity, increased resorptions and late fetal deaths, decreased numbers of live fetuses, and reduced fetal body weight per litter were observed. The number and percentage of malformed fetuses per litter were elevated at the three highest dose levels. A single oral administration of DEHP (0.1 ml/kg) on day 7 of gestation to ddY-strain SPF (specific pathogen free) mice decreased the number and the body weights of live fetuses (Tomita et al., 1982b). In a study by Yagi et al. (1980), DEHP was given orally to SPF mice on days 6, 7, 8, 9 or 10 of gestation. When 5.0 or 10.0 ml/kg was given on day 7 there were no live fetuses, whereas 2.5 ml/kg administered on the same day resulted in 14% live embryos and 1.0 ml/kg gave 40% live embryos. The percentages of live embryos when 10.0 ml/kg was given on day 8, 9 or 10 of gestation were 18, 92, and 95%, respectively. Gross and skeletal abnormalities occurred in fetuses given 2.5 and 7.5 ml/kg on day 7 or 8. The abnormalities included exencephaly, open eyelid, and club-foot. In a study by Shiota & Mima (1985), groups of ICR mice were given DEHP by stomach intubation on days 7, 8, and 9 of gestation. The DEHP doses (in olive oil) were 250, 500, 1000, and 2000 mg/kg, and the mice were sacrificed on day 18. In the two highest dose groups, the numbers of resorptions and malformed fetuses were significantly increased. Fetal weights were significantly depressed. The most frequent malformations were anencephaly and exencephaly. When doses of up to 8000 mg/kg were given by intraperitoneal injections on days 7,8, and 9 of gestation, no effects were noted. DEHP is highly embryotoxic and terato-genic in mice when given orally but not when given intraperitoneally. The monoester MEHP, at oral doses of 225, 450, and 900 mg/kg per day, produced significant signs of maternal toxicity when given to pregnant Wistar rats on days 6-15 of gestation (Ruddick et al., 1981). In the highest dose group a 73% mortality was observed in the dams. There was a dose-related decrease in the number of litters and in the litter weights of live pups. Oral dosing with 0.1 or 1.0 g MEHP/kg on day 7 of gestation led to increased incidence of early embryonic deaths in SPF mice (ddY strain), but dosing on day 8 or 9 had less effect (Yagi et al., 1980; Tomita et al., 1986). The fetuses had reduced body weight, and there was a higher incidence of gross abnormalities, compared to controls, when the higher dose level was given on day 8 or 9. The mice dosed on day 8 produced fetuses with a high incidence of skeletal effects. Intravenous injections of MEHP (11.38 mg/kg) to rabbits on days 6-17 of gestation gave a high incidence of resorptions (Thomas et al., 1979). The incidence of fetal anomalies was similar to that in controls. When Nikonorow et al. (1973) administered DEHP (0.34 or 1.7 g/kg per day) to female Wistar rats by gavage for 3 months prior to mating, there was an increase in the number of resorptions but no effects on fetal weights or the incidence of skeletal anomalies. In utero administration of DEHP (1000 mg/kg body weight) to rats daily from days 6 to 15 of gestation resulted in retardation of fetal growth and an increase in fetal liver weight. There were significant quantities of DEHP and decreased mitochondrial enzyme activities in the fetal liver. These results indicate an effect on fetal liver cell bioenergetics (Srivastava et al., 1989). The mechanism of DEHP or MEHP teratogenicity is not known. Teratogenic activity could result from zinc deficiency, which is known to produce such effects (Swenerton & Hurley, 1971). 7.6 Mutagenicity and related end-points The possible genotoxic effect of DEHP has been thoroughly investigated in several different short-term tests. The effects of the major metabolites of DEHP, i.e. MEHP and 2-ethylhexanol, as well as phthalic acid and phthalic anhydride, have also been studied. 7.6.1 Mutation Studies on the possible mutagenic effect of DEHP have been performed in bacteria, fungi, and in cultured mammalian cells. Drosophila melanogaster has also been used and results from a few in vivo studies on mice have been reported. 220.127.116.11 Bacteria Many studies have been performed using a variety of strains of Salmonella typhimurium and DEHP doses of up to 10 mg/plate. Incubations both with and without exogenous activation systems have been performed. S9 mix from rats induced by Aroclor 1254 has frequently been used, but other species and other inducers have also been used to produce metabolic activation systems. With one exception (Tomita et al., 1982a) these test results have all been negative (Kirby et al., 1983; Yoshikawa et al., 1983; Zeiger et al., 1985; Agarwal et al., 1985b), and in a IPCS collaborative study (Ashby et al., 1985) all five laboratories reported negative results. Bacteria other than S. typhimurium have also been used; negative results were obtained with E. coli WP2 at doses of up to 2 mg per plate (Yoshikawa et al., 1983). The major metabolites of DEHP have also been tested for mutagenic activity in bacteria. Concentrations of up to 3.333 mg per plate for MEHP and phthalic anhydride (Zeiger et al., 1985) and 2 mg/plate for 2-ethylhexanol and phthalic acid (Agarwal et al., 1985b) have yielded negative results in strains of Salmonella (see also Kirby et al., 1983; Yoshikawa et al., 1983). However, Tomita et al. (1982a) reported a significant increase in TA100 revertants following exposure to either DEHP or MEHP (both with and without S9). It should be noted, however, that MEHP mutagenicity is only demonstrable within a narrow range of concentration, because it has a sterilizing effect at high concentration and shows no mutagenic activity at low concentration. Tomita et al. (1982a) also detected dose-dependent (0.4 and 0.5 mg MEHP/plate) DNA damage in a B. subtilis Rec assay, while DEHP, phthalic acid, and 2-ethylhexanol were all negative. In this study MEHP also gave a positive result in E. coli WP2 b/r. Negative results were obtained when pooled urine from rats, treated with 2000 mg DEHP/kg per day for 15 days, was tested for genotoxic activity. A direct plating procedure was used with S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, both with and without S9 and ß-glucuronidase/aryl sulfatase as the activation system. When 2-ethylhexanol was tested according to the same protocol, the result was also negative (Divincenzo et al., 1985). 18.104.22.168 Fungi The induction of mutations by DEHP has been studied in various fungal species. In the IPCS collaborative study on in vitro assay systems (Ashby et al., 1985), DEHP was considered to be negative in six out of seven assays. Positive results were obtained with Saccharomyces cerevisiae, both with and without S9 activation, at lowest effective concentrations of 1541 mg/litre and 3081 mg/litre, respectively. However, other laboratories using different strains of S. cerevisiae or Schizosaccharomyces pombe reported negative results at a maximum tested concentration of 5000 mg/litre. 22.214.171.124 Mammalian cells Mouse lymphoma cells (L5178Y), Chinese hamster V79 cells, and human lymphoblasts have been used to study the mutagenic effect of DEHP in cultured mammalian cells. Several investigators obtained negative results, but a few positive results have also been reported. In the IPCS collaborative study (Ashby et al., 1985), only one out of ten investigators reported a positive response. Mouse lymphoma cells were exposed to DEHP without S9, and two concentrations (7.5 and 20 mg/litre) gave positive results. In a separate study (Kirby et al., 1983), where MEHP, 2-ethylhexanol, and DEHP were tested in the mouse lymphoma cell assay, all three substances were found to be non-mutagenic. The concentrations used were 0.016-1.0 ml/litre (without S9) and 0.067-5.0 ml/litre (with S9) for DEHP, and 0.013-1.0 ml/litre for MEHP and 2-ethylhexanol. 126.96.36.199 Drosophila DEHP has also been tested for mutagenicity in Drosophila melanogaster using the sex-linked recessive lethal (SLRL) test and various somatic recombination and mutation assays. SLRLs were not induced by DEHP (20 µg/g) administered by injection (Yoon et al., 1985). Negative results for DEHP were also reported in the SLRL mutation assay on Drosophila melanogaster larvae (Zimmering et al., 1989). In the IPCS study (Ashby et al., 1985), DEHP gave a positive response in the unstable eye mosaic test at a dose of 6.1 g/litre, in two separate experiments, but neither lower nor higher concentrations produced any response. No activity was seen in the wing spot test with a single dose of 6.1 g/litre. It was concluded that DEHP exhibits marginally positive mutagenicity (Ashby et al., 1985). 7.6.2 DNA damage Various end-points, such as unscheduled DNA synthesis (UDS) and single strand breaks, have been used to detect DNA damage induced by DEHP in a variety of mammalian test systems. In the IPCS study (Ashby et al., 1985), negative results were obtained when single strand breaks were measured, either by alkaline elution in hepatocytes (up to 3.907 g/litre) or alkaline sucrose sedimentation in Chinese hamster ovary (CHO) cells (up to 39 g/litre). UDS in either isolated hepatocytes or cultured HeLa cells was investigated by four different laboratories. One investigator detected a positive response using isolated hepatocytes, but, since this result was only statistically significant at one dose and not dose related, the consensus was that DEHP does not cause UDS. In a study by Butterworth et al. (1984), DEHP did not induce DNA repair in primary rat hepatocytes. Similarly, neither DEHP nor MEHP induced any DNA repair in primary human hepatocytes from three different subjects. In this study, concentrations as high as 0.01 mol DEHP/litre and 5 x 10-4 mol MEHP/litre were used and exposure continued for 18 h. No induction of DNA repair or increased alkaline elution of DNA was seen in hepatocytes from either female or male F-344 rats treated with DEHP in vivo. UDS was not induced in male rats treated with 500 mg DEHP/kg by gavage 2, 12, 24 or 48 h before sacrifice. Furthermore, treatment of male rats with 150 mg/kg per day by gavage for 14 days and treatment of female rats with dietary DEHP (12 g/kg) for 30 days resulted in peroxisome proliferation, but no UDS was induced. Similar in vivo/in vitro results in studies of UDS in hepatocytes were reported by Kornbrust et al. (1984). No UDS was observed in primary rat hepatocytes exposed in vitro to 10-5 to 10-2 mol DEHP/litre or in vivo by a single gavage dose of 5 g/kg 2, 15 or 24 h prior to the isolation of hepatocytes. A dietary concentration of 20 g DEHP/kg led to a marked proliferation of peroxisomes after 4 weeks. Neither this treatment nor the additional administration of a single gavage dose of 5 g/kg (15 h before sacrifice) to animals fed the 20-g/kg diet for either 8 weeks or for 4 weeks with or without pretreatment with 3-amino-1,2,4-triazole (to inhibit endogenous catalase activity) induced any detectable DNA repair in hepatocytes. Indeed, the administration of DEHP and some other proliferators has been shown to increase the levels of 8-hydroxy-deoxyguanosine in rat liver DNA (Takagi et al., 1990). 