INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY ENVIRONMENTAL HEALTH CRITERIA 153 Carbaryl This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organisation, or the World Health Organization. First draft prepared by Professor F. Kaloyanova (National Center of Hygiene and Medical Ecology Sofia, Bulgaria) and Dr. P.P. Simeonova (University of Sofia, Bulgaria) Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization World Health Orgnization Geneva, 1994 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 Hexachlorobutadiene. (Environmental health criteria: 153) 1. Carbaryl - adverse effects 2. Carbaryl - toxicity 3. Environmental exposure I.Series ISBN 92 4 157153 5 (NLM Classification QU 98) 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 1994 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 impression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of every 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 1. SUMMARY AND EVALUATION, CONCLUSIONS AND RECOMMENDATIONS 1.1. Summary and evaluation 1.1.1. Identity, properties, and analytical methods 1.1.2. Production and uses 1.1.3. Environmental transport, distribution, and transformation 1.1.4. Environmental levels and human exposure 1.1.5. Kinetics and metabolism 1.1.6. Effects on organisms in the environment. 1.1.7. Effects on experimental animals and in vitro test systems 22.214.171.124 Reproduction 126.96.36.199 Mutagenicity 188.8.131.52 Carcinogenicity 184.108.40.206 Effects on different organs and systems 220.127.116.11 Primary mechanism of toxicity 1.1.8. Effects on humans 1.2. Conclusions 1.2.1. General population exposure 1.2.2. Subpopulations at high risk 1.2.3. Occupational exposure 1.2.4. Environmental effects 1.3. Recommendations 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, 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 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION 4.1. Volatilization and air transportation 4.2. Water 4.2.1. Hydrolysis 4.2.2. Photolysis 4.2.3. Degradation by microorganisms 4.2.4. Persistence in surface water 4.2.5. Removal from water 4.2.6. Persistence in sea water 4.2.7. Bioaccumulation/biomagnification 4.3. Soil 4.3.1. Adsorption, desorption 4.3.2. Transformation 18.104.22.168 Photolysis in soil 4.3.3. Biotransformation in soil 4.3.4. Degradation by microorganisms 4.3.5. Persistence in soil 4.3.6. Interaction with other physical, chemical, or biological factors 4.3.7. Vegetation 22.214.171.124 Uptake and transformation in plants 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1. Environmental levels 5.1.1. Air 5.1.2. Water 5.1.3. Soil 5.1.4. Food and animal feed 126.96.36.199 Fruit, vegetables, and grain 188.8.131.52 Animal products 184.108.40.206 Animal feed crops 5.1.5. Other products 5.1.6. Terrestrial organisms 5.2. General population exposure 5.2.1. Exposure through the food 5.2.2. Exposure during insect control 5.3. Occupational exposure during manufacture, formulation, or use 6. KINETICS AND METABOLISM 6.1. Absorption 6.2. Distribution 6.3. Metabolism 6.3.1. In vitro studies on animal tissues 6.3.2. In vivo studies on animals 6.3.3. Metabolic transformation in plants 6.3.4. In vitro studies with human tissues 6.3.5. In vivo studies on humans 6.4. Elimination and excretion in expired air, faeces, urine, milk, and eggs 7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT 7.1. Microorganisms 7.1.1. Soil microorganisms 7.1.2. Aquatic microorganisms 7.2.1. Aquatic invertebrates 7.2.2. Fish 220.127.116.11 Acute toxicity 18.104.22.168 Short-term and long-term toxicity 7.2.3. Amphibians 7.3. Terrestrial organisms 7.3.1. Worms 7.3.2. Insects 7.3.3. Birds 7.3.4. Mammals 7.4. Effects on the population and ecosystem 8. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS 8.1. Single exposures 8.1.1. Oral toxicity 8.1.2. Acute inhalation toxicity 8.1.3. Dermal toxicity 8.1.4. Other routes of exposure 8.2. Skin and eye irritation, sensitization 8.2.1. Skin and eye irritation 8.2.2. Sensitization 8.3. Short- and long-term oral exposure 8.4. Short- and long-term inhalation toxicity 8.5. Reproduction and developmental toxicity 8.5.1. Mammalian reproductive toxicity studies 22.214.171.124 Mouse 126.96.36.199 Rat 188.8.131.52 Gerbil 8.5.2. Mammalian developmental toxicity studies 184.108.40.206 Mouse 220.127.116.11 Rat 18.104.22.168 Guinea-pig 22.214.171.124 Rabbit 126.96.36.199 Dog 188.8.131.52 Pig 184.108.40.206 Monkey 8.5.3. Reproductive and developmental toxicity studies in non-mammalian species 220.127.116.11 Fish 18.104.22.168 Amphibian 22.214.171.124 Birds 8.5.4. Appraisal 8.6. Mutagenicity of carbary and N-nitrosocarbaryl 8.6.1. Genotoxicity assays in vitro 126.96.36.199 Primary DNA damage 188.8.131.52 Gene mutation assay 184.108.40.206 Chromosomal aberration assays and sister chromatid exchange 8.6.2. Genotoxicity in vivo 220.127.116.11 Host-mediated assay 18.104.22.168 Drosophila melanogaster and other insects 22.214.171.124 Chromosomal aberrations and sister chromatid exchange 126.96.36.199 Dominant lethal assays in rodents 8.6.3. Other end-points 188.8.131.52 Cell transformation 184.108.40.206 Aneuploidy induction 8.6.4. Appraisal 8.7. Carcinogenicity 8.7.1. Carcinogenicity studies of carbaryl in rodents 220.127.116.11 Mouse 18.104.22.168 Rats 22.214.171.124 Overall appraisal of carbaryl carcinogenicity 8.7.2. Carcinogenicity studies of N-nitrosocarbaryl 126.96.36.199 Rats 188.8.131.52 Mice 184.108.40.206 Overall evaluation of the carcinogenicity of N-nitrosocarbaryl 8.7.3. Carcinogenicity of ß-carbaryl 8.8. Special studies 8.8.1. Neurotoxicity 8.8.2. Effects on the immune system 220.127.116.11 Appraisal on immunotoxicology 18.104.22.168 In vivo studies 22.214.171.124 In vitro studies 8.8.3. Effects in blood 8.8.4. Effects on the liver and other organs 8.8.5. Effects on serum glucose 8.8.6. Interactions with the drug metabolizing enzyme system 8.8.7. Effects on the endocrine system 8.8.8. Other studies 8.9. Factors modifying toxicity, toxicity of metabolites 8.9.1. Factors modifying toxicity 8.9.2. Toxicity of metabolites 8.9.3. N-nitrosocarbaryl 8.10. Mechanism of toxicity - mode of action 8.10.1. Inhibition of cholineresterase activity 9. EFFECTS ON HUMAN BEINGS 9.1. General population exposure 9.1.1. Acute toxicity, poisoning incidents 9.1.2. Controlled human studies 9.1.3. Long-term exposure 9.2. Occupational exposure 9.2.1. Epidemiological studies 10. PREVIOUS EVALUATION BY INTERNATIONAL BODIES REFERENCES APPENDIX RESUME ET EVALUATION, CONCLUSIONS ET RECOMMANDATIONS RESUMEN Y EVALUACION, CONCLUSIONES Y RECOMENDACIONES WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR CARBARYL Members Dr C.D. Carrington, Food and Drug Administration (FDA) Washington, DC, USA (Chairman). Dr N. Chernoff, US Environmental Protection Agency, Research Triangle Park, North Carolina, USA Dr T.S.S. Dikshith, VIMTA Labs Ltd, Hyderabad, India Professor F. Kaloyanova, National Center of Hygiene and Medical Ecology, Sofia, Bulgaria (Rapporteur) Professor Yu.I. Kundiev, Institute for Occupational Health, Kiev, Ukraine (Vice-Chairman) Dr D. Osborn, Institute of Terrestrial Ecology, Monks Wood Experimental Station, Huntingdon, United Kingdom Professor C. Ramel, University of Stockholm, Stockholm, Sweden Professor Shou-Zheng Xue, Shanghai Medical University, Shanghai, The People's Republic of China Observers Dr S. Kozlen, Rhône-Poulenc, Lyon, France (Representative from Rhône-Poulenc) Dr P.G. Pontal, Rhône-Poulenc, Lyon, France (Representative from ECETOC) Mr D. Demozay, Rhône-Poulenc, Lyon, France (Representative from GIFAP) Secretariat Dr K.W. Jager, International Programme on Chemical Safety, WHO, Geneva, Switzerland (Secretary) 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 Director of the International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland, in order that they may be included in corrigenda, which will appear in subsequent volumes. * * * A detailed data profile and a legal file can be obtained from the international Register of Potentially Toxic Chemicals, Case postale 356, 1219 Châtelaine, Geneva, Switzerland (Telephone No. 9799111). * * * This publication was made possible by grant number 5 U01 ES02617-14 from the National Institute of Environmental Health Sciences, National Institutes of Health, USA. * * * NOTE: The proprietary information contained in this document cannot replace documentation for registration purposes, because the latter has to be closely linked to the source, the manufacturing route, and the purity/impurities of the substance to be registered. The data should be used in accordance with paragraphs 82-84 and recommendations paragraph 90 of the Second FAO Government Consultation (1982). ENVIRONMENTAL HEALTH CRITERIA FOR CARBARYL A WHO Task Group on Environmental Health Criteria for Carbaryl met at the World Health Organization, Geneva, from 21 to 25 September 1992. Dr K.W. Jager, of the IPCS, welcomed the participants on behalf of the Director IPCS and the three IPCS cooperating organizations (UNEP/ILO/WHO). The Group reviewed and revised the draft criteria monograph and made an evaluation of the risks for human health and the environment from exposure to carbaryl. The first draft was prepared by Professor F. Kaloyanova of the National Center of Hygiene and Medical Ecology and Dr P.P. Simeonova, Medical Faculty, University of Sofia, Bulgaria, who also prepared the second draft, incorporating comments received following circulation of the first drafts to the IPCS contact points for Environmental Health Criteria monographs. Dr K.W. Jager of the IPCS Central Unit was responsible for the scientific content of the document, and Mrs M.O. Head of Oxford, England, for the editing. The fact that Rhône-Poulenc Agro, Lyon, France, made available to the IPCS and the Task Group its proprietary toxicological information on the product under discussion is gratefully acknowledged. This allowed the Task Group to make its evaluation on a more complete data base. The efforts of all who helped in the preparation and finalization of this publication are gratefully acknowledged. 