7.6.3 DNA binding In an in vivo study by Albro et al. (1982), radioactivity from carbonyl-labelled DEHP did not associate with purified protein, RNA or DNA from rat liver. Label from 2-ethyl-(1-14C)-hexyl-labelled DEHP or MEHP appeared to associate strongly with purified DNA, but this was not the case with label from free 14C-labelled 2-ethylhexanol. According to Albro (1987), although there is incorporation into normal nucleosides, there is no evidence of alkylation. In a similar study (von Däniken et al., 1984; Lutz, 1986), DEHP radiolabelled in different positions was administered orally to female F-344 rats with or without pretreatment with unlabelled DEHP (10 g/kg diet) for 4 weeks. The administration of 14C-carboxylate-labelled DEHP resulted in no measurable DNA radioactivity, whereas radioactivity was clearly measurable after the administration of DEHP, 14C- or 3H-labelled in the alcohol moiety, or of 2-ethyl(1-14C)hexanol. HPLC analysis showed that the normal nucleosides had incorporated radiolabel, but fractions expected to contain carcinogen-modified nucleoside adducts did not contain any radioactivity. DNA isolated from the livers of male F-344 rats administered 2000 mg DEHP/kg daily by gavage for 3 days was analysed for possible carcinogen-DNA adducts by the 32P-postlabelling technique (Gupta et al., 1985). No adducts were detected in the DNA, which also was the case when DNA from hepatocytes exposed to 10-3 mol DEHP/litre in vitro for 4 h was analysed. 7.6.4 Chromosomal effects Chromosomal effects of DEHP have mainly been studied in vitro, although some studies on the induction of micronuclei in the peripheral blood erythrocytes of mice have been published. DEHP did not induce any increase in the level of sister-chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells, treated for 1 h, either with or without S9, at levels of up to 0.01 mol/litre (Douglas et al., 1986). On the other hand, MEHP has been reported to induce SCE in V79 cells treated with 25 or 50 mg/litre for 24 h or 1500 mg/litre for 3 h (Tomita et al., 1982a). This metabolite also induced chromosomal aberrations in CHO cells and RL4 cells (from rat liver), but only at cytotoxic concentrations in the CHO cells (1.0 and 1.3 mmol/litre, with or without S9). MEHP was less toxic to RL4 cells and concentrations between 2.0 and 5.0 mmol/litre gave a dose-related increase in chromosomal aberrations (Phillips et al., 1986). According to the authors, observations on changes in CHO cell structure and permeability and on the haemolytic effects of phthalate monoesters suggest that MEHP cytotoxicity may be due primarily to an action on cell membranes. The induction of aneuploidy by DEHP was investigated both in mammalian cells and in fungi in the IPCS study (Ashby et al., 1985). The mammalian assays gave positive responses at 50 mg/litre in a fibroblast cell line from Chinese hamster liver and at levels of between 25 and 50 mg/litre in Chinese hamster primary liver cells. Two out of four studies using fungi were also positive and the consensus was that DEHP is capable of inducing aneuploidy in vitro in both fungi and mammalian cells. In a study by Ahmed et al. (1990), male Wistar rats were fed for alternate 7-day periods with a diet containing 20 g DEHP/kg and a control diet. The rats were examined after 3 days on the DEHP diet or after 7 days on the control diet. An analysis of nuclear size gave results consistent with an increase in tetraploid hepatocytes after treatment with DEHP, which was reversed when the rats returned to the control diet. 7.6.5 Cell transformation DEHP-induced cellular transformation has been studied in several different experimental systems. In a test programme (Astill et al., 1986), the BALB/3T3 cell transformation assay was used both with and without rat primary hepatocytes. Both DEHP (0.875-1 µl/litre) and the two metabolites MEHP and 2-ethylhexanol gave negative results. On the other hand, the majority of transformation tests in the IPCS study (Ashby et al., 1985) were positive for DEHP. Negative results were obtained with BALB/c-3T3 cells, while a study measuring the enhancement of viral transformation of Syrian hamster embryo (SHE) cells was considered to be inconclusive. Positive responses were obtained by four other investigators using SHE cells at 1-300 mg/litre (two different laboratories), embryonic mouse fibroblasts at 1000 mg/litre with S9 and 10 mg/litre without S9, or retrovirus-infected Fischer rat embryo cells at 2000 mg/litre (the highest dose tested). In a separate study (Tomita et al., 1982a), both DEHP (7.5 and 15 g/kg) and MEHP (375 and 750 mg/kg) induced morphological transformation, as well as chromosomal aberrations, in SHE cells after transplacental administration. In a study by Diwan et al. (1985), anchorage-independent growth of JB6 mouse epidermal cells was enhanced by DEHP at concentrations of 500 to 20 000 ppm/ml (the Task Group noted that the unit used in Diwan's paper is ppm/ml, but it should read ppm or mg/litre). Ward et al. (1986) used the same model system and reported that both DEHP (1.3-51 x 10-6 mol/litre) and MEHP (2-5 x 10-8 mol/litre) were effective, while 2-ethylhexanol (4-77 x 10-7 mol/litre) was without effect. The morphological transformation in the SHE cell system induced by DEHP, MEHP, and other hepatic peroxisome proliferators seemed not to be correlated with increased peroxisomal ß-oxidation, increased production of oxidative radicals or peroxisome proliferation (Mikalsen et al., 1990a; Mikalsen et al., 1990b; Mikalsen et al., 1990c). A few studies on DEHP-induced inhibition of metabolic cooperation, which may be indicative of the promoting potential of a substance, have been reported. Metabolic cooperation in Chinese hamster V79 cells was not inhibited by DEHP at non-cytotoxic concentrations, i.e. 3 x 10-4 mol/litre (0.12 mg/litre) or less (Kornbrust et al., 1984). In the IPCS study (Ashby et al., 1985), one investigator reported inhibition in V79 cells at non-cytotoxic concentrations of DEHP (25-200 mg/litre in two separate experiments and 5-25 mg/litre in another), while another investigator, using V79 cells in a microassay method, detected a slight (but non-significant) increased inhibition at doses of between 10-5 and 2 x 10-4 mol/litre (3.9-78 mg/litre). In a study by Malcolm & Mills (1989) on V79 cells, DEHP inhibited intercellular communication (gap junctions) at non-cytotoxic concentrations (10-30 mg/litre). 7.6.6 In vivo effects Putman et al. (1982) dosed male Fischer-344 rats orally for 5 days with DEHP (5.0, 1.7, and 0.5 g/kg per day), MEHP (0.14, 0.05, and 0.01 g/kg per day), and 2-ethylhexyl (0.21, 0.07, and 0.02 g/kg per day), and bone marrow metaphase cells were examined. No significant increases were observed in gap breaks or structural rearrangements. In addition, the mitotic index was unaffected by treatment. A micronucleus assay on mouse (B6C3F1) peripheral blood erythrocytes (intraperitoneal doses of 0.6, 3.0, and 6.0 g/kg per day for 5 days), sampled at 0, 2, and 4 weeks after the last treatment, yielded negative results (Douglas et al., 1986). A sperm morphology assay in B6C3F1 mice and Sprague-Dawley rats at the same dose levels also gave negative results (Douglas et al., 1986). However, Agarwal et al. (1986b) reported increases in sperm morphology changes in adult F-344 rats given 20 g DEHP/kg for 60 consecutive days. Two studies have yielded negative results in dominant lethal tests (Hamano et al., 1979; Rushbrook et al., 1982). Hamano et al. (1979) administered MEHP and DEHP orally, and the observation period was six weeks. Rushbrook et al. (1982) dosed ICR/SRM mice orally for 5 days with DEHP (2465, 4930, and 9860 mg/kg per day), MEHP (50, 100, and 200 mg/kg per day) or 2-ethylhexanol (250, 500, and 1000 mg/kg per day). Each male was mated weekly with virgin females for 8 consecutive weeks, and females were evaluated for pregnancy, live fetuses, and early and late fetal deaths. All data were within the normal ranges. DEHP and its major metabolites have also been tested for their potential to induce micronuclei. Exposure to 0.6, 3.0 or 6.0 g DEHP/kg per day for 5 days over a 4-week period did not induce micronuclei in the peripheral blood erythrocytes of B6C3F1 male mice (Douglas et al., 1986). Negative results were also obtained in another mouse micronucleus test after either a single or daily doses of 5 g DEHP/kg. In this study MEHP and 2-ethylhexanol also gave negative results (Astill et al., 1986). 7.7 Carcinogenicity In a carcinogenicity study (NTP, 1982; Kluwe et al., 1982), groups of 50 male and 50 female Fischer-344 rats were fed diets containing 6 or 12 g DEHP/kg diet, and 50 male and 50 female B6C3F1 mice were fed diets containing 3 or 6 g DEHP/kg for 103 consecutive weeks. Concurrent controls (50 of each sex and species) were fed a diet without the addition of DEHP. Food and water were supplied ad libitum. All animals were given the control diet for 1-2 weeks after 103 weeks of treatment and were then killed and examined both grossly and microscopically. The administered concentrations of DEHP were estimated to be equal to, or one half of, the maximum tolerated doses. Under the conditions studied, DEHP caused an increased incidence of hepatocellular carcinomas in female rats and male and female mice, and an increased incidence of hepatocellular carcinomas and neoplastic nodules in female rats (Table 8). Twenty of the 57 hepatocellular carcinomas in the DEHP-treated mice (sexes and doses combined) had metastasized to the lung. The figure of nine hepatocellular carcinomas in control male mice is considered to be within the normal range (NTP, 1982). The reported decreased incidence of tumours of the thyroid, pituitary, and testis could be related to an increased endocrine activity of the pituitary gland (NTP, 1982). The carcinogenicity of DEHP in rats has been confirmed in two further studies. Rao et al. (1990) found a 78.5% incidence in a group of 14 male Fischer-344 rats fed a diet containing 20 g DEHP/kg for up to 108 weeks, whereas the incidence in the 10 controls was 10%. Popp et al. (1987) found either hepatocellular carcinomas or neoplastic nodules in 6 out of 20 animals after female Fischer-344 rats were exposed for 2 years to a diet containing 12 g/kg. Table 8. Carcinogenic effects of DEHP on the livera Control Low dose High dose P valueb Hepatocellular carcinoma Male rats 1/50 1/49 5/49 < 0.05 Female rats 0/50 2/49 8/50 < 0.005 Male mice 9/50 14/48 19/50 < 0.05 Female mice 0/50 7/50 17/50 < 0.0001 Neoplastic nodules Male rats 2/50 5/49 7/49 n.s. Female rats 0/50 4/49 5/50 < 0.05 Hepatocellular adenoma Male mice 6/50 11/48 10/50 n.s Female mice 1/50 5/50 1/50 n.s a From: NTP (1982) b Probability level in Codiran-Armitage test for linear trend when P < 0.05; otherwise not significant (n.s.) Two other long-term studies on DEHP have been performed by Carpenter et al. (1953) and Harris et al. (1956), but, due to the small numbers of animals used, the studies are inadequate to assess the carcinogenic potential. DEHP has been investigated in two life-time studies on Syrian hamsters (Schmezer et al., 1988). In one study, groups of 25 male and 25 female 6-week-old hamsters were assigned to each of six groups: untreated control; 3 g DEHP/kg body weight given intraperitoneally once every week for 18 weeks; the same dose once every two weeks for 18 weeks; the same dose once every four weeks for 32 weeks; the same dose once every four weeks for 32 weeks plus N-dimethylnitrosamine (NDMA) at 1.67 mg/kg body weight given orally once per week; and the same dose of NDMA without DEHP treatment. NDMA increased the tumour rate for malignant liver tumours, mainly haemangioendotheliomas. Co-administration of DEHP with NDMA neither increased nor decreased the tumour rates. No significant differences of tumour rates were observed in groups treated with DEHP alone compared with controls. Schmezer et al. (1988) also investigated the life-time, whole- body exposure of Syrian hamsters to DEHP vapour alone in air or in combination with orally administered NDMA. The low doses of DEHP (15 µg/m3, resulting in a total exposure of about 7.5 µg/kg body weight) in this study render it inadequate for the assessment of the long-term effects of DEHP alone. In combination with NDMA, this low level of DEHP was associated with highly significant (P < 0.001) decreases in hepatic haemangioendothe-lioma and fibrosarcomas combined in males and females combined. This unexpected result should be further investigated to evaluate its significance. 