1. SUMMARY AND EVALUATION, CONCLUSIONS AND RECOMMENDATIONS 1.1 Summary and evaluation 1.1.1 Identity, properties, and analytical methods Carbaryl is the common name for the carbamic acid derivative 1-naphthyl N-methylcarbamate. The technical grade product is a white crystalline solid, with a low volatility; it is poorly soluble in water, which is stable to light and heat, but easily hydrolysed in alkaline media. The FAO has established a minimum specification of 98% purity, with an impurity limit of 0.05% for ß-naphthyl N-methylcarbamate. Carbaryl and its metabolites are analysed using numerous analytical procedures, such as thin-layer chromatography, spectro-photometry, gas chromatography, high pressure liquid chromatography, and chemical ionization mass spectrometry. Detection limits of below one nanogram are achievable and recovery is usually more than 80%. 1.1.2 Production and uses Carbaryl has been used for about 30 years as a contact and ingestion insecticide with some systemic properties and controls a wide range of pests. The principal production plant is in the USA. Carbaryl is processed by more than 290 formulators into over 1500 different products. 1.1.3 Environmental transport, distribution, and transformation Under most conditions, carbaryl is not persistent in the environment. In water, the hydrolysis half-life is dependent on the temperature, pH, and the initial concentration, and varies from several minutes to several weeks. The major degradation product is 1-naphthol. Accumulation of carbaryl, expressed as a bioconcentration factor in the aquatic environment, has been studied in freshwater fish and found to be in the range of 14-75. Carbaryl is adsorbed more readily on soils with a high organic content than on sandy soils. At the usual application rates, under "good agricultural practice", dissipation is rapid, with a half-life of 8 days to 1 month under normal conditions. Carbaryl may occasionally be carried by rainfall and soil cultivation from the surface into the subsoil (one metre from the surface). Carbaryl contaminates vegetation, either during spraying, or by migrating through contaminated soil into plants. The degradation of carbaryl in the environment is determined by the extent of the volatilization, photodecomposition, and chemical and microbial degradation occurring in soil, water, and plants. The rate of decomposition is more rapid under hot climatic conditions. 1.1.4 Environmental levels and human exposure Food represents the major source of carbaryl intake for the general population. Residues in total dietary samples are relatively low, ranging from trace amounts to 0.05 mg/kg. In the USA, the daily intake during the first years of carbaryl application was 0.15 mg/day per person (in 7.4% of the composites); this decreased to 0.003 mg/day per person in 1969 (in only 0.8% of the composites). During the period of application, carbaryl may be found, occasionally, in surface water and reservoirs. The general population can be exposed to carbaryl during pest control operations in both the home and recreation areas. Workers can be exposed to carbaryl during its manufacture, formulation, packing, transportation, storage, and during and after application. Concentrations in the working-air environment during production varied from <1 mg/m3 to 30 mg/m3. Significant dermal exposure may occur in industrial and agricultural workers if protective measures are inadequate. 1.1.5 Kinetics and metabolism Carbaryl is rapidly absorbed in the lungs and digestive tract. In human volunteers, dermal absorption of 45% of an applied dose in acetone occurred in 8 h. However, in vitro dermal penetration data and toxicity data indicate that dermal absorption usually occurs at a much lower rate. The principal metabolic pathways of carbaryl are ring hydroxylation and hydrolysis. As a result, numerous metabolites are formed and subjected to conjugation with the formation of water-soluble sulfates, glucuronides, and mercapturates, excreted in the urine. Hydrolysis results in the formation of 1-naphthol, carbon dioxide, and methylamine. Hydroxylation produces 4-hydroxycarbaryl, 5-hydroxycarbaryl, N-hydroxymethylcarbaryl, 5-6-dihydro-5-6- dihydroxycarbaryl, and 1,4-naphthalendiol. The principal metabolite in humans is 1-naphthol. Under normal exposure conditions, the accumulation of carbaryl in animals is unlikely. Carbaryl is excreted primarily via the urine, since the product of its hydrolysis, 1-naphthol, is mainly detoxified to water-soluble conjugates. Enterohepatic cycling of carbaryl metabolites is also considerable, especially after oral administration. The hydrolysis product, N-naphthol carbamic acid, is spontaneously decomposed to methylamine and carbon dioxide. The methylamine moiety is later demethylated to carbon dioxide and formate, the latter being excreted mainly in the urine. Carbaryl metabolites are also present in a small percentage of the absorbed doses in saliva and milk. 1.1.6 Effects on organisms in the environment LC50 values for crustacea vary from 5 to 9 µg/litre (water fleas, mysid shrimps), 8 to 25 µg/litre (scud), and 500 to 2500 µg/litre (crayfish). Aquatic insects have a similar range of sensitivity. Plecoptera and Ephemeroptera (stoneflies and mayflies) are the most sensitive groups. Molluscs are less susceptible with EC50s in the range of a few mg/litre. For fish, most LC50 values are between 1 and 30 mg/litre. Salmonids are the most sensitive group. The acute toxicity for birds is low. The LD50 for waterfowl and game birds is >1000 mg/kg. The most susceptible bird tested is the red-winged blackbird (LD50= 56 mg/kg). There was no evidence of field effects on birds in forest areas sprayed with 1.1 kg carbaryl/ha. Carbaryl is very toxic for honey-bees and earthworms. The oral LD50 for the former is 0.18 µg/bee (about 1-2 mg/kg). There are indications that carbaryl may temporarily influence the species composition of both terrestrial and aquatic ecosystems. For instance, one study showed that effects on certain terrestrial invertebrate communities may persist for at least 10 months following a single application. 1.1.7 Effects on experimental animals and in vitro test systems The acute toxicity, expressed as the LD50, varies considerably according to species, formulation, and vehicle. Estimates of the oral LD50 for the rat range from 200 to 850 mg/kg. Cats are more sensitive with an LD50 of 150 mg/kg. Pigs and monkeys are less sensitive with an LD50 of >1000 mg/kg. The maximum achievable aerosol concentration of carbaryl of 792 mg a.i./m3 during a 4-h exposure resulted in the mortality of one out of five female rats. Carbaryl aerosols, at concentrations of 20 mg/m3, decreased cholinesterase activity (ChEA) in cats during single 4-h exposures, but this concentration did not have any observable effects in rats. Carbaryl is a mild eye irritant and has little or no sensitizing potential. During long-term studies, the NOEL was 10 mg/kg body weight (200 mg/kg diet) for rats, and 1.8 mg/kg body weight (100 mg/kg diet) for dogs. The long-term inhalation NOEL for cats is 0.16 mg/m3. Carbaryl has a low cumulative potential. 126.96.36.199 Reproduction Carbaryl has been shown to affect mammalian reproduction and perinatal development adversely in a number of species. Effects on reproduction include impairment of fertility, decreased litter size, and reduced postnatal viability. Developmental toxicity is seen as increased in utero death, reduced fetal weight, and the occurrence of malformation. With the exception of a small number of studies, all adverse reproductive and developmental effects were noted only at doses that caused overt maternal toxicity, and, in a number of cases, the maternal animal was more sensitive to carbaryl than the conceptus. The maternal toxic effects included lethality, decreased growth, and dystocia. Data indicate that the reproductive and developmental processes of mammals are not especially sensitive to carbaryl compared with the susceptibility of the adult organism. 188.8.131.52 Mutagenicity Carbaryl has been evaluated for its potential mutagenicity in a number of in vitro and in vivo tests, in bacterial, yeast, plant, insect, and mammalian systems, testing a variety of end-points. The available evidence indicates that carbaryl does not have any DNA-damaging properties. There have been no reports of confirmed induction of mitotic recombination, gene conversion, and UDS in prokaryotes ( H. influenzae, B. subtilis) and eukaryotes ( S. cerevisiae, A. nidulans, cultured human lymphocytes, and rat hepatocytes) in vitro. Negative results were obtained in tests for gene mutations in a large number of bacterial assays, with the exception of two cases. In several studies of gene mutations in mammalian cells in vitro, carbaryl produced only one equivocal positive result in a cell culture study. However, the study had several shortcomings and the result has not been confirmed in any other comparable studies. Chromosomal damage with high dosages of carbaryl has been reported in vitro in human, rat, and hamster cells, and in plants. No such effects have been observed in mammalian tests in vivo, even at doses as high as 1000 mg/kg. Carbaryl has been shown to induce disturbances in the spindle fibre mechanism in plant and mammalian cells in vitro. The relevance of plant assays for extrapolation to humans is unclear. It can be concluded that the available data-base does not support the presumption that carbaryl poses a risk of inducing genetic changes in either the somatic or the germinal tissue of humans. The nitrosated product of carbaryl, N-nitrosocarbaryl, is capable of inducing mitotic recombination and gene conversion in prokaryotes ( H. influenzae, B. subtilis) and eukaryotes ( S. cerevisiae) in vitro, and gives positive results in E. coli spot tests. Furthermore, experimental results indicate that N-nitrosocarbaryl binds to DNA, causing alkali-sensitive bonds and single-strand breakage. Nitrosocarbaryl has not been established as a clastogen in vivo (bone marrow and germ cells), even at high toxic doses. 