7.8 Special studies Since DEHP lacks genotoxic activity in most test systems, it has been suggested that the carcinogenic effect is exerted during the promotion phase of hepatocarcinogenicity. DEHP has therefore been tested in several initiation/promotion experiments in rats and mice where the end-point has been the number and/or volume of altered liver cell foci. As expected, DEHP lacked initiating activity in these studies (Ward et al., 1986; Popp et al., 1987). DEHP appears to be a tumour promotor in mouse liver. In male B6C3F1 mice given an intraperitoneal initiating dose of diethylnitrosamine (80 mg/kg), DEHP at levels of 6 and 12 g/kg diet caused accelerated growth of foci and increased incidence of hepatocellular adenomas (Ward et al., 1983; Ward et al., 1986). However, in a 6-month study, DEHP at a level of 12 g/kg diet did not appear to promote altered foci in male F-344 rats given an intraperitoneal initiating dose of diethylnitrosamine of 150 mg/kg (Popp et al., 1987). In other promotion studies DEHP appeared to accelerate the regression or inhibit the appearance of some kinds of foci in rats (DeAngelo & Garrett, 1983; DeAngelo et al., 1984). However, other studies have shown that peroxisome proliferators can promote the development of altered foci and tumours in rat liver. These results are somewhat different from those of other liver tumour promotors with respect to the histological phenotype of the foci and the relatively low frequency of the foci (Cattley & Popp, 1989). Thus, further studies are required to demonstrate that DEHP can promote altered foci in rat liver. 7.9 Mechanisms of hepatotoxicity Together with a number of structurally diverse chemicals, including certain hypolipidaemic drugs, herbicides, and chlorinated solvents, DEHP has been shown to produce hepatic peroxisome proliferation in rats and mice (Reddy & Lalwani, 1983; Lock et al., 1989). This proliferation is accompanied by liver enlargement, stimulation of replicative DNA synthesis and cell division, and the induction of peroxisomal and microsomal fatty acid oxidizing enzyme activities (Reddy & Lalwani, 1983; Marsman et al., 1988; Lock et al., 1989). The increase in microsomal fatty acid oxidation is due to the induction of a cytochrome P-450 IVA1 (Lock et al., 1989). Peroxisome proliferation and induction of peroxisomal and microsomal fatty acid oxidizing enzyme activities can be observed in primary hepatocyte in vitro cultures, as well as in the intact animal (Elcombe & Mitchell, 1986; Lake et al., 1986). Both in vivo and in vitro studies with rat hepatocytes have demonstrated wide compound potency differences (Reddy et al., 1986; Barber et al., 1987; Lake et al., 1988). Compared to some other compounds, DEHP is not considered to be a particularly potent peroxisome proliferator in rodent liver (Reddy et al., 1986; Barber et al., 1987). Possible mechanisms of peroxisome proliferation include a perturbation of hepatic lipid metabolism and/or the presence of a receptor protein (Lalwani et al., 1987; Lock et al., 1989; Issemann & Green, 1990). Certain peroxisome proliferators, including DEHP, have been shown to increase the incidence of liver tumours in rats and mice (Reddy & Lalwani, 1983; Reddy et al., 1986; Butterworth et al., 1987). Generally, there is a good correlation between the potency of a compound to produce peroxisome proliferation in rat and mouse liver and its hepatocarcinogenic properties (Reddy et al., 1986). However, the magnitude of peroxisome induction does not necessarily always correlate with the carcinogenic response when different compounds are compared (Marsman et al., 1988). Since the peroxisome proliferators known to date are non-genotoxic carcinogens, Reddy and coworkers (Reddy & Lalwani, 1983; Rao & Reddy, 1987; Reddy & Rao, 1989) have suggested that liver tumour formation arises from a sustained oxidative stress to the hepatocytes due to an imbalance in the production and degradation of hydrogen peroxide (H2O2). DEHP administration results in increased peroxisomal production of H2O2 in hepatocytes (Tomaszewski et al., 1986). There is an imbalance in H2O2 production and degradation due to the fact that catalase is induced to a much lesser extent than peroxisomal ß-oxidation enzymes. In addition, the level of cytosolic glutathione peroxidase is reduced on prolonged exposure to DEHP (Lake et al., 1987; Marsman et al., 1988; Conway et al., 1989b; Tamura et al., 1990). Treatment with peroxisome proliferators also results in a reduction of superoxide dismutase and glutathione S-transferase activities (Ciriolo et al., 1982; Goel et al., 1986; Lake et al., 1987). It has been suggested that the increased level of H2O2 in hepatocytes, either directly or via other reactive oxygen species (e.g., hydroxyl radical), damages intracellular membranes and/or DNA (Reddy & Rao, 1989). Indeed chronic administration of DEHP and other peroxisome proliferators results in increased lipid peroxidation and lipofuscin deposition in rat hepatocytes (Mitchell et al., 1985b; Goel et al., 1986; Cattley et al., 1987; Lake et al., 1987; Reddy & Rao, 1989; Conway et al., 1989b). Oxygen radicals may damage DNA (Bridges, 1985), leading to increased levels of 8-hydroxydeoxy-guanosine and other modified bases in DNA (Reddy & Rao, 1989). Indeed, the administration of DEHP and some other peroxisome proliferators has been shown to increase levels of 8-hydroxyde-oxyguanosine in rat liver DNA (Kasai et al., 1989; Takagi et al., 1989, 1990, 1991). Apart from a role in peroxisome proliferation, leading to oxidative stress, recent studies on DEHP have demonstrated a role in the stimulation of replicative DNA synthesis in the hepatocarcinogenicity of peroxisome proliferators (Butterworth et al., 1987; Smith-Oliver & Butterworth, 1987; Marsman et al., 1988). Increased cellular division may result in spontaneous mutational events or promotional effects (Bridges, 1985; Butterworth et al., 1987), including the promotion of spontaneously initiated cells found in the livers of aging rodents (Schulte-Hermann et al., 1989). A comparative study of the hepatic effects of Wy-14643 ([4-chloro-6 (2,3-xylidino) 2-pyrimidinylthio]acetic acid), a potent peroxisome proliferator, and DEHP indicated that, although both compounds produced a similar induction of peroxisome proliferation at the dose levels administered, only Wy-14643 produced liver tumours within one year (Marsman et al., 1988; Conway et al., 1989b). However, analysis of these data demonstrates that Wy-14643 produces both a more marked increase in lipofuscin deposition (presumably reflecting oxidative stress) and a sustained stimulation of replicative DNA synthesis. These data suggest a role for both oxidative stress and increased cell replication in the hepatocarcinogenicity of DEHP and other peroxisome proliferators. The mechanism of hepatocarcinogenicity induced by peroxisome proliferators could include both enhanced cell replication and DNA damage induced by oxygen radicals. Thus, at dose levels that do not produce either significant peroxisome proliferation or cell replication, it is unlikely that DEHP or other peroxisome proliferators would elicit hepatic tumour formation. In this context data on threshold values for liver effects in sensitive species (e.g., the rat and mouse), together with an assessment of species differences in response (see below), should be considered. The value for the subchronic liver effects of DEHP (e.g., peroxisome proliferation and DNA synthesis) in the rat appears to be around 50 mg/kg per day (Lake et al., 1984, 1991; Mitchell et al., 1985b; Tomaszewski et al., 1986). Furthermore, a two-year study of DEHP in female rats demonstrated no effects at 0.3 g/kg diet, a value which corresponds well to that noted above (Cattley et al., 1987; Popp et al., 1987). Many studies have shown dramatic species differences in response to peroxisome proliferators including DEHP (ECETOC, 1992). For example, DEHP threshold values for peroxisome proliferation of 25 and 250 mg/kg, respectively, were found in rats and hamsters after 14 days of daily gastric intubation (Lake et al., 1984). Several other studies showing species differences in the response to DEHP have been reported. One experiment demonstrated a large increase in hepatic peroxisomes after 14 days of oral DEHP administration (2000 mg/kg per day) to rats, but the same treatment of marmosets resulted in no peroxisome proliferation (Rhodes et al., 1986). A limited study in cynomolgus monkeys also revealed no evidence of peroxisome proliferation due to DEHP (Short et al., 1987). In addition, DEHP did not elicit peroxisome proliferation in guinea-pigs (Osumi & Hashimoto, 1978; Mitchell et al., 1985a). Some studies have indicated that intrinsic species differences in hepatocellular sensitivity exist. For example, MEHP and MEHP metabolite VI (mono(2-ethyl-5-oxohexyl) phthalate), which is a proximate peroxisome proliferator in the rat, were good peroxisome proliferators in cultured rat hepatocytes, but had little or no effect in guinea-pig, marmoset or human hepatocytes (Mitchell et al., 1985a; Elcombe & Mitchell, 1986). More recently, Bichet et al. (1990) and Butterworth et al. (1989) have also demonstrated the inability of MEHP to elicit peroxisome proliferation in human hepatocyte cultures. Studies using a variety of other peroxisome proliferators (e.g., clofibric acid, beclobric acid, methylclofenapate, trichloroacetic acid, ciprofibrate, and benzbromarone) have also failed to demonstrate peroxisome proliferation in cultured human hepatocytes, in spite of a good response in rat hepatocytes (Elcombe, 1985; Allen et al., 1987; Elcombe & Styles, 1989; Butterworth et al., 1989; Bichet et al., 1990; Blaauboer et al., 1990). Species differences in cytochrome P-450 IVA1 induction and stimulation of replicative DNA synthesis have been less well studied. However, MEHP and metabolite VI did not induce P-450 IVA1-mediated lauric acid hydroxylation in vivo in marmosets (Rhodes et al., 1986) or in cultured marmoset or human hepato-cytes (Elcombe & Mitchell, 1986). In conclusion, it appears that the livers of rats and mice are exquisitely sensitive to peroxisome proliferators, including DEHP, while those of guinea-pigs, monkeys, and humans show minimal or no response (ECETOC, 1992). 8. EFFECTS ON HUMANS 8.1 General population exposure Two adults who swallowed 5 or 10 g DEHP experienced no untoward effects apart from mild gastric disturbances and moderate diarrhoea with 10 g DEHP (Shaffer et al., 1945). Three cases of nonspecific hepatitis were described among 27 haemodialysis patients with terminal renal failure. The PVC blood tubing used released DEHP at a concentration of 10-20 mg/litre of perfusate. The symptoms and signs of hepatitis disappeared rapidly when the use of tubing that did not contain DEHP was resumed (Neergaard et al., 1971). In three pre-term infants artificially ventilated with PVC respiratory tubes, unusual lung disorders (opacification of the lung) were observed during the fourth week of life. It was assumed by the authors that these lung disorders were causually related to the exposure to DEHP released from the respiratory tubes (Roth et al., 1988). 8.2 Occupational exposure There are very few data on the effects of occupational exposure specifically to DEHP. Two studies reported symptoms and signs of polyneuropathy among 47 out of 147 workers at a PVC-processing plant in the USSR and 12 out of 23 workers at a plant for phthalate production in Italy. The workers were exposed to a mixture of phthalates and DEHP was a minor constituent, at least in the USSR plant. Furthermore, tricresyl phosphate (a neurotoxin) was a component of the incombustible materials produced in 10-20% of machines assigned to various workers (Milkov et al., 1973). In the Italian study there was no corresponding unexposed control group and the authors concluded that no definite conclusion could be drawn from the study because of the limited number of workers examined (Gilioli et al., 1978). The total phthalate air concentrations recorded varied between 1.7 and 66 mg/m3 in the USSR and 1 and 60 mg/m3 in Italy (Milkov et al., 1973; Gilioli et al., 1978). In a study involving a Swedish PVC-processing factory, peripheral nervous system symptoms and signs were investigated among 54 male workers exposed mainly to DEHP, diisodecylphthalate, and some butylbenzylphthalate. The workers were divided into three groups of approximately equal size and with mean phthalate exposures of 0.1, 0.2, and 0.7 mg/m3. Some workers displayed various peripheral nervous system symptoms and signs, but these were not related to the level of exposure. None of the workers reported symptoms indicating work-related obstructive lung disease. Conventional lung function tests also showed no association with exposure (Nielsen et al., 1985). However, several biochemical parameters showed significant associations with exposure. There was a slight decrease in the haemoglobin level with longevity of employment as well as with exposure in the last year. The serum ý-1-antitrypsin level increased slightly with length of employment and the serum immunoglobulin A level rose with rising exposure during the last year (Nielsen et al., 1985). One case of occupational asthma due to DEHP was reported in worker at a PVC-processing plant (Brunetti & Moscato, 1984). Diagnosis was made by exposing the patient to DEHP vapour in an inhalation chamber; this evoked a dual asthmatic reaction. The action was inhibited by prior administration of sodium chromoglycate. A study of blood lipids, serum activities of liver enzymes, and routine haematological tests was carried out among workers at a German plant for DEHP production. The results were negative and uninformative due to very low exposure levels (below 0.16 mg/m3) and the lack of a control group (Thiess et al., 1978b). Thiess & Flieg (1978) investigated the frequency of chromosome aberrations in 10 workers who were employed from 10 to 30 years in this DEHP production plant. The exposure levels were very low (0.09-0.16 mg/m3), and there was no increase in chromosome aberrations compared to the control group. A mortality study of 221 workers exposed to DEHP in the same plant was also conducted. Eight deaths occurred in the cohort compared with expected values of 15.9 and 17.0 from the city and county data, respectively (Thiess et al., 1978a). 9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD 9.1 Toxicity to microorganisms Bringmann & Kühn (1980) calculated toxic thresholds from cell multiplication inhibition tests and found thresholds of > 400 mg/litre for the bacterium Pseudomonas putida, 10 mg/litre for the alga Scenedesmus quadricaudaia, and 19 mg/litre for the protozoan Entosiphon sulcatum. Mathur (1974b) reported that DEHP added to soil (3 g/kg) inhibited respiration, whereas addition of the same amount of DEHP to soil preincubated for 14 weeks with DEHP or dioctyl phthalate had no effect on respiration rate. The experiments were conducted at 22 °C since no degradation of DEHP occurred at 4 °C or 10 °C. In an intermittent flow-through hydrosoil microcosm, DEHP (at 1 and 100 mg/litre) produced no significant effect on the numbers or physiological activity of the microflora monitored, nor on any of the overall activities that were studied (Mutz & Jones 1977). Larsson et al. (1986) studied the effect of DEHP on the microbial activity in sediments. Sediment cores were taken, together with the overlying water, from a eutrophic lake and the sediment was spiked with between 25 and 400 mg DEHP/kg at a level of 5 mm below the surface. Uncontaminated sediment samples had a significantly higher oxygen uptake than sediment containing DEHP. Microbial activity, as estimated from decreased oxygen saturation in the soil column, correlated inversely with increasing levels of DEHP in the sediment. 9.2 Toxicity to aquatic organisms Many of the toxicity values given in this section are above the water solubility of DEHP, which ranges from 0.3 to 0.4 mg/litre (see section 2.2). DEHP adsorbs strongly to sediment, food, dissolved organic carbon, and even the testing vessels. Many of the toxicity tests are also based on nominal concentrations. Therefore, the actual exposure of the organisms is very difficult to determine and care must be taken when interpreting the results. 9.2.1 Invertebrates Acute toxicity to aquatic invertebrates is summarized in Table 9. Table 9. Toxicity of DEHP to aquatic invertebratesa Organism Age Temperature Hardnessb pH Duration LC50 Reference (° C) (h) (mg/litre)d Water flea < 24 h 17 48 0.133 Passino & Smith (1987) (Daphnia pulex) Water flea < 24 h 21-23 173 7.4-9.4 24 > 68 Leblanc (1980) (Daphnia magna) < 24 h 21-23 173 7.4-9.4 48 11 Leblanc (1980) Crayfish 96 > 10 Mayer & Sanders (1973) (Orconectes nais) Harpacticoid adult 20-22 7c 7.8 96 > 300 Linden et al. (1979) (Nitocra spinipes) Scud 96 > 32 Sanders et al. (1973) (Gammarus pseudolimnaeus) Midge larvae 21-23 270 7.4 48 > 18 Streufert et al. (1980) (Chironomus plumosus) a All experiments were performed using static conditions (water unchanged for duration of test) b Hardness is expressed as mg calcium carbonate per litre. The tests were performed in fresh water except where stated otherwise c Salinity (%) d Nominal values Brown & Thompson (1982a) found no mortality of Daphnia magna over an exposure period of 48 h and at DEHP dose levels up to 320 µg/litre. However, at levels of 180 µg/litre or more, the daphnids were seen to float in the surface layer. The authors suggested that this observation was connected with the solubility of DEHP (which tends to precipitate out at > 180 µg/litre, possibly onto the daphnids, thereby causing them to float). Stephenson (1983) found no mortality in Gammarus pulex exposed for up to 120 h to a DEHP concentration (400 µg/litre) in excess of its solubility. Laughlin et al. (1978) exposed grass shrimps (Palaemonetes pugio) to DEHP concentrations of up to 1 mg/litre (well above the solubility limit of the ester) and found no significant difference in mortality between the control and treated shrimps during the 28-day exposure period. DEHP had no significant effect on the development rate of larvae from hatching to moult. No apparent effects were noted when mussels (Mytilus edulis) were exposed to 50 µg/litre for 28 days (Brown & Thompson, 1982b). Hobson et al. (1984) found no significant mortality of penaeid shrimps (Penaeus vannamei) fed a diet containing up to 50 g DEHP/kg for 14 days, this representing 4% of the body weight per day. Histological examination revealed no changes and no significant dose- related effects were observed on moulting. Sanders et al. (1973) and Mayer & Sanders (1973) exposed Daphnia magna for a complete life cycle (21 days) to 3, 10 or 30 µg DEHP/litre in an intermittent-flow system. Reproduction was significantly inhibited at all concentrations (60, 70, and 80% inhibition at the three dose levels, respectively). In contrast, Brown & Thompson (1982a) found no effect on the reproduction of Daphnia magna at concentrations of up to 100 µg/litre and suggested that the difference in the numbers of young born to the controls in the two studies, i.e. 11 offspring per parent (Mayer & Sanders, 1973) compared with 170 per parent (Brown & Thompson, 1982a), could account for these conflicting results. When Knowles et al. (1987) exposed Daphnia magna to DEHP concentrations of between 12 and 811 µg/litre for up to 21 days under flow-through conditions (the reproduction rate was approximately 200 offspring per adult), survival was not affected at levels of < 158 µg/litre. At 811 µg/litre, however, survival was significantly reduced after both 7 and 21 days. The mean number of young per surviving adult was also reduced at this dose level. The levels of major biochemical components such as protein, RNA, DNA, and glycogen were all significantly reduced after 7 days at 811 µg/litre, but all except glycogen were unaffected after 21 days. The authors concluded that the maximum acceptable toxicant concentration (MATC), based on survival and reproduction, was between 158 and 811 µg/litre. A no-observed- effect level (NOEL) of 72 µg/litre was identified both by DNA content and RNA/DNA ratio at day 7 and by surfacing of Daphnia at day 0. Thuren & Woin (1991) exposed the freshwater amphipod Gammarus pulex to DEHP at concentrations of 100 or 500 µg/litre for 10 days under flow-through conditions. There was a 5 day pre- and post- exposure period. The overall locomotor activity of G. pulex was significantly decreased at the higher exposure level, and the effect persisted throughout the post-exposure period. No significant effects were observed at the lower dose level. Flow-through chronic toxicity tests in both sand and hydrosoil showed that DEHP concentrations as high as 360 µg/litre (sand) and 240 µg/litre (hydrosoil) had no significant effects on the growth or development of midge larvae (Chironomus plumosus) over a 35-day period. The continuous exposure of first-generation midge eggs in sand substrate to mean DEHP concentrations of between 140 and 360 µg/litre had no significant effect on hatchability (Streufert et al., 1980). Woin & Larsson (1987) found that dragonfly larvae ( Aeshna sp.) caught significantly fewer prey (Chaborus larvae) per attempt when exposed to sediment DEHP concentrations of approximately 600 mg/kg for between 3 and 9 weeks. 