184.108.40.206 Carcinogenicity Carbaryl has been studied for its carcinogenic potential in numerous studies on rats and mice. The results of most of these studies were negative, but the studies were old and did not meet contemporary standards. However, new studies on mice and rats, which meet modern standards, are in progress.a The latest IARC evaluation (IARC, 1987) concluded that there were no data on cancer in humans and that the evidence of carcinogenicity in experimental animals was inadequate. Carbaryl could not be classified as to its carcinogenicity for humans (Group 3). N-nitrosocarbaryl has been shown to induce tumours locally in rats (either sarcoma at the site of injection or forestomach squamous cell carcinoma, when given by the oral route). Given the human chemistry of carbaryl, the risk of N-nitrosocarbaryl carcinogenicity in humans from carbaryl exposure can be judged as negligible. aThese studies have not yet been reviewed by the IPCS. The company performing these studies has indicated that there is a significant increase in tumors at the highest dose in both species. 220.127.116.11 Effects on different organs and systems (a) Nervous system The effects of carbaryl on the nervous system are primarily related to cholinesterase inhibition and are usually transitory. The effects on the central nervous system were studied in rats and monkeys. Oral doses of 10-20 mg carbaryl/kg for 50 days were reported to disrupt learning and performance in rats. In a small study on pigs, carbaryl (150 mg/kg body weight in the diet for 72-82 days) was reported to produce a number of neuromuscular effects. Reversible leg weakness was noticed in chickens given high doses of carbaryl. No evidence of demyelination was observed in the brain, sciatic nerve, or in spinal cord sections examined microscopically. Similar effects were not observed in long-term rodent studies. (b) Immune system Carbaryl, when administered in vivo, at doses causing overt clinical signs, has been reported to produce a variety of effects on the immune system. Many of the effects described were detected at doses close to the LD50. Most studies on rabbits and mice at doses permitting survival have not produced significant effects on the immune system. Shortcomings of several of these studies were a lack of consistency and, sometimes, overt contradiction between results, which prevents the description of a defined immunotoxic mechanism. (c) Blood Carbaryl has been reported to affect coagulation, but there are conflicts about the direction of the effect. In glucose-6-phosphate dehydrogenase-deficient sheep erythrocytes, carbaryl produced a dose-dependent increase in methaemoglobin (Met-Hb) formation. Human serum albumin reacted in vitro with the ester group of carbaryl. Carbaryl binds free blood amino acids. (d) Liver Disturbances have been reported in the carbohydrate metabolism and protein synthesis and detoxification function of the liver in mammals. Carbaryl is a weak inducer of hepatic microsomal drug-metabolizing activity. Phenobarbital sleeping time is shortened. The hepatic levels of cytochrome P-450 and b5 are increased. Changes in liver metabolism may account in part for the three-fold increase of the carbaryl LD50 in carbaryl-pretreated rats. (e) Gonadotropic function Carbaryl has been reported to increase the gonadotropic function of the hypophysis of rats. 18.104.22.168 Primary mechanism of toxicity Carbaryl is an inhibitor of cholinesterase activity. This effect is dose-related and quickly reversible. There was no aging of the carbamylated cholinesterase. All identified metabolites of carbaryl are appreciably less active cholinesterase inhibitors than carbaryl itself. 1.1.8 Effects on humans Carbaryl is easily absorbed through inhalation and via the oral route and less readily by the dermal route. Since the inhibition of cholinesterase (ChE) is the principal mechanism of carbaryl action, the clinical picture of intoxication is dominated by ChE inhibition symptoms, such as: increased bronchial secretion, excessive sweating, salivation, and lacrimation; pinpoint pupils, bronchoconstriction, abdominal cramps (vomiting and diarrhoea); bradycardia; fasciculation of fine muscles (in severe cases, diaphragm and respiratory muscles also involved); tachycardia; headache, dizziness, anxiety, mental confusion, convulsions, and coma; and depression of the respiratory centre. Signs of intoxication develop quickly after absorption and disappear rapidly after exposure ends. In controlled studies on human volunteers, single doses of less than 2 mg/kg were well tolerated. A single dose of 250 mg (2.8 mg/kg) produced moderate ChE inhibition symptoms (epigastric pain and sweating) within 20 min. Complete recovery occurred within 2 h of treatment with atropine sulfate. In cases of occupational overexposure to carbaryl, mild symptoms are observed long before a dangerous dose is absorbed, which is why severe cases of occupational intoxication with carbaryl are rare. During agricultural application, dermal exposure may play an important role. No local irritative effect is usually observed, however, the appearance of a skin rash after accidental splashing with carbaryl formulations has been described. There are conflicting data about the effects of carbaryl on sperm count and changes in sperm morphology in plant workers. No adverse effects on reproduction have been reported. The most sensitive biological indicator of carbaryl exposure is the appearance of 1-naphthol in the urine and a decrease in ChE activity in the blood. Levels of 1- naphthol in the urine can be used as a biological indicator, if there is no 1-naphthol in the working environment. During occupational exposure, 40% of the urine samples contained more than 10 mg total 1-naphthol/litre. In one case of acute intoxication, 31 mg/litre was found in the urine. The hazard level is >10 mg/litre and the symptomatic level 30 mg 1-naphthol/litre urine ( Data sheet on carbaryl, WHO, 1973, VBC/DS/75.3). Measurement of the ChE activity can be a very sensitive test for monitoring, provided that measurement is carried out soon after exposure. 1.2 Conclusions The hazards of carbaryl for human beings are judged to be low, because of its low vapour pressure, rapid degradation, rapid spontaneous recovery of inhibited cholinesterase, and the fact that symptoms usually appear well before a dangerous dose has accumulated in the body. Good carcinogenicity studies, which meet modern standards, are not yet available. 1.2.1 General population exposure Residue levels of carbaryl in food and drinking-water, which remain after its normal use in agriculture, are far below the acceptable daily intake (ADI) (0.01 mg/kg body weight per day) and are not likely to produce health hazards in the general population. 1.2.2 Subpopulations at high risk Use of carbaryl for public health purposes in the home or in recreation areas may create overexposure, if the rules for its application are neglected. 1.2.3 Occupational exposure By enforcing reasonable work practices, including safety precautions, personnel protection, and proper supervision, occupational exposure during the manufacture, formulation, and application of carbaryl will not create hazards. Undiluted concentrations must be handled with great care, because improper work practices may cause skin contamination. Air concentrations in the workplace should not exceed 5 mg/m3. 1.2.4 Environmental effects Carbaryl is toxic for honey-bees and earthworms. It should not be applied to crops during flowering. With normal use, carbaryl should not cause environmental concern. Carbaryl is adsorbed on soil to a great extent and does not readily leach into ground water. It is rapidly degraded in the environment and therefore is not persistent. Use of carbaryl should not result in harmful short-term effects on the ecosystem. 1.3 Recommendations * The handling and application of carbaryl should be accomplished with the care given to all pesticides. Instructions for proper usage, provided on the package containing the chemical, should be carefully followed. * The manufacture, formulation, use, and disposal of carbaryl should be carefully managed to minimize contamination of the environment. * Regularly exposed worker populations should receive periodic health evaluations. * The application of carbaryl should be timed to avoid effects on non-target species. * Carcinogenicity studies that meet modern standards should be conducted. 