9.2.2 Fish The acute toxicity to fish is summarized in Table 10 and the toxicity to the embryo-larval stages of fish is shown in Table 11. DEHP caused no deaths among one-year-old rainbow trout (Salmo gairdneri) exposed to between 1 and 1000 mg/litre for 48 h (Silvo, 1974) or juvenile Atlantic salmon (Salmo salar) exposed to 100 mg/litre for 96 h (Zitko, 1972). Mehrle & Mayer (1976) reported no effects on growth or survival of adult fathead minnows (Pimephales promelas) exposed to between 1 and 62 µg/litre for 56 days. They also exposed rainbow trout eggs to DEHP levels of 5, 14, and 54 µg/litre for 12 days prior to hatching, but there was no significant effect on hatchability. The resulting fry were continuously exposed to DEHP for a further 90 days. The two highest concentrations caused a significant, but not dose related, increase in the mortality of sac fry within 5 days of hatching. After yolk absorption (at 24 days), DEHP caused no significant mortality or effects on growth and development for the remainder of the exposure period. Table 10. Toxicity (96-h LC50 values) of DEHP to fish Organism Size Stat/ Temperature Hardnessb pH LC50 Reference flowa (° C) (mg/litre)c Bluegill sunfish > 10 Mayer & Sanders (1973) (Lepomis macrochirus) 0.32-1.2 g stat 21-23 32-48 6.7-7.8 > 770 Buccafusco et al. (1981) Fathead minnow > 10 Mayer & Sanders (1973) (Pimephales promelas) 0.055-0.25 g 24-26 44-46 7-8 > 0.33d Defoe et al. (1990) Channel catfish > 10 Mayer & Sanders (1973) (Ictalurus punctatus) Rainbow trout fingerling stat 14-16 540 Hrudey et al. (1976) (Oncorhynchus mykiss) > 10 Mayer & Sanders (1973) a Stat = static conditions (water unchanged for duration of test) b Hardness is expressed as mg calcium carbonate per litre. The tests were performed in fresh water except where stated otherwise c Nominal values unless stated otherwise d Measured value Table 11. Toxicity of DEHP to the embryo-larval stages of fisha Organism Stat/flowb Hardnessc pH LC50 (mg/litre) 95% confidence limits Channel catfish stat 90-115 7.6-8.1 0.69 0.55-0.86 (Ictalurus punctatus) Redear sunfish stat 90-115 7.6-8.1 6.18 4.65-8.04 (Lepomis microlophus) Largemouth bass flow 45-55 7.5-8.0 42.1 28.8-77.8 (Micropterus salmoides) flow 190-225 7.5-8.0 32.9 19.8-58.9 Rainbow trout flow 45-55 7.5-8.0 139.5 123.2-165.2 (Oncorhynchus mykiss) flow 190-225 7.5-8.0 149.2 125.8-203.8 a From: Birge et al. (1978). Exposure was initiated 2 to 6 h after spawning (except in the case of rainbow trout where exposure was initiated 15 min after fertilisation) and continued until 4 days after hatching. The hatching times were 22 days for rainbow trout (13.5-14.3 °C), 3 days for catfish (29-31 °C), and 3-4 days for bass and sunfish (20-24 °C). b Stat = static conditions, but water renewed every 12 h; flow = flow-through conditions (DEHP concentration in water continuously maintained) c Hardness is expressed as mg calcium carbonate per litre. The tests were performed in fresh water except where stated otherwise. DeFoe et al. (1990) exposed rainbow trout (Oncorhynchus mykiss) and Japanese medaka (Oryzias latipes) to DEHP using concentrations of up to 0.502 mg/litre for a 90-day trout embryo-larval test and 0.554 mg/litre for a 168-day larval test on medaka. No significant adverse effects were observed on trout hatchability, survival or growth, but there was a significant reduction in the growth of Japanese medaka. Mayer & Sanders (1973) studied the effects of DEHP on the reproduction of zebra fish (Brachydanio rerio) and guppies (Lebistes reticulatus). During the 90-day dietary exposure, zebra fish were fed on 50 or 100 mg DEHP/kg food, and guppies were fed 100 mg/kg. Although the treated zebra fish spawned more frequently than the control fish, the latter produced more eggs per spawn. Fry survival was significantly reduced by DEHP. Treated guppies produced fewer fry per adult and had an 8% incidence of abortions, whereas there were no abortions in the control group (no statistics were given). In a study of fish exposed to DEHP, Mayer et al. (1977) found reduced vertebral collagen levels after 150 days at a DEHP concentration of 3.7 µg/litre in adult brook trout (Salvelinus fontinalis), after 127 days at 11 µg/litre in fathead minnow, and after 90 days at 5 µg/litre in rainbow trout. However, there was no effect on fish growth. Pfuderer & Francis (1975) found that DEHP had no effect on the heart rate of goldfish when the fish were exposed to 200 mg/litre for 10 min. 9.2.3 Amphibians The acute toxicity of DEHP to tadpoles of Fowler's toad and the leopard frog is given in Table 12. When Larsson & Thuren (1987) exposed eggs of the moorfrog (Rana arvalis) to sediment concentrations of between 10 and 800 mg DEHP/kg (fresh weight), the number of tadpoles hatching decreased with increasing exposure concentration. The percentage hatch was 90% for the controls eggs, 50% at 150 mg DEHP/kg, and < 30% at > 400 mg/kg. The lowest DEHP concentration that caused a significant decrease in the number of tadpoles hatching was 25 mg/kg. After hatching, the survival of tadpoles was unaffected. There were no delays in hatching and no abnormalities in the developing tadpoles exposed to the various DEHP concentrations. Table 12. Toxicity of DEHP to frog and toad tadpolesa Organism LC50 (mg/litre) 95% confidence limits Fowler's toad 3.88 3.08-4.84 (Bufo fowleri) Leopard frog 4.44 3.65-5.37 (Rana pipiens) a From: Birge et al. (1978). Exposure occurred under static conditions, but water was renewed every 12 h. It was initiated 2 to 6 h after spawning and continued until 4 days after hatching. The hatching times varied between 3 and 4 days, and so the exposure period varied between 7 and 8 days. The temperature was 20-24 °C, hardness 90-115 mg CaCO3/litre, and pH 7.6-8.1. Wams (1987) quoted an unpublished Dutch report which stated that an exposure of 2 mg DEHP/litre caused a reduction in the growth rate of clawed toad (Xenopus laevis) larvae. 9.3 Toxicity to terrestrial organisms 9.3.1 Plants When Herring & Bering (1988) grew spinach and pea plants from seed for 14 to 16 days in soil containing 100 mg DEHP/kg, there was no effect on plant growth, as measured by height. The effect of DEHP on seed germination was observed by placing seeds in petri dishes containing a solution of 100 mg/litre. A reduction of 40% to 50% in the number of seeds germinating was found. Lokke & Rasmussen (1983) reported that DEHP had no visible effect on Sinapis alba, Brassica napusor or Achillea millefolium when sprayed in the field at concentrations of up to 87.5 kg/ha. According to Stanley & Tapp (1982), DEHP at a level of 1000 mg/kg soil had no effect on the growth of rape (Brassica rapa) and only a slight effect on that of oats (Avena sativa). 9.3.2 Earthworms In a series of contact toxicity tests, red earthworms (Eisenia foetida) were exposed to DEHP via filter paper in glass vials. An LD50 could not be calculated because DEHP was not toxic even at the highest dose of 25 mg/cm2 (Neuhauser et al., 1986). 9.3.3 Insects Al-Badry & Knowles (1980) found DEHP to be non-toxic to female houseflies (Musca domestica) when applied topically or by injection at a concentration of 20 µg/fly (equivalent to 1000 mg/kg). When it was topically applied simultaneously with various organophosphates, an antagonistic interaction was apparent; mortality of 85% to 95% was reduced to less than 10% by the DEHP. However, when DEHP was topically applied 30 min before exposure to an organophosphate concentration that caused 10% to 30% mortality, the resulting interaction was synergistic with mortality rising to over 60% and in most cases to over 80%. 9.3.4 Birds Hill et al. (1975) found no mortality among 10-day-old ring- necked pheasants or mallard fed up to 5000 mg DEHP/kg for 5 days followed by 3 days on a normal diet. When Wood & Bitman (1984) fed broiler hens a diet containing 2000 mg DEHP/kg (226 mg DEHP/hen per day) for 4 weeks, egg production and body weights were significantly decreased. Ishida et al. (1982) reported the cessation of egg production and an abnormality of the ovaries in laying hens fed 5 or 10 g DEHP/kg diet for up to 230 days. In a study by O'Shea & Stafford (1980), starlings (Sturnus vulgaris) were fed on a diet containing 25 or 250 mg DEHP/kg for a 30-day period. At both dose levels, the treated birds gained significantly more body weight than controls during the exposure period, but at the lower dose level food consumption was significantly reduced compared to controls. When Peakall (1974) fed ring doves (Streptopelia risoria) on a diet containing 10 mg/kg, there were no effects on the eggshell thickness, ashed egg weight, rate of water loss, surface area or permeability of the eggs laid. 10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT 10.1 Evaluation of human health risks 10.1.1 Exposure levels DEHP concentrations of up to 300 ng/m3 have been measured in urban air, but usually the levels in ambient air are well below 100 ng/m3. Data on occupational exposure levels are limited. Concentrations of up to 4.1 mg/m3 have been reported but they are usually below 1 mg/m3. Total phthalate exposure levels have been reported to be between 0.1 and 60 mg/m3. Clinical treatment, including transfusion, haemodialysis, extracorporal circulation, and artificial respiration, may lead to DEHP exposure. The exposure to DEHP from drinking-water and food is low. 10.1.2 Toxic effects Dose-dependent kinetics of DEHP or its metabolites and species differences in the metabolism have been confirmed in several studies. In animals some inhalation studies have been performed. However, no consistent findings are reported. The oral and intraperitoneal LD50 values exceed 25 g/kg, indicating that DEHP has low acute toxicity. In rats and mice, DEHP administration produces hepatic hyperplasia and hypertrophy, which is characterized by peroxisome proliferation. The threshold dose level for hepatic peroxisome proliferation in the rat is approximately 50 mg/kg per day. High DEHP dose levels (12 g DEHP/kg diet in rats; 6 g DEHP/kg diet in mice) in a feeding study resulted in an increased incidence of hepatic tumours in rats and mice. Marked species differences in DEHP-induced hepatomegaly and peroxisome proliferation exist. Rats and mice are very responsive, Syrian hamsters less responsive, and guinea-pigs, marmosets, and cynomolgus monkeys non-responsive. Studies with primary hepatocyte cultures have shown an excellent in vitro/in vivo correlation concerning responsiveness to DEHP/DEHP metabolites. Several studies have demonstrated the non-responsive nature of cultured human hepatocytes to DEHP metabolites and other peroxisome proliferators. The induction of peroxisome proliferation and cell replication is strongly associated with the development of hepatic tumours in rats and mice. Thus, the available data suggest that species which do not respond to the hepatic effects of DEHP are unlikely to be susceptible to the development of hepatic tumours. Testicular atrophy is one of the most consistent effects of DEHP in in vivo studies on a variety of experimental animal species. Such effects have been observed in rats fed 6 g DEHP/kg diet and mice fed 3 g/kg diet, and they are more pronounced in young animals. Hamsters and marmosets appear to be more resistant to the testicular effects of DEHP. When both male and female CD-1 mice were treated with 1 g DEHP/kg diet, a significant reduction in fertility was observed. A dietary level of 20 g DEHP/kg throughout gestation produced an increased incidence of resorptions in rats but no malformations. In the mouse, however, 1 g/kg throughout pregnancy increased the incidence of embryolethality and abnormalities. Sensitivity was greatest on days 7-9 of gestation. The dose level (0.5 g/kg) that induced fetotoxicity in mice did not induce maternal toxicity. Results from several different genotoxicity tests indicate that DEHP and its major metabolites do not exhibit any direct genotoxic effect in either bacteria or in vitro mammalian cells. This has been confirmed in in vivo binding studies, which indicated that DEHP and its metabolites do not interact covalently with DNA. However, it has been established that DEHP has the potential of inducing aneuploidy in fungi as well as in in vitro mammalian cells. There are no consistent results from dominant lethal studies. Few data on the human health effects of DEHP exposure have been reported. There have been a few studies on workers exposed to phthalate mixtures, but no consistent health effects that could be directly related to DEHP have been reported. 