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS 2.1 Identity Structural formula Molecular formula: C12H11N02 Common name: Carbaryl (BSI) CAS chemical name: 1-naphthalenylmethylcarbamate (9CI) CAS registry number: 63-25-2 RTECS registry number: FC5950000 Common synonyms: alpha-naftyl- N-methylkarbamat, alpha-naphthalenyl methylcarbamate, alpha-naphthyl methylcarbamate, alpha-naphthyl N-methylcarbamate, carbamic acid, methyl-, 1-naphthyl ester, N-methyl-alpha-naphthyl-urethan, N-methyl-1-naftyl- carbamaat, N-methyl-1-naphthyl-naphthyl carbamate, N-methyl-1-naphthyl-carbamat, N-methylcarbamate de 1-naphtyle, N-metil-1-naftil-carbammato, 1-naphthol N-methyl-carbamate, 1-naphthyl methylcarbamate, 1 naphthyl- N-methyl-karbamat The most commonly used chemical name is 1-naphthyl- N- methyl-carbamate. Common trade names: Arilat, Arilate, Arylam, Atoxan, Bercema, Caprolin, Carbacine, Carbatox, Carbavur, Carbomate, Carpolin, Denapon, Dicarbam, Dyna-carbyl, Karbaryl, Karbatox, Karbosep, Menaphtam, Monsur, Mugan, Murvin, Oltitox, Panam, Pomex, Prosevor, Ravyon, Seffein, Sevimol, Sevin, Vioxan The most commonly used trade name is Sevin. Previous codes: Compound 7744, ENT 23,969, ENT 23969, Experimental insecticide 7744, Germain's HSDB 952, NAC, NMC 50, Union Carbide 7744 Purity: The technical product is principally manufactured in the USA; however, there are other minor sources in other parts of the world. The technical product manufactured in the USA is produced to a minimum purity of 99% w/w carbaryl with a <0.05% w/w content of the 2-naphthyl carbamate isomer (sometimes known as "beta-carbaryl"). FAO specifies a minimum purity of 98% w/w carbaryl with <0.05% w/w content of the 2-naphthyl carbamate isomer. 2.2 Physical and chemical properties Some of the physical properties of carbaryl are listed in Table 1. Pure carbaryl is a white crystalline solid without odour. The explosion limit for dust (finely divided particles) in air is 20.3 g/m3 (approximately 2500 ppm). It is non-corrosive (Weston, 1982). The volatility may increase 4-fold when the relative humidity is increased from 8 to 80%. Table 1.Physical properties Melting point (°C) 142 Boiling point (°C) decomposing Solubility in water (30 °C) 40 mg/litre Specific density (20 °C) 1.23 Relative vapour density - Vapour pressure 1.17 x 10-6-3.1 x 10-7 mmHg at 24-25 °C Flash point 193 °C Octanol/water partition 1.59-2.3 coefficient (log Kow) Flammability (explosive) limits - Relative molecular mass 201 The solubility of carbaryl increases with temperature (Bowman & Sans, 1985). In sea water at 18 °C, the solubility is 31 mg/litre (Karinen et al., 1967). Carbaryl is soluble to some extent in most organic solvents, and it is soluble in corn oil. It is lipophilic (Kanazawa, 1981). It is stable to light and heat (up to 70 °C) and acids, but easily hydrolysed by alkaline materials (Dittert & Higuchi, 1963). It is a strong oxidizer. The quality of carbaryl depends upon the purity of the precursor, 1-naphthol. The amount of the 2-naphthylcarbamate isomer found as a contaminant in the final product is directly related to the purity of this precursor. 1-Naphthol, free of 2-naphthol (undetectable), is produced in the USA today through the catalytic conversion of naphthalene. However, 1-naphthol produced by other manufacturing processes may contain 2-naphthol as a byproduct. High pressure liquid chromatography (HPLC) has been used to determine 1- and 2-naphthol in their mixtures in ratios 500:1, in order to check for traces of contamination in samples of a commercially important insecticide. The results have been summarized in Table 2 (Argauer & Warthen, 1975). Table 2.2-Naphthol recovered from carbaryl samplesa Sample and size Amount of 2-naphthyl Amount of methylcarbamate added 2-naphthol for recovery check found USA produced 250 mg Union Carbide 99.66% active None Undetectable 500 mg Ortho Sevin 50% wettable powder None Undetectable 570 mg Union Carbide 44% aqueous slurry None Undetectable 310 mg Union Carbide 80% wettable powder None Undetectable 310 mg Union Carbide 80% wettable powder 2.5 mg 1.7 mg 310 mg Union Carbide 80% wettable powder 0.50 mg 0.33 mg Foreign origin 250 mg Sample A- technical None 2.3 mg 250 mg Sample B- technical None 14 mg 500 mg Sample C- 50% wettable powder None 12.4 mg 500 mg Sample D- 50% wettable powder None 1.3 mg aFrom: Argauer & Warthen (1975). 2.3 Conversion factors 1 ppm = 8.22 mg/m3 of air; 1 mg/m3 = 0.12 ppm. 2.4 Analytical methods The methods used to determine carbaryl are summarized in Table 3. They vary considerably in relation to the equipment used. As an alternative to chemical analysis, Bowman et al. (1982) used a simple bioassay system procedure to determine foliar residues for a safe re-entry period and to screen food for residues. Daphnia and Hyalella were used as highly sensitive test organisms. The Joint FAO/WHO Codex Alimentarius Commission has given recommendations for the methods of analysis to be used for the determination of carbaryl residues (FAO/WHO 1986a). Table 3. Methods of analysis Medium Sampling preparation Analytical Detection limit Comments Reference methods Meat extraction by paper test based 0.5 mg/kg Filatov & Brytskov acetone on ChE determination; (1972) butyryl choline as a substrate and phenol red as an indicator Air thin-layer 0.2 ng qualitative Wagner (1973) chromatography 0.5-50 ng quantitative Air (carbaryl absorption in UV spectro- 1 mg/kg carbaryl lambda max. Klisenko (1965) and methanol photometry 281 nm 1-naphthol 1-naphthol) lambda max. 296 nm Soil plant extraction by thin-layer 0.1 µg recovery 80-95% Kovaleva & hexane, acetone, chromatography sensitivity 0.02 mg/kg Talanov (1978a) chloroform Cow's milk and spectrophotometry 0.002 mg/litre - milk recovery 79-81% Hurwood (1967) tissues p-nitro-benzene diazonium fluoborate 0.02 mg/kg - tissues fortification 9.5-5 µg coupling method Urine 1-naphthol extraction with gas chromatography 0.02 mg/litre recovery 89-95% Shafik et al. (1971) benzene tritium detector Table 3 (continued) Medium Sampling preparation Analytical Detection limit Comments Reference methods Air of working absorption in sodium gas chromatography, Krechniak & Foss environment hydroxide solution electron capture (1981) with simultaneous detector hydrolysis to 1-naphthol; derivatization by 1-fluoro-2-4 dinitro-benzene Spinach Chicory sulfuric acid for electron capture 0.2 mg/kg in fortified Tilden & van hydrolysis to for chromatography crop extract; 20 pg for Middelem (1970) methylamine salt; pure standards 4-bromobenzoyl chloride used to produce derivative 4-bromo-N-methyl benzamide Water carbaryl extraction with gas chromatography 0.2 µg/litre recovery for carbaryl, Deuel et al. (1985) and 1-naphthol dichloromethane H-3 source electron 100%; for naphthol, 90% capture detector or 63Ni detector Soil carbaryl extraction with 0.01 mg/kg for recovery for carbaryl, and 1-naphthol 20% diethylester fortified soil 89.8%; for 1-naphthol, in dichloromethane samples 79.4% Table 3 (continued) Medium Sampling preparation Analytical Detection limit Comments Reference methods Air ambient series of gas HPLC with ultraviolet Currier et al. (1982) scrubbers charged detection at 220 nm with methanol; particulate deposit on Teflon discs Pads methanol ethanol HPLC 50 ng/pad (103.2 cm2) sensitive, inexpensive Bogus et al. (1985) extraction; selective procedure absorption and elution of reversed phase solid support Post-mortem extraction procedure HPLC ultraviolet recovery: blood and Duck & Woolias (1985) specimens on Extrelut column; detection reversed urine 99%; liver and protein precipitation phase stomach tissue 95% with acetonitrile Foliage surface extraction HPLC recovery: at 5 ng/kg Pieper (1979) by trichloromethane fortification level Grass, etc. extraction by CH3CN grass 89.5%; geranium 86.5% Water extraction by CH2Cl2 aspen 75%; Douglas fir 49.8%; at 0.1 mg/litre level Table 3 (continued) Medium Sampling preparation Analytical Detection limit Comments Reference methods Soil extraction by soil 103%; acetonitrile + water stream water 99.7%; sediment 101% Water, soil, extraction by thin-layer 0.5 mg/kg qualitative carbaryl and 1-naphthol Klisenko et al. (1972) cow's milk, benzene ( n-hexane, chromatography tissue (liver, diethyl ether) kidney, heart, lungs, etc.) Vegetables and extraction by HPLC Recovery 83-97% at Ting et al. (1984) fruits methanol (10% in 0.5 mg/kg fortification petroleum ether) or level. Detailed acetonitrile clean-up description of the on a florasil column method is given Water and Serum single extraction by HPLC 0.5 ng/ml Analysis time is Strait et al. (1991) methanol after 1 ml 10 min Recovery in of C18 solid-phase water > 99% extraction columns Plants methanol and liquid chromatography 0.01 mg/kg Recovery varies from Krause (1985a,b) mechanical reverse phase; LC 86 to 121% ultrasonic column using homogenizer used acetonitrile - water for extraction mobile phase Table 3 (continued) Medium Sampling preparation Analytical Detection limit Comments Reference methods Marion-berries extracted by Luke chemical ionization Recommended when GC Cairns et al. (1983); procedure (Luke et mass spectrometry retention data are al., 1975, 1981) with both methane inconclusive cited by Cairns and ammonia as (1983); eluted reagent gases through florisil using 50% mixed petroleum ethers Honey-bees extraction by gas chromatography, 0.03-0.10 mg/kg Recovery 95-100% Kendrick et al. (1991) diethyl ether nitrogen-phosphorous clean-up using ionization detector silica cartridge 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE Carbaryl was first synthesized in 1953 and, in 1958, the Union Carbide Corporation began its commercial production. Carbaryl is now processed by more than 290 formulators into 1537 different registered products (Harry, 1977, unpublished report). Carbaryl is formulated as a 50 and 85% wettable powder, 1.75-50% dust, oil-and water-based 4% liquid suspension, and 5 and 10% granules and baits. Annual production is of the order of 10 000 tonnes. It is produced by the reaction of 1-naphthol with methylisocyanate. Carbaryl is widely used, in many countries, as a broad spectrum contact and ingestion insecticide with some systemic properties, and is recommended for use at 0.25-2.7 kg active ingredient per hectare to control various insect pests. Up to 10 kg/ha per season can be used on tree fruits. In the USA, carbaryl is registered for the control of about 560 different pests. It is used on more than 115 food and fibre crops, trees, and ornamentals. About 40% of the quantity used is applied to cotton (Kuhr & Dorough, 1976; Mastro & Cameron, 1976; Payne et al., 1985). In combination with other substances, or alone, carbaryl is used as a plant regulator for apple thinning (Looney & McKellar, 1984; Looney & Knight, 1985). In veterinary practice, carbaryl is used on cattle, poultry, and pets, especially to control flies, mosquitos, ticks, and lice, some of which are vectors of disease. Carbaryl (5% formulation Carbacide) is used to treat human body louse infestation (Sussman et al., 1969). 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION 4.1 Volatilization and air transportation Carbaryl has a low volatility and a low air-water partition coefficient. Thus, only limited evaporation can be anticipated after treatment. Some traces of carbaryl in the air and in fog, resulting from spray drift, may be detected at a certain distance from the treated areas. A maximum level of 0.09 mg/m3 in the air has been reported. The dimensionless air-water partitioning constant (Henry's Law Constant) for carbaryl has been evaluated to be 5.3x10-6 (Schomburg et al., 1991). Deuel et al. (1985) studied the persistence of carbaryl in paddy water. Results indicated that no measurable dissipation could occur as a result of volatilization. In contrast to the results from Deuel, using the BAM model, Lee et al. (1990) calculated that 50 days after treatment, 0.63% of the carbaryl applied to soil could have been volatilized and 78.84% degraded. 4.2 Water 4.2.1 Hydrolysis In pure, sterilized water, kept in the dark, the persistence of carbaryl is pH-dependent. Carbaryl is rather stable in acidic conditions. Its half-life at pH 7 is 10-16 days, and at pH 8 it is 1.3-1.9 days only. At pH levels higher than 8, its half-life is in the range of a few hours, or even less (Aly & El Dib, 1971a). Carbaryl is an example of an N-substituted carbamate that hydrolyses readily in water. In this mechanism, an acid-base equilibrium is established and the conjugate carbamate undergoes an elimination type reaction to give an unstable carbamic acid that decomposes to the primary amine and CO2. Initially, the principal non-biological degradation pathway of carbaryl in water, however, involves base-catalysed hydrolysis to 1-naphthol (Khasawinah, 1977). Aly & El Dib (1971a,b, 1972) conducted studies to determine the physical factors that may influence the degradation of carbamates, including carbaryl, in aquatic systems. Hydrolysis of carbaryl in alkaline medium was a function of hydroxyl ions in solution and was first order with respect to these ions. Carbaryl was so sensitive to hydroxide ions that, at high base concentrations, the liberation of 1-naphthol was too fast (few minutes) to be measured by conventional methods. Carbaryl was stable to hydrolysis at the acid pH range of 3-6. At pH 7, a rise in the hydrolysis rate was observed, which increased with increase in pH. Carbaryl was very susceptible towards hydrolysis in aqueous solutions at neutral and alkaline pH values. A series of kinetic studies was carried out at different temperatures (3-33 °C) to study the temperature dependence of the rate of hydrolysis. Results showed that an increase in temperature resulted in an increase in the reaction velocity. Wauchope & Haque (1973) reported that, in weak acidic solutions, carbaryl and 1-naphthol were stable for several weeks, in the dark or under laboratory light. In basic solutions, the basic form of 1-naphthol (1-naphthoxide) was light sensitive. 1-Naphthoxide ion was transformed into 2-hydroxy-1,4-naphthoquinone; this was confirmed by mass spectrometry. Khasawinah (1977) carried out the hydrolysis of carbaryl in the dark, in aqueous buffer solutions at temperatures of 25 and 35 °C. Increasing the pH and temperature of the buffer accelerated the disappearance of carbaryl and the formation of 1-naphthol. Rate constants (at 20 °C) were determined for the purely chemical hydrolysis of carbaryl in water containing solvents at pH values ranging from 4 to 8 (Chapman & Cole, 1982). The solvent composition (water/ethanol = 99/1) was close to pure water and the solutions were sterilized to ensure that only chemical reactions were taking place. The half-lives for carbaryl were calculated from pseudo-first order disappearance rate constants (Table 4). Fisher & Lohner (1986) conducted tests on the environmental fate of carbaryl as a function of pH. In both a microcosm and abiotic studies, greater amounts of carbaryl were detected in water at pH 4, than at pH 6 or 8. Hydrolysis of labelled carbaryl in aqueous solution was conducted by Carpenter (1990) under dark conditions. When degradation occurred, the major degradation product of significance was 1-naphthol. No other degradation product accounted for more than 2% of the radioactivity and no volatile products were generated during the hydrolysis reaction. The test systems were sterile and the transformation/degradation mechanism was purely chemical hydrolysis. Carbaryl half-lives in aqueous solution observed by different authors are summarized in Table 4. Table 4. Half-life of carbaryl at different pH values and temperatures in aqueous solution (days) pH Temperature References 3 4 5 6 7 8 9 10 (°C) 10.5 1.3 0.1 0.01 Aly & El Dib (1971a, 1972) 0.12 0.01 25° Wauchope & Haque (1973) stable stable 0.14 25° Khasawinah (1977) stable 29 0.02 35° 1500 15 0.15 27° Wolfe et al. (1978) 2100a 406 14 1.9 20° Chapman & Cole (1982) 104 71.6 1.4 Fisher & Lohner (1986) 171.4 16.5 25° Larkin & Day (1986) stable 11.6 0.13 Carpenter (1990) 12.5 a pH 4.5 4.2.2 Photolysis There is sufficient evidence to suggest that photodecomposition will account for some loss of carbaryl in clear surface waters exposed to sunlight for long periods. In turbid waters, light penetration is greatly reduced, thus photolysis will play only a minor role in the decomposition of carbaryl. The primary effect of ultraviolet light radiation (UVR) seems to be cleavage of the ester bond, however, other modifications in the carbaryl molecule occur. Crosby et al. (1965) studied the photodecomposition of carbaryl and found several other cholinesterase inhibitory substances, in addition to 1-naphthol, indicating that these substances retained the intact carbamate ester group, and that irradiation resulted in changes at other positions in the molecule. Both UVR and natural sunlight caused decomposition of carbaryl, however, the extent of photodecomposition was not the same under the different conditions of irradiation. Intense UV irradiation generally resulted in the formation of a greater number of degradation products. It is expected that the effects of natural sunlight UVR (292-400 nm) on the photodegradation rate and the nature of the degradation products would differ from those of the shorter wavelength irradiations used above. The effect of UVR on the photodegradation of carbaryl was studied by Aly & El Dib (1971b, 1972). Generally, the concentration of carbaryl decreased with time, however, the photolysis rate gradually decreased. Photodecomposition proceeded at increasing rates as the pH values of solutions increased. After an exposure time of 60 min, the decomposition rates of carbaryl at pH 5, 7, and 8 were 50, 57, and 78%, respectively. The half-life at pH 8 was <20 min while, at pH 5, it was approximately 80 min. The main decomposition product after 5 min of exposure in all irradiated solutions was 1-naphthol. However, its concentration also decreased as the irradiation time was increased, indicating that 1-naphthol also underwent photolysis, as soon as it appeared in solution. The photodecomposition of 1-naphthol was also affected by the pH of the medium. The half-lives of carbaryl and 1-naphthol were 39 and 60 min, respectively, at pH 7 and 8, and 43 min at pH 8. The photochemistry of carbaryl was studied by Addison et al. (1975) in aerated and pure ethanol, cyclohexane, isopropyl alcohol, and tert-butanol. Irradiation of carbaryl produced 1-naphthol and small amounts of naphthamides, naphthalene, and ß-napthyl-1-naphthol. In cyclohexane, 1-naphthol was the only decomposition product. Khasawinah (1977) conducted a photolysis study on labelled carbaryl in an aqueous solution buffered at pH 6. The study terminated before the actual half-life was reached. The half-life was calculated to be 40-50 days. The author stated that under field conditions it is expected that carbaryl will photodegrade slowly and that photodegradation does not play a major role in the environmental degradation of carbaryl. The direct photolysis half-life of carbaryl in sunlight was 6.6 days in distilled water (Wolfe et al., 1978). On the basis of the results of the methods of Zepp et al. (1976) and Zepp & Cline (1977), the calculated half-life for direct