10.1.3 Conclusion DEHP causes reproductive and hepatocarcinogenic effects in rats and mice. Testicular atrophy is the main reproductive effect in rats and mice, and young animals are more susceptible than older ones to this effect. The induction of hepatic peroxisome proliferation and cell replication are strongly associated with the liver carcinogenic effect of certain non-genotoxic carcinogens including DEHP. However, marked differences have been observed among animal species with respect to DEHP-induced peroxisome proliferation. Currently there is not sufficient evidence to suggest that DEHP is a potential human carcinogen. 10.2 Evaluation of effects on the environment 10.2.1 Exposure levels DEHP exists widely in the environment, being released during production, processing, usage, and disposal. Transport in the air is the major route by which it enters the environment. It can be deposited by dry or wet deposition and has been found to leach into ground water. DEHP is readily photodegraded in the atmosphere. In the laboratory, aerobic biodegradation has been shown to occur under certain circumstances. However, in the environment aerobic biodegradation seems to be slow and anaerobic degradation is even slower. Based on a measured log Pow of about 5 and measured bioaccumulation factors for biota and sediment, it can be seen that DEHP is highly bioaccumulative. In rivers and lakes, DEHP concentrations of up to 4 µg/litre have been measured. Due to its hydrophobic nature, DEHP adsorbs readily to soil, sediment, and particulate matter. Levels of up to 70 mg/kg dry weight have been found in river sediments, but near to a discharge point levels of up to 1480 mg/kg dry weight have been reported. 10.2.2 Toxic effects Most acute toxicity studies on aquatic organisms show DEHP to be of low toxicity. However, one study suggested a greater sensitivity for the water flea (Daphnia pulex), the nominal 48-h LC50 being 133 µg/litre. In a 21-day study on Daphnia magna, the NOEL for biochemical and behavioural effects was 72 µg/litre. A significant increase in mortality was recorded in trout fry exposed to 14 µg DEHP/litre from 12 days prior to hatching. At concentrations of 3.7 to 11 µg/litre, reductions in vertebral collagen were reported in three species of fish exposed for 150 days. Zebra fish fry survival and guppy reproduction were adversely affected at DEHP concentrations in food of 50 and 100 mg/kg, respectively, during a 90-day exposure period. In two separate studies, DEHP concentrations of 25 mg/kg (wet weight) of sediment significantly reduced microbial activity and the number of frog tadpoles hatching. Available studies indicate that the acute toxicity of DEHP to algae, plants, earthworms, and birds is low. There are no data relating to effects upon wild mammals. 10.2.3 Conclusion There is no documented information that DEHP presents any hazard, based on acute exposure to fish and daphnids. However, a reduction of microbial activity in sediment at environmental levels of DEHP was reported. A comparison between environmental levels and the concentrations that produce effects in prolonged studies, especially early life-stage tests on fish and amphibians, indicates that a hazard for the environment, particularly via water and sediment, cannot be excluded. Adverse effects on organisms are likely in areas with highly contaminated water and sediments which are near to point emission sources. Although few relevant studies have been reported, the acute toxicity of DEHP to algae, plants, earthworms, and birds appears to be low. 11. RECOMMENDATIONS FOR PROTECTION OF HUMAN HEALTH AND THE ENVIRONMENT a) Current disposal practises should be improved. b) Measures must be undertaken to reduce the release of DEHP to the environment. c) Medical devices and products that contribute to the body burden of DEHP must be scrutinized to reduce exposure to DEHP via the intravenous route. 12. FURTHER RESEARCH More research is needed on the effects of DEHP on the ecosystem of sediments and on the subject of DEHP leaching, particularly from landfill to ground water. Since DEHP occurs in the air, including workplace air, further inhalation studies are needed. Epidemiological surveys on exposed populations should be encouraged. Further studies are needed on the mechanism of peroxisome- proliferator-induced hepatocarcinogenicity, with emphasis on human health effects. 13. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES DEHP was one of the substances discussed by a working group of the International Agency for Research on Cancer in 1981 (IARC, 1982). The evaluation by the group was that there was sufficient evidence for the carcinogenicity of DEHP in mice and rats, but that no adequate epidemiological study was available. The working group concluded that DEHP is possibly carcinogenic to humans and placed it in group 2B (IARC, 1987). DEHP was evaluated at the 28th meeting of the Joint FAO/WHO Expert Committee on Food Additives. The recommendation was to reduce human exposure to DEHP from food-contact materials to the lowest level technologically attainable (WHO/FAO, 1984a,b). During the 33rd meeting of the Joint FAO/WHO Expert Committee on Food Additives, DEHP was again evaluated and the recommendation was the same: DEHP in food should be reduced to the lowest level attainable. 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Identité, propriétés physiques et chimiques et méthodes d'analyse Le phtalate de di(2-éthylhexyle) est un ester de l'acide benzènedicarboxylique qui se présente, à la température ambiante, sous la forme d'un liquide huileux, incolore à jaune. Il est peu soluble dans l'eau (0,3-0,4 mg/litre) et encore moins dans l'eau salée. Il est miscible à la plupart des solvants organiques courants. Sa volatilité est relativement faible (tension de vapeur: 8,6 x 10-4Pa). On a mis au point, pour le dosage du DEHP dans différents milieux, un grand nombre de méthodes de prélèvement et d'analyse. On fait de plus en plus appel à des méthodes sensibles telles que la chromatographie en phase gazeuse, la chromatographie en phase liquide à haute performance et la spectrométrie de masse. La contamination due au matériel en plastique utilisé lors des prélèvements et de l'analyse complique le dosage du DEHP à faibles concentrations. 2. Sources d'exposition humaine et environnementale Le DEHP présent dans l'environnement est, dans sa presque totalité, d'origine artificielle. La production mondiale de DEHP augmente depuis quelques décennies et s'élève actuellement à environ un million de tonnes par an. Les Etats-Unis d'Amérique et l'Europe entrent chacun pour un tiers dans cette production. Le DEHP est le plastifiant le plus largement utilisé pour augmenter la fluidité des résines puisqu'il représente 50% de l'ensemble des esters phtaliques utilisés comme plastifiants. Il peut représenter jusqu'à 40% en poids ou davantage de la matière plastique. Le DEHP est utilisé dans la fabrication du chlorure de polyvinyle (PVC) utilisé dans l'industrie du bâtiment et de l'emballage ainsi que pour la fabrication d'accessoires à usage médical. On l'utilise également en plus petites quantités pour la préparation de peintures industrielles ainsi que comme diélectrique dans les condensateurs. Les produits contenant du plastique qui sont mis au rebut peuvent être éliminés soit par incinération soit par enfouissement dans une décharge. Lors de l'incinération à basse température, une proportion importante du produit peut se dissiper dans l'atmosphère. On n'a pas très bien étudié ce qu'il advient du DEHP déposé dans les décharges et il n'est pas possible à ce stade de tirer des conclusions définitives. 3. Transport, distribution, et transformation dans l'environnement C'est principalement en étant transportés dans l'atmosphère que les phtalates pénètrent dans l'environnement. A partir de là, le DEHP tombe sur le sol à moins d'être éliminé par les précipitations. Le coefficient de partage du DEHP entre l'octanol et l'eau est élevé, de sorte que l'équilibre entre l'eau et un sol ou un sédiment riche en substances organiques s'établit aux dépens de la phase aqueuse. Le DEHP s'adsorbe facilement sur les particules organiques et du sol. Bien que peu soluble dans l'eau, le DEHP peut se rencontrer en quantités relativement élevées dans les eaux de surface en raison de son adsorption sur les particules organiques et de son interaction avec les substances organiques en solution. L'adsorption s'effectue plus particulièrement sur les particules de petite taille et elle est favorisée par la présence d'eau salée. Dans l'atmosphère, le DEHP subit une photodégradation rapide mais son hydrolyse dans l'environnement est pratiquement inexistante. Plusieurs microorganismes terricoles effectuent la dégradation aérobie du DEHP. Toutefois dans l'environnement, cette dégradation microbienne s'effectue avec lenteur. La biodégradation commence par l'hydrolyse du mono-ester qui est transformé en acide phtalique. Il y a ensuite dégradation en pyruvate et succinate par ouverture du cycle puis en CO2 et H2O, comme dans la dégradation métabolique de l'acide benzoïque. La dégradation aérobie dépend de la température. Au dessous de 10 °C la dégradation est peu importante. A température supérieure, la biodégradation se produit dans la partie supérieure du sol mais, à plus grande profondeur elle est pratiquement inexistante du fait des conditions d'anaérobiose. La dégradation anaérobie, pour autant qu'elle existe, est beaucoup plus lente que la dégradation aérobie. Le DEHP est très lipophile et modérément persistant. Le degré de bioaccumulation dépend de la capacité de l'organisme qui l'absorbe à métaboliser la substance. On a montré qu'il s'accumulait en forte proportion chez divers invertébrés aquatiques, les poissons et les amphibiens. Après application de DEHP sur des feuilles de végétaux, on n'a constaté qu'une faible perte de matière sur une période de 15 jours. Le DEHP présent dans le sol ou dans les boues d'égout n'est que peu fixé par les plantes. 