photolysis was about 50 h in a clear water body, near the surface. The annual variation of the photolysis half-life of carbaryl according to season of the year was calculated. As the intensity of the sunlight increases, so do photolysis rates. Carbaryl absorbs UV-B radiation most strongly, and, thus, can also be photolysed under overcast conditions (cloudy days). UVR is absorbed by water, and the photolysis rate decreases as the water deepens. In spring and summer, when carbaryl is applied, the rate of photolysis is about four times that in winter months. In distilled water under the June midday sunlight at pH 5.5, the half-life of carbaryl was 45 h. Deuel et al. (1985) studied the photodecomposition of carbaryl in deionized water. They confirmed that carbaryl could be photodecomposed in an aquatic environment. The influence of different aqueous systems (rivers, lakes, and seawater) on the photochemical degradation of some carbamate insecticides in Greece was studied by Samanidou et al. (1988). In lake and sea water, carbaryl was almost completely degraded by sunlight within 4 and 2 days, respectively, in the presence of oxygen. One day of UV irradiation in river, lake, and sea water, respectively, resulted in 98%, 87%, and 99% degradation. The high concentration of suspended matter in river and lake water influences the absorption of sunlight and consequently the degradation of carbaryl. Das (1990a) exposed sterile water, buffered at pH 5, with labelled carbaryl to artificial sunlight (548.8 watts/m2) for 360 h; 65% of the carbaryl had disappeared by the end of the 360-h period. The major degradation product was 1-naphthol. In control test solutions, incubated in the dark, changes in carbaryl concentrations were insignificant. 4.2.3 Degradation by microorganisms A review of the chemical and microbial degradation of carbaryl in aquatic systems has been published by Paris & Lewis (1973). The rate of hydrolysis of carbaryl in neutral and slightly basic conditions was so rapid that differences reported between sterilized and non-sterile water were usually were minor. Thus, it is generally considered that the microbial degradation of carbaryl in natural water plays only a secondary role in comparison with chemical hydrolysis. The bacterial decomposition of 1-naphthol by ring cleavage was reported in farm pond water (Hughes & Reuszer, 1970). They studied bacterial populations in pond water containing drainage water from carbaryl-treated fields. Their data were the earliest to show that bacteria can adapt to live on carbaryl and that when this occurs one strain dominates. They also showed that there may be a minimum period of time before bacteria can degrade carbaryl and a minimum concentration below which bacteria will not multiply rapidly enough to cause degradation. Ahlrichs et al. (1970) found bacteria that could break the ring structure of carbaryl, however data concerning the role of microorganisms in the elimination of insecticides from surface water were variable. In a study by Hughes (1971) a bacterium ( Flavobacterium sp.) was isolated from pond water, which degraded 1-naphthol to o-hydroxycinnamic acid, salicylic acid, and an unidentified product. In this study, the bacteria cleaved the naphthalene ring, since both hydroxycinnamic acid and salicylic acid each have only a single phenol ring. Aly & El Dib (1972) studied the biodegradation of carbaryl in Nile River water in 5-gallon containers, which was buffered to maintain a pH of 7.2 and held at 25±2 °C under aerobic conditions. The concentration of carbaryl in water decreased progressively with time and 89% of the added amount of carbaryl (4.75 mg/litre) degraded in 6 days. 1-Napthol, which appeared as a degradation product, did not result only from the chemical hydrolysis of carbaryl, since a sterile buffered solution showed negligible hydrolysis. 1-Naphthol was produced mainly as a result of the biological activity of microorganisms in river water. Subsequent additions of increasing concentrations of carbaryl disappeared in shorter periods of time, and there was no build-up of 1-naphthol. Carbaryl disappeared more rapidly in Nile River water containing sewage. Thus, the authors considered that natural waters and sewage contain microorganisms capable of degrading carbaryl and 1-naphthol. A number of marine microorganisms, including algae, bacteria, fungi, and yeasts, were tested by Sikka et al. (1973, 1975) for their ability to metabolize carbaryl or 1-naphthol. None of them was able to degrade carbaryl to a significant extent. Only a very small amount of carbaryl was metabolized to form water-soluble metabolites by the algae Cyclotella nana and Dunaliella tertiolecta. 1-Naphthol was degraded to water- and ether-soluble metabolites by Culcitalna achraspora, Halosphaeria mediosetigera, Humicola alopallonella, Aspergillus fumigatus, Serratia marina, Spirillum sp., and Flavobacterium sp. The organisms differed greatly in their ability to convert carbaryl to water-soluble products, and also in their ability to degrade 1-naphthol. Overall, 1-naphthol appeared more susceptible to degradation than carbaryl, and filamentous fungi appeared to possess a greater ability to degrade 1-naphthol than bacteria or yeast. Bacteria isolated from river water were also capable of degrading 1-naphthol (Bollag et al., 1975; Czaplicki & Bollag, 1975). After 60 h of incubation with 14C-labelled 1-naphthol, it was possible to trap 44% as CO2 and 22% was recovered. The release of labelled CO2 clearly indicated that complete biodegradation of carbaryl had taken place via rupture of the naphthyl ring. However, 15-20% of radioactivity remained in the growth medium. This suggests that at least 2 different pathways may be involved in the degradation of 1-naphthol by these bacteria. The radioactivity in the growth medium was partitioned and the dominant product was identified as 4-hydroxy-1-tetralone, which suggests an alternative pathway that involves hydroxylation of the naphthyl ring in the 4-position and conversion of an aromatic ring to an aliphatic cyclic compound. Walker et al. (1975a) confirmed this pathway with a soil pseudomonad. Paris et al. (1975) found that, in heterogeneous bacterial cultures in water, bacteria did not significantly degrade carbaryl but did utilize 1-naphthol, produced from hydrolysis, as a carbon source. Products of the bacterial degradation of the 1-naphthol were 1,4-naphthoquinone and 2 unidentified compounds. Hydrolysis and photolysis contributed significantly to the degradation of carbaryl, since half-lives were short compared with those of biolysis. Within 7 days of incubation in river water, 92% of carbaryl or 1-naphthol disappeared (Prima et al., 1976); 68% was removed by biochemical degradation, and 24% by a physico-chemical process. Liu et al. (1981) measured the rate of carbaryl degradation at pH 6.8 both with, and without, bacteria obtained from lake sediment. Without the lake sediment inoculum, the half-life of carbaryl was 8.3 days under aerobic conditions and 15.3 days under anaerobic conditions. With bacterial metabolism, half-lives of 6.8 and 5.8 days, respectively, were measured. After the addition of cometabolites (glucose and peptone), the half-lives were further reduced to 3.8 and 4.2 days, respectively. Thus, bacterial degradation played a much greater role in the degradation of carbaryl under anaerobic conditions. Microbial activity was an important factor in the breakdown of carbaryl in water from a pond and creek (Szeto et al., 1979). Autoclaving prior to the addition of carbaryl and incubating for 50 days increased the recovery from 39 to 57% (creek water) and from 28 to 58% (pond samples containing sediment). Boethling & Alexander (1979) studied the degradation of carbaryl in stream water (pH 7.5-8.6) at extremely low concentrations. They reported that, at initial concentrations of 30 and 300 mg/litre, more than 60% of the carbaryl was degraded to CO2 within 4 days, but 10% or less was converted to CO2 at 0.3 mg/litre and 0.0003 mg/litre. At these two latter concentrations, CO2 was generated at rates not exceeding 3% of the starting material per day. The authors concluded that laboratory tests on bio-degradation are usually conducted with concentrations of chemicals higher than those found in rivers, lakes, and marine waters and, therefore, will not accurately predict the environmental behaviour of microorganisms. Sharom et al. (1980) compared the persistence of carbaryl in natural water, distilled water, sterilized natural water, and sterilized distilled water. Carbaryl disappeared from all four types of water, which was considered to show that chemical processes played a major role, and biological processes, a secondary role, in the degradation of carbaryl in water. Chaudhry & Wheeler (1988) maintained a Pseudomonas sp. isolated from a pesticide waste disposal site on a medium containing normal and radiolabelled carbaryl. Pseudomonas sp. degraded carbaryl and the authors concluded that Pseudomonas sp. may have potential as biological treatments for waste and groundwater. 4.2.4 Persistence in surface water The agricultural use of carbaryl may indirectly produce residues in surface water and in sediment, following application, as result of drift or from soil-bound particles. In general, carbaryl is not expected to persist in the aquatic environment. Although it is stable to hydrolysis in acidic water, at the pH of most surface fresh waters (7-8.2), it is highly susceptible to hydrolysis. Biologically-mediated degradation and photolysis are secondary mechanisms; sediment and humic substances also influence the persistence of carbaryl in aquatic systems. Half-lives of carbaryl in natural water, calculated from experimental results, are summarized in Table 5. In one case, with exceptionally cold and acidic conditions, the half-life was in the range of 70 days. In most cases, half of the carbaryl degraded in a few days, or even in less than one day. Table 5. Percentage of carbaryl degraded in water, a certain number of days after treatment, and the corresponding approximate half-lives Origin of water Days % Half-life References after (days) treatment laboratory 7 95 1 Eichelberger & Lichtenberg (1971) pond < 0.5 Romine & Bussian (1971) containers 6 89 2 Aly & El-Dib (1972) microcosm < 5 Kanazawa (1975) river 7 92 2 Prima et al. (1976) lab. pond water 42 80-82 18 Szeto et al. (1979) lab. + sediment < 2 lab. creek water 42 60-63 30 lab. + sediment < 7 lab. drainage 28 100 4 Sharom et al. (1980) field, drainage 6 100 1 Osman & Belal (1980) brooks, rivers, 1 Stanley & Trial (1980) streams stream 17 100 3 Ott et al. (1981) Table 5. Contd Origin of water Days % Half-life References after (days) treatment lab. sewage 42 100 11 Odeyemi (1982) lab. fresh water 60 100 < 15 rice irrigation 10 80 4 Thomas et al. (1982) pond 1 60 100 8 Gibbs et al. (1981, 1984) pond 2 138 77 2-3 rice fieldDeuel et al. (1985) microcosm (pH 4-8) 7 31-73 13-4 Fisher & Lohner (1986) stream < 0.1 Sundaram & Szeto (1987) stream 1-3 100 < 1 Springborn (1988b) rice irrigation < 1 Springborn (1988a) ponds (18 °C) 4 100 1 Hanazato & Yasuno (1989) ponds (4 °C) 10 30 20 45 95 10 ponds (4-20 °C) 0.4-0.9 Hanazato & Yasuno (1990a,b) aRounded. The behaviour and persistence of carbaryl in water from a pond and creek, with and without sediment, were studied under simulated conditions in the laboratory by Szeto et al. (1979). At 9 °C, carbaryl was less persistent in pond water (pH 7.5-7.8) than in creek water (pH 7-7.1). Carbaryl degraded to 18-20% of the initial amount after 42 days in pond water samples and to 37-40% of the initial amount in creek water samples after 50 days. The higher pH of pond water compared with creek water may have contributed to this effect. The presence of sediment did not affect the rate of loss of carbaryl, but approximately 50% of the remaining carbaryl was found in the sediment. Microbial activity was a major factor in the degradation of carbaryl in this study. Eichelberger & Lichtenberg (1971) measured the persistence of carbaryl in river water at room temperature and pH 7.3-8. From an initial concentration of 10 µg/litre, only 5% could be detected after 1 week and none was detected at 2 weeks. At the time of disappearance of carbaryl, 1-naphthol could not be detected. In Egypt, Osman & Belal (1980) mentioned that residues present in irrigation and drainage canals following application of carbaryl disappeared from the water 6 days after spraying. The persistence of carbaryl was tested by Odeyemi (1982) who incubated water samples treated wtih 45 mg/kg under tropical greenhouse conditions (Nigeria). According to colorimetric measurements, carbaryl disappeared after 42 days of incubation in sewage water, and after 60 days in fresh water samples. The impact of an experimental aerial application of carbaryl (Sevin-4-oil) on woodland ponds in Northern Maine (USA) was studied by Gibbs et al. (1981, 1984). Carbaryl was applied at the rate of 840 g/ha. Maximum residues levels of 734 µg/litre were detected in the water, and about 4860 µg/kg (dry weight) in the sediment. In one pond, carbaryl was not found after 62 days. Data from another pond showed residues equivalent to 23% of the initial residue after 138 days, and 13% of the initial residues persisting after 375 days. Investigators noted a rapid movement of carbaryl into bottom sediments with persistence up to 16 months in this compartment, compared with 14 months in the water. The anaerobic state of the organic substrate and acidic conditions in one pond may have contributed to greater persistence. The lower residue levels in the other pond were attributed to a greater flow of water. Hanazato & Yasuno (1990a,b) conducted studies in experimental outdoor concrete ponds in Japan. Water received three treatments with carbaryl in order to produce a minimal concentration of 0.5 mg/litre on 12, 19, and 20 October. The water temperature, which was 20 °C at the start of the experiment, declined steadily to 4 °C in early December and then did not change. The pH increased from 7.3 on the third day after the start of the experiment to about 9 on the 13th day. It remained between 8-9 until the end of the experiment. The concentration of carbaryl in water decreased exponentially and it was no longer detected 4 days after the first and second treatments and 11 days after the third treatment. The half-lives for the three treatments were respectively 0.36, 0.40, and 0.86 days. The corresponding times for 90% degradation were, respectively, 1.18, 1.32, and 2.84 days. Carbaryl was added to 60 litres of water and 15 kg of soil held in 110-litre, plastic, garbage containers, buried partially in open ground (Junk et al., 1984). Carbaryl was studied at the high and low concentrations of 4 g/litre and 0.2 g/litre, respectively. Additional variables studied included aeration (1 litre/min) and peptone nutrients (0.1% by weight). Data obtained from this experiment demonstrated that soil and water in an inexpensive container provide satisfactory conditions for the containment of pesticides so that chemical and biological degradation can occur. Hydrolysis of carbaryl was rapid. Deuel et al. (1985) studied the persistence of carbaryl and the 1-naphthol metabolite in paddy water under flooded rice cultivation conditions. Persistence was evaluated with respect to time, application rates (1.1 and 5.6 kg/ha), and irrigation scheme (intermittent or continuous). Results showed that application rate and time of sample collection had a significant influence on the carbaryl residues recovered in paddy water during the 3-year study. They were found to be greater in plots under intermittent irrigation, but only in 2 of the 3 years. Carbaryl residues in water were greatest in years when rainfall occurred within 24 h of foliar application. Using intermittent treatment data, carbaryl was determined to dissipate to half the initial residue level within 48-59 h. 4.2.5 Removal from water As already mentioned, Chaudhry & Wheeler (1988) proposed that Pseudomonas sp. may have potential as a biological treatment for waste and groundwater. According to Miles et al. (1988a,b), the degradation rates of N-methylcarbamate insecticides, including carbaryl and its metabolite 1-naphthol, were more rapid in chlorinated water than in pure water. Half-lives of carbaryl were as follows: carbaryl control pH 7:10.3 days carbaryl control pH 8: 1.2 days carbaryl chlorinated pH 7: 3.5 days carbaryl chlorinated pH 8: 0.05 days. A separate experiment with 1-naphthol in chlorinated water showed that this product is highly unstable with a half-life of the order of minutes. A water source contaminated with carbaryl and treated by chlorination will have lower concentrations of the insecticide in the effluent. Mason et al. (1990) studied the removal of carbaryl from drinking-water by the disinfectants, Cl2, ClO2, and O3. Carbaryl did not react with chlorine or with ClO2, but reacted very rapidly with O3. Therefore removal/degradation of carbaryl can be achieved using ozonization. 4.2.6 Persistence in sea water Under laboratory conditions, the persistence of carbaryl in sea water is slightly higher than that in freshwater and, according to temperature, lighting, and microbial presence, its half-life varies from a few days to about one month. The salt content of natural waters (ionic strength) may affect the rate of hydrolysis of carbamates (Christenson, 1964). Thus, carbaryl is expected to be more stable to hydrolysis in waters with a high salt content than in freshwater. Carbaryl may enter marine systems when it is used to control oyster pests and predators, such as oyster drills, mud shrimp, ghost shrimp, and star fish (Loosanoff et al., 1960a,b; Lindsay, 1961; Loosanoff, 1961; Snow & Stewart, 1963; Haydock, 1964; Haven et al., 1966). However, Hurlburt et al. (1989) stated that tidal action diluted the insecticide rapidly and that larvae are unlikely to be subjected to high concentrations of carbaryl for more than several hours. The persistence of carbaryl in estuarine water was studied in the laboratory by Karinen et al. (1967). Samples of water containing 5, 10, or 25 mg carbaryl/litre and 3% NaCl were buffered at pH 8 and kept in aquaria at 8 °C. In the absence of mud, the concentration of carbaryl decreased 50% in 38 days, with most of the decrease accounted for by the production of 1-naphthol. In another experiment, naphthyl-14C and carbonyl-14C carbaryl (6.8-8.7 mC) were used with cold carbaryl at total concentrations of 15 and 25 mg/litre, respectively, and maintained at 20 °C. About 50% of the carbaryl was hydrolysed in 4 days and, after 17 days, the carbaryl had almost disappeared with 43% converting to 1-naphthol. Fluorescent light slightly accelerated the hydrolysis of carbaryl to 1-naphthol. When mud was added to the aquarium system, both carbaryl and 1-naphthol in sea water declined to less than 10% of the initial concentration in 10 days. Both compounds were adsorbed by the mud, where decomposition continued at a slower rate than in sea water. 