4. Concentrations dans l'environnement et exposition humaine Le DEHP est très répandu dans l'environnement et on le retrouve dans la plupart des échantillons prélevés dans l'air, dans les précipitations, l'eau, les sédiments, le sol et les biotes. C'est généralement dans les zones industrielles que les teneurs sont les plus fortes. On a relevé des concentrations allant jusqu'à 300 ng/m3 dans l'air des villes et en atmosphère polluée. Dans l'atmosphère surmontant les océans, on a fait état de concentrations allant de 0,5 à 5 ng/m3 et les précipitations de ces régions en contenaient jusqu'à 200 ng/litre. L'étude d'échantillons de précipitations prélevés dans un secteur situé à proximité d'une usine produisant des plastifiants, a montré que la vitesse de dépot à sec était de 0,7 à 4,7 µg/m2 et par jour. Dans les rivières et les lacs, on a trouvé des concentrations de DEHP allant jusqu'à 4 µg/litre, les teneurs les plus élevées étant relevées au point de décharge d'effluents industriels. Dans la mer, la concentration est inférieure à 1 µg/litre, les concentrations les plus fortes étant observées dans les estuaires. En raison de son caractère hydrophobe, le DEHP est facilement absorbé par le sol, les sédiments et les particules. Dans les sédiments de cours d'eau, des concentrations allant jusqu'à 70 mg/kg (de poids à sec) ont été signalées, concentrations qui peuvent monter jusqu'à 1480 mg/kg (de poids à sec) au voisinage des points de décharge. Dans les biotes, la concentration du DEHP varie de moins de 1 à 7000 µg/kg. On le rencontre dans divers types d'aliments: poisson, coquillages, oeufs et fromages. L'exposition moyenne a été estimée à environ 300 µg par personne et par jour aux Etats-Unis d'Amérique en 1974 et à 20 µg par personne et par jour au Royaume-Uni en 1986. Les transfusions de sang et autres traitements utilisant du matériel en plastique peuvent entraîner l'exposition involontaire des malades au DEHP. C'est ainsi qu'on a relevé dans le poumon de malades ainsi traités, des teneurs allant de 13,4 à 91,5 mg/kg de poids à sec. Les quelques données disponibles indiquent que sur les lieux de travail, la concentration de DEHP est généralement inférieure à 1 mg/m3. 5. Cinétique et métabolisme Des données disponibles au sujet de l'administration par voie orale indiquent que le DEHP est hydrolysé dans l'intestin par la lipase pancréatique. Les métabolites qui se forment, c'est-à-dire le phtalate de mono-2-éthylhexyle (MEHP) et le 2-éthylhexanol sont rapidement absorbés. Après administration de DEHP marqué au 14C (2,9 mg/kg) par voie orale à des rats, on a récupéré 50% de la dose initiale dans l'urine et la bile de ces animaux. Il semble que la biodisponibilité d'une dose orale de DEHP soit plus élevée chez les jeunes animaux. Après administration par voie orale, le DEHP est largement hydrolysé dans l'intestin de certains animaux, par exemple les rats, et se répartit dans l'organisme, principalement sous forme de phtalate de monoéthylhexyle (MEHP). Toutefois chez les primates et l'homme, l'hydrolyse est beaucoup moins importante. Il semble que ce soit principalement au niveau du foie que s'effectue la métabolisation du MEHP et du 2-éthylhexanol. On a identifié plusieurs autres métabolites, l'omega et omega-1-oxydation étant les principales voies métaboliques. Un ou plusieurs des produits résultant de l'omega- oxydation peuvent encore être métabolisés par ß-oxydation. On a constaté que l'omega-oxydation s'effectuait selon une cinétique non linéaire. Le métabolisme du DEHP offre des différences considérables selon les espèces; par exemple, l'omega-oxydation est moins importante chez l'homme que chez le rat. Une dose de DEHP de 2,9 mg/kg, administrée par voie orale à des rats, a été récupérée une semaine plus tard presque intégralement dans les matières fécales et l'urine des animaux. La bile et l'urine constituent les principales voies d'excrétion. Lors d'une étude sur l'homme, 15 à 25% d'une dose orale (0,45 mg/kg de DEHP) ont été excrétés sous forme de MEHP et la majeure partie des produits d'excrétion étaient constitués de métabolites oxydés. 6. Effets sur les mammifères de laboratoire et les systèmes d'épreuve in vitro La DL50 par voie orale du DEHP est d'environ 25 à 34 g/kg, selon l'espèce, mais cette valeur est plus faible dans le cas du MEHP. Lors d'études d'alimentation sur des rats et des souris, on a constaté que des doses quotidiennes de DEHP supérieures à 3 g/kg entraînaient la mort dans les 90 jours et qu'une dose de 0,4 g/kg réduisait le gain de poids en l'espace de quelques jours. Dans d'autres études, c'est une dose de 6,3-12,5 g/kg de nourriture qui a entraîné une réduction du poids corporel. Lors d'études à long terme, on a observé chez les animaux traités une hépatomégalie accompagnée d'une augmentation du poids relatif des reins. Dans l'une de ces études, on observait également une hypertrophie cellulaire au niveau du lobe antérieur de l'hypophyse. Un certain nombre d'études ont permis de mettre en évidence une atrophie testiculaire, apparaîssant au bout de quelques jours, et liée à l'administration de DEHP (doses dans l'alimentation allant de 10-20 g/kg). Les jeunes rats semblent plus vulnérables et les rats et les souris plus sensibles que les hamsters et les ouistitis. On a constaté que cette atrophie était réversible. Le MEHP exerce des effets toxiques in vitro sur les cellules de Sertoli. Le DEHP de même que le MEHP ont des propriétés tératogènes. Des malformations ont été observées à des doses de 0,5-2 g/kg de nourriture chez la souris et, à des doses supérieures à 10 g/kg, on a observé des effets embryotoxiques. Les tests de mutagénicité et autres manifestations du même genre ont donné des résultats négatifs dans la plupart des études. Le DEHP peut provoquer la transformation des cellules et on a montré qu'il était cancérogène à des doses respectives de 6 et 12 g/kg de nourriture chez le rat et de 3 et 6 g/kg de nourriture chez la souris. On a constaté une augmentation, liée à la dose, des tumeurs hépatocellulaires chez les deux sexes de l'une et l'autre espèce. La prolifération des peroxysomes hépatiques et la réplication cellulaire sont fortement liées aux effets cancérogènes sur le foie de certains produits non génotoxiques comme le DEHP. Toutefois on a observé des différences importantes entre les espèces animales en ce qui concerne la prolifération des peroxysomes induite par le DEHP. Contrairement à ce qui se passe dans le cas des hépatocytes de rat, les métabolites du DEHP ne provoquent pas de prolifération des peroxysomes dans les cultures d'hépatocytes humains. 7. Effets sur l'homme On ne dispose que de données très limitées sur les effets que le DEHP peut exercer sur l'homme. On a fait état chez deux sujets qui avaient reçu 5 ou 10 g de DEHP respectivement, d'effets qui se limitaient à de légers troubles gastriques. 8. Effets sur les autres êtres vivants en laboratoire et dans leur milieu naturel Dans la plupart des études, les valeurs nominales de la CL50 obtenues lors des tests de toxicité aiguë dépassent 10 mg/litre, ce qui indique que le DEHP est peu toxique. Toutefois ces valeurs sont supérieures à la solubilité du DEHP dans l'eau (0,3-0,4 mg/litre). D'après une étude, une daphnie, Daphnia pulex, serait particulièrement sensible, la CL50 à 48 heures étant dans son cas de 0,133 mg/litre. La seule épreuve de toxicité aiguë où l'on ait mesuré des concentrations de DEHP a été effectuée sur un poisson, Pimephales promelas, et elle a donné une CL50 à 96 heures supérieure à 0,33 mg/litre. Lors d'études de longue durée, on a obtenu une dose sans effet nocif observable de 72 µg/litre pour Daphnia magna. Pour les poissons adultes, cette dose était supérieure à 72 µg/litre. Une exposition à une concentration de 14 µg/litre, pendant 12 jours avant l'éclosion, a provoqué une importante augmentation de la mortalité parmi des alevins de truites. Des concentrations comprises en 3,7 et 11 µg/litre ont entraîné une réduction de la teneur en collagène vertébral chez les poissons. La présence de DEHP dans la nourriture à raison de 50 mg/kg a eu des effets nocifs sur la survie des alevins de Melambaphes zebra. A la concentration de 25 mg/kg en poids dans les sédiments, on a constaté une réduction de l'activité microbienne et du nombre d' éclosions chez les tétards. Le DEHP est peu toxique pour les algues, les végétaux, les lombrics et les oiseaux. 9. Evaluation Le DEHP exerce des effets cancérogènes sur le foie et affecte le système reproducteur chez le rat et la souris. Le principal effet au niveau des gonades consiste, chez le rat et la souris, en une atrophie des testicules qui affecte davantage les jeunes animaux. La prolifération des peroxysomes hépatiques et la réplication cellulaire sont fortement liées à l'effet cancérogène qu'exercent sur le foie certains produits non génotoxiques au nombre desquels figure le DEHP. Toutefois, on a observé de fortes différences selon les espèces animales pour ce qui est de la prolifération des peroxysomes induite par le DEHP. On ne possède pas actuellement de preuves suffisantes permettant de conclure que le DEHP est potentiellement cancérogène pour l'homme. Il n'existe pas de données attestées selon lesquelles le DEHP serait dangereux pour la faune, si l'on s'en tient aux résultats obtenus lors de l'exposition de poissons et de daphnies. Toutefois, on a observé une réduction de l'activité microbienne dans les sédiments aux doses auxquelles le DEHP est présent dans l'environnement. Si l'on compare les doses de DEHP présentes dans l'environnement aux concentrations qui sont susceptibles de produire des effets lors d'études de longue durée, notamment sur les larves de poissons et d'amphibiens, on ne peut exclure l'existence d'un risque pour l'environnement, les effets s'exerçant notamment par l'intermédiaire de l'eau et des sédiments. Des effets nocifs sur les êtres vivants sont possibles dans les secteurs où l'eau et les sédiments sont fortement pollués en raison de leur proximité des points de décharge. Bien que peu d'études valables aient été effectuées, il semble que le DEHP présente une faible toxicité aiguë pour les algues, les végétaux, les lombrics et les oiseaux. RESUMEN 1. Identidad, propiedades físicas y químicas y métodos analíticos El di(2-etilhexil) ftalato (DEHF) es un éster del ácido bencenodicarboxílico que a temperatura ambiente es un líquido oleoso entre incoloro y amarillo. Su solubilidad en agua es baja (0,3-0,4 mg/litro) y aún más baja en agua salada. Es miscible con la mayor parte de los disolventes orgánicos normales. La volatilidad del DEHF es relativamente baja (8,6 x10-4 Pa). Se han perfeccionado numerosos métodos de muestreo y análisis para la determinación del DEHF en diferentes medios. Cada vez se utilizan más métodos de gran sensibilidad, como la cromatografía de gases, la cromatografía líquida de alto rendimiento y la espectrometría de masas. El análisis de concentraciones bajas de DEHF se complica por la contaminación debida al material de plástico utilizado en el muestreo y el análisis. 2. Fuentes de exposición humana y ambiental Casi todo el DEHF presente en el medio ambiente procede de fuentes antropogénicas. La producción mundial de DEHF ha ido aumentando durante los últimos decenios y en la actualidad es de alrededor de 1 x 106 toneladas al año. Un tercio del total se produce en los Estados Unidos y otro tercio en Europa. El DEHF es el plastificante más utilizado (representa el 50% de todos los plastificantes a base de ésteres de ftalato) para ablandar las resinas. A él corresponde el 40% (p/p) o más del total de plásticos. El DEHF se emplea en la fabricación de cloruro de polivinilo (PVC) utilizado en los edificios, en la construcción, en embalajes y en componentes de aparatos médicos. Se utiliza en cantidades más pequeñas en las pinturas industriales y como fluido dieléctrico en los condensadores. Los productos plastificados de desecho se pueden eliminar por incineración o terraplenado. Durante la incineración a baja temperatura, se puede liberar un porcentaje elevado del DEHF a la atmósfera. No se ha estudiado bien el destino medioambiental del DEHF depositado en terraplenes, por lo que no se pueden sacar conclusiones definitivas. 3. Transporte, distribución y transformación en el medio ambiente El transporte en el aire es la vía más importante de incorporación de los ftalatos al medio ambiente. De la atmósfera, el DEHF precipita o es lavado por la lluvia. El DEHF tiene un coeficiente de reparto octanol-agua elevado, de manera que el equilibrio entre el agua y un suelo o sedimento rico en materia orgánica está desplazado a favor de este último. Las partículas orgánicas del suelo lo adsorben fácilmente. Aunque la solubilidad del DEHF en agua es baja, la cantidad presente en las aguas de superficie puede ser mayor debido a la adsorción a las partículas orgánicas y a la interacción con la materia orgánica disuelta. Las pequeñas partículas lo adsorben más fácilmente, y el proceso se potencia en el agua salada. La fotodegradación atmosférica del DEHF es rápida; en cambio, su hidrólisis química en el medio ambiente es prácticamente inexistente. Se ha observado que varios microorganismos del suelo llevan a cabo la degradación aerobia. Sin embargo, se ha informado que la degradación microbiana del DEHF en el medio ambiente es lenta. El proceso de biodegradación comienza con la hidrólisis para formar el monoéster, que se transforma luego en ácido ftálico. La degradación con ruptura del anillo para dar primero piruvato y succinato y después CO2 y H2O es análoga a la vía metabólica del ácido benzoico. La degradación aerobia es dependiente de la temperatura. Por debajo de los 10 °C es escasa. A temperaturas más altas se produce biodegradación en la capa más superficial del suelo, pero es prácticamente inexistente en las capas más profundas, donde las condiciones son de anaerobiosis. La degradación anaerobia, si existe, es mucho más lenta que la aerobia. El DEHF es muy lipófilo y moderadamente persistente. El grado de bioacumulación depende de la capacidad de los organismos para metabolizarlo. Se ha observado un grado elevado de acumulación en diversos invertebrados acuáticos, peces y anfibios. Cuando se aplicó DEHF a hojas de plantas, la pérdida fue pequeña durante un período de 15 días. Se ha observado que su absorción por las plantas a partir del suelo o de los fangos cloacales es muy baja. 4. Niveles medioambientales y exposición humana El DEHF está muy ampliamente distribuido en la naturaleza y se encuentra en la mayor parte de las muestras, por ejemplo de aire, precipitación, sedimento, suelo y biota. Las concentraciones más altas aparecen generalmente en las zonas industrializadas. En el aire urbano y contaminado se han encontrado concentraciones de hasta 300 ng/m3. Se han comunicado de niveles comprendidos entre 0,5 y 5 ng/m3 en el aire de zonas oceánicas, y la lluvia de esas zonas contenía hasta alrededor de 200 ng/litro. En muestras de precipitación procedentes de una zona cercana a una fábrica de plastificantes se observó que la tasa sedimentación seca era de 0,7 a 4,7 µg/m2 al día. Se han encontrado concentraciones de DEHF en ríos y lagos de hasta 4 µg/litro; los niveles más altos están asociados a los puntos de descarga de efluentes industriales. La concentración en el mar es inferior a 1 µg/litro, y los niveles más altos se detectan en los estuarios. Debido a su carácter hidrófobo, el DEHF se adsorbe muy fácilmente al suelo, los sedimentos y la materia particulada. En los sedimentos fluviales se han comunicado niveles de hasta 70 mg/kg (peso seco), y cerca de los puntos de descarga ha llegado a ser de 1480 mg/kg (peso seco). La concentración de DEHF en la biota oscila entre menos de 1 y 7000 µg/kg. Se ha detectado en diversos tipos de alimentos, como pescado, marisco, huevos y queso. En 1974 se estimó en los Estados Unidos una exposición media de 300 µg/persona al día y en el Reino Unido en 1986 de 20 µg/persona al día. Las transfusiones de sangre y otros tipos de tratamiento médico en los que se utilizan aparatos de plástico pueden dar lugar a una exposición humana involuntaria al DEHF. En el tejido pulmonar de algunos pacientes se han detectado concentraciones comprendidas entre 13,4 y 91,5 µg/kg (peso seco). Los escasos datos disponibles indican que las concentraciones de DEHF en el lugar de trabajo suelen estar por debajo de los 1 mg/m3. 5. Cinética y metabolismo Los datos disponibles sobre la administración oral indican que la lipasa pancreática hidroliza el DEHF en el intestino. Los metabolitos formados, que son el mono (2-etilhexil) ftalato (MEHF) y el 2-etilhexanol, se absorben rápidamente. Cuando se administró por vía oral a ratas DEHF marcado con 14C (2,9 mg/kg), se recuperó en la orina o la bilis más del 50%. La biodisponibilidad de una dosis oral de DEHF parece ser más elevada en las ratas jóvenes que en las más viejas. El DEHF administrado por vía oral se hidroliza en el intestino de ciertos animales, por ejemplo las ratas, y se distribuye sobre todo como monoetilhexil ftalato (MEHF). Sin embargo, la hidrólisis es mucho menor en los primates y el ser humano. El MEHF se liga a las proteínas del plasma. El hígado parece ser el principal órgano metabolizador del MEHF y del 2-etilhexanol. Se han identificado algunos otros metabolitos, siendo las principales vías metabólicas la omega-y la omega-1-oxidación. Uno o varios productos de la omega-oxidación pueden metabolizarse ulteriormente por ß-oxidación. En la omega-oxidación se han observado cinéticas no lineales. En el metabolismo del DEHF se registran considerables diferencias entre las especies; por ejemplo, la omega-oxidación es menor en el hombre que en las ratas. Una semana después de administrar una dosis oral de DEHF (2,9 mg/kg) se recuperó casi el 100% en las heces y la orina de ratas. La bilis y la orina son las principales vías de excreción. En un estudio humano, del 15 al 25% de la dosis oral (0,45 mg/kg) de DEHF se excretó en forma de MEHF; los metabolitos oxidados constituían la parte más importante de los productos de excreción. 6. Efectos en los mamíferos de laboratorio y en sistemas de ensayo in vitro La DL50 del DEHF por vía oral es de alrededor de 25-34 g/kg, según las especies, pero el valor para el MEHF es más bajo. En estudios de alimentación en ratas y ratones, las dosis de DEHF superiores a 3 g/kg al día causaron muertes en un plazo de 90 días, y con una concentración de 0,4 g/kg al día se redujo el aumento de peso a los pocos días. En otros estudios, con dosis de 6,3-12,5 g/kg de alimentos se produjo una reducción del peso corporal. En los animales tratados en estudios de larga duración se ha observado hepatomegalia y un aumento del peso relativo de los riñones. En un estudio se encontraron también células hipertróficas en el lóbulo anterior de la hipófisis. En varios estudios se ha detectado atrofia testicular, evidente al cabo de pocos días, relacionada con la administración de DEHF (concentraciones de 10-20 g de DEHF/kg de alimentos). Las ratas más jóvenes parecen ser más susceptibles que las viejas, y las ratas y los ratones parecen ser más sensibles que los titís y los hámsters. Se ha observado que la atrofia es reversible. El MEHF tiene efecto tóxico in vitro en las células de Sertoli. Tanto el DEHF como el MEHF muestran propiedades teratogénicas. Con concentraciones de 0,5 a 2 g/kg de alimentos se observaron malformaciones en ratones, y con niveles superiores a los 10 g/kg se apreciaron efectos embriotóxicos. Las pruebas de mutagenicidad y puntos finales asociados han dado resultados negativos en la mayor parte de los estudios. El DEHF puede inducir la transformación celular y se ha demostrado que con dosis de 6 y 12 g/kg de alimentos en ratas y de 3 y 6 g/kg de alimentos en ratones tiene efectos carcinógenos. En ambos sexos de las dos especies se produjo un aumento de tumores hepatocelulares dependiente de la dosis. La inducción de la proliferación de peroxisomas hepáticos y de la replicación celular está fuertemente vinculada al efecto carcinógeno en el hígado de determinados compuestos carcinógenos no genotóxicos, entre los que figura el DEHF. Sin embargo, se han observado notables diferencias entre distintas especies animales con respecto a la proliferación de peroxisomas inducida por el DEHF. A diferencia de lo que ocurre con los hepatocitos de rata, los metabolitos del DEHF no inducen proliferación de peroxisomas en cultivos de hepatocitos humanos. 7. Efectos en el ser humano La información disponible sobre los efectos del DEHF en el ser humano es muy escasa. Se han comunicado dos casos de trastornos gástricos leves, con dosis de 5 y 10 g de DEHF, pero sin ningún otro efecto nocivo. 8. Efectos en otros organismos en el laboratorio y en el medio ambiente En la mayoría de los estudios de toxicidad aguda se han obtenido unos valores nominales para la CL50 que se sitúan por encima de los 10 mg/litro, por lo que el índice de toxicidad del DEHF es bajo. Sin embargo, esos niveles son superiores a los correspondientes a su solubilidad en agua (0,3-0,4 mg/litro). En un estudio se detectó en la pulga de agua Daphnia pulex una sensibilidad mayor, con un valor nominal de la CL50 a las 48 h de 0,133 mg/litro. La única prueba de la toxicidad aguda realizada con concentraciones medidas de DEHF se hizo en el pez Pimephales promelas y puso de manifiesto una CL50 > 0,33 mg/litro a las 96 h. En estudios prolongados, el nivel sin observación de efectos (NOEL) en Daphnia magna fue de 72 µg/litro. En peces adultos se determinó un NOEL > 62 µg/litro. Una exposición a 14 µg/litro desde 12 días antes de la eclosión produjo un significativo aumento de la mortalidad de los alevines de trucha. Las concentraciones entre 3,7 y 11 µg/litro provocaron una reducción del colágeno vertebral en peces. Con concentraciones de 50 mg/kg de alimentos, el DEHF tiene efectos adversos sobre la supervivencia de los alevines de Brachydanio rerio. Concentraciones de 25 mg/kg (p/p) en el sedimento redujeron de manera considerable la actividad microbiana y el número de renacuajos que nacían. La toxicidad aguda del DEHF en algas, plantas, lombrices de tierra y aves es baja. 9. Evaluación El DEHF tiene efectos reproductivos y hepatocarcinogénicos en ratas y ratones. El principal efecto sobre la reproducción en ratas y ratones es la atrofia testicular; los animales jóvenes son más susceptibles que los viejos. La inducción de la proliferación de peroxisomas hepáticos y de la replicación celular está estrechamente relacionada con el efecto carcinogénico en el hígado de determinados compuestos carcinógenos no genotóxicos, incluido el DEHF. Sin embargo, se han observado notables diferencias entre las distintas especies de animales con respecto a la proliferación de peroxisomas inducida por el DEHF. En la actualidad no hay pruebas suficientes que indiquen que el DEHF sea un posible carcinógeno para el ser humano. No hay información documentada de que el DEHF constituya un riesgo, tomando como base la exposición aguda de peces y dáfnidos. Sin embargo, se ha informado de una reducción de la actividad microbiana en los sedimentos con los niveles de DEHF presentes en el medio ambiente. La comparación entre los niveles medioambientales y las concentraciones que producen efectos en estudios prolongados, especialmente las pruebas en las fases vitales tempranas de peces y anfibios, indica que no se puede descartar un cierto peligro para el medio ambiente, sobre todo por conducto del agua y los sedimentos. Es probable que se produzcan efectos adversos en los organismos en zonas con agua y sedimentos muy contaminados que están próximos a las fuentes de emisión. Aunque son pocos los estudios que se conocen al respecto, la toxicidad aguda del DEHF en algas, plantas, lombrices de tierra y aves parece ser baja.