1-Naphthol persisted in mud for a short period of time, but carbaryl could be detected for approximately 3 weeks. The naphthol moiety of the carbaryl molecule was converted to more persistent products. The experiment with labelled carbaryl demonstrated degradation by hydrolysis of carbamate and oxidation of the naphthyl ring to produce 14CO2 and 14CH4. In a preliminary field study, estuarine mud flats were treated with carbaryl at 11.2 kg/ha (dose used to control pests in oysters beds); carbaryl could be detected in the mud for up to 42 days. 1-Naphthol concentrations were found only after the first day, which may indicate that hydrolysis proceeds slowly in mud. The fate of 1-naphthol was studied in simulated marine systems by Lamberton & Claeys (1970). 1-Naphthol was unstable in the alkaline environment of sea water, since light and microorganisms enhanced its degradation to CO2 and other products. 1-Naphthol was relatively stable in an oxygen-free aqueous solution. Odeyemi (1982) incubated water samples treated with 45 mg carbaryl/litre under tropical greenhouse conditions (Nigeria). According to colorimetric measurements, about 77% of the insecticide had disappeared after 63 days of incubation in seawater, whereas it had completely disappeared after 42 days of incubation in sewage water, and after 60 days in freshwater samples. 4.2.7 Bioaccumulation/biomagnification Because of its low persistence in water, and its even faster degradation by living organisms, carbaryl has very low bio-accumulation properties and presents no risk of biomagnification under practical conditions. Kanazawa (1975) studied the uptake and excretion of carbaryl by Pseudorasbora parva. Fish were reared for 30 days in an aquarium containing carbaryl at about 1 mg/litre. In water, 95% of the carbaryl was degraded in 6 days. One day after treatment, residues of carbaryl in P. parva reached 7.5 mg/kg, which was the maximum uptake. Kanazawa et al. (1975) studied the distribution and metabolism of 14C-1-naphthyl-labelled methylcarbamate (carbaryl) in an aquatic model ecosystem containing 10 kg soil, 80 litre water, and catfish ( Ictalurus punctatus), crayfish ( Procambarus sp.), daphnids ( Daphnia magna), snails (Physa sp.), algae ( Oedogonium cardiacum) and duckweed ( Lemna minor). After 20 days, 31% of the radioactivity was lost, 67.85% remained in the soil (about 45% unextractable residues), 1.1% was present in water, and only 0.1% was recovered in organisms. Bioaccumulation ratios were calculated from radioactivity recovery and were likely to be low in the case of animals. In the case of plants, they were apparently higher, but as they are based only on radioactivity, including normal synthesis reutilizing 14CO2 and not on real carbaryl measurement, they should be considered as an overestimation of the true bioconcentration factor for carbaryl (Table 6). A flow-through bioconcentration study was conducted with bluegill sunfish ( Lepomis macrochirus) exposed to 0.093 mg carbaryl/litre for 28 days followed by a depuration phase in which bluegill were held for 14 days in untreated water (Chib, 1986a; Chib et al., 1986). Analysis of fillet, whole fish, and visceral portions indicated a rapid uptake of carbaryl. Bioconcentration factors ranged from 14x to 75x, for fillet and viscera, respectively. Data suggest that fish stopped accumulating carbaryl after day 7 of uptake, indicating that a steady state had been reached. Table 6. Accumulation of 14C radioactivity expressed as carbaryl equivalenta by various aquatic organismsb Carbaryl Concentration in water 1.66 (1 day) µg/litre 9.45 (22 days) Concentration in soil 2.43 (start) mg/kg 2.55 (22 days) Organisms mg/kg BARc Algae 37.9 4000 Duckweed 34.2 3600 Snail 2.81 300 Catfish 1.33 140 Crayfish 2.48 260 aCarbaryl not actually measured. bFrom: Kanazawa et al. (1975). cBioaccumulation ratio calculated by dividing mg/kg of dried tissues by mg/litre in water at harvest. By the end of the 14-day depuration period, over 90% of the accumulated carbaryl was eliminated. The persistence and accumulation of radiolabelled carbaryl (technical, 99%), administered in either food or water (flow-through), was studied in catfish ( Ictalurus punctatus) over a 56-day period (Korn, 1973). Fish were removed periodically for whole-body residue analysis. Intact carbaryl was not distinguished from 1-naphthol. Accumulation from dietary carbaryl (2.8 mg/kg per week) was 11 and 9 mg/kg tissue at 3 and 56 days, respectively. Fish accumulated 1% or less of the available pesticide via dietary exposure. The mean total accumulation of carbaryl (and/or metabolite residues) after 56 days of exposure to water containing 0.25 mg carbaryl/litre was 11 mg/kg. Fish exposed via the water retained 0.0001% carbaryl. Fish exposed to 2.8 mg/kg per week via diet eliminated residues rapidly after they were placed on a carbaryl-free diet for 28 days. However, residues remained constant for 28 days in fish previously exposed to 0.25 mg carbaryl/litre water for 56 days. The authors speculated that the greater persistence of carbaryl residues from the water might be due to the persistence in fish of 1-naphthol. The low accumulation and rapid elimination of carbaryl in fish were confirmed in other laboratory work (Kanazawa, 1975, 1981). The biodegradation of carbaryl, was studied by Bogacka & Groba (1980) in a model system simulating the river-water and aqueous ecosystem conditions. The rate of pesticide decay in water depended on the initial concentration, temperature, and the kind of model. Lowering the temperature inhibited this process. The accumulation of carbaryl in bottom sediments or in aquatic organisms (algae, snails, and fish) was not observed at concentrations of 10 or 50 µg/litre. Trace quantities of 1-naphthol could be detected occasionally. 4.3 Soil 4.3.1 Adsorption, desorption Carbaryl is adsorbed on soil. Most Koc values were in the range of 100-600, which corresponds to medium to strong adsorption. Only in some soils from Eastern Australia was carbaryl apparently less tightly bound (Koc 90-220). Taking into account both the adsorption/desorption properties and the short persistence of carbaryl in soil, it can be calculated that the compound has low to moderate mobility in soil and will remain in the top layers, when applied under actual agricultural conditions (normal dose rates). Leenheer & Ahlrichs (1971) stated that fast adsorption rates for carbaryl on soil particles were related to organic matter (OM) and were of the order expected for physical adsorption. Carbaryl desorption and movement in the soil was studied in 1-m soil columns in glass tubes, which had a drain (LaFleur, 1976a). Desorption by added water was rectilinear for carbaryl (soil range 1-200 µmol/kg). The movement of carbaryl in the column was a function of the percentage of the organic matter. In soil containing 5.16% organic matter, carbaryl reached the 40-cm section of the column only. When the organic matter content of the soil was low (0.22-0.57%), about one-half of the added carbaryl moved through the column and was found in the effluent, after addition of a volume of water that was equal to six months' rainfall. Briggs (1981) also reported that increasing levels of organic matter in soils resulted in an increase in the adsorption of carbaryl. On the basis of the comparative mobility of carbaryl on soil thin-layer chromatography (TLC) plates using 4 US soils (silt loam, loam, Norfolk sandy loam, and silty clay loam), Chib & Andrawes (1985) concluded that the mobility of carbaryl is low to moderate. With water elution equivalent to about 580 mm of rainfall, the major portion of carbaryl was in the top 10 cm of soil, thus exhibiting low mobility. Only 0.6% of the applied 14C was eluted with the water. Approximately 70% of the applied carbaryl was degraded to volatile gases during the 30-day incubation. Aly et al. (1980) studied the adsorption of carbaryl at different temperatures on Ca-bentonite and on two Egyptian soils (Nile alluvial and a highly calcareous soil). Carbaryl adsorption increased as the temperature decreased. Ca-bentonite exhibited the highest degree of adsorption followed by the Nile alluvial soil and the calcareous soil. The factors that contributed most to the total adsorption of carbaryl were the soil contents of clay, calcium carbonate, and organic matter. The adsorption-desorption and mobility of carbaryl in 3 soils, and in a stream sediment were studied by Sharom et al. (1980). The order of adsorptive capacity was organic soil (75.3% OM, pH 6.1) > sediment (2.8% OM, pH 6.6) > sandy loam (2.5% OM, pH 6.8) > sand (0.7% OM, pH 7). Desorption occurred in greatest amounts from sand > sandy loam > sediment. Leachability studies were consistent with the adsorption/desorption results and a water solubility of 40 mg/litre. Carbaryl leached more from the sand than from the organic soil. In another adsorption/desorption study on carbaryl on sand, sandy loam, silt loam, silty clay loam, and on aquatic sediment using batch equilibrium techniques, Chib (1985a) concluded that carbaryl binds strongly to soil/sediment matrices. This depends on the carbaryl concentration in solution (the more in solution, the more on soil) and on the organic content of soil. Desorption isotherms for carbaryl at low concentrations were nearly flat with all soils, indicating that carbaryl was tightly bound to the substrates and difficult to move. The transport of carbaryl in the soil was also studied under natural conditions. High-volume rainfall occurring shortly after carbaryl has been applied to a field can generate the low-level transport of the pesticide to non-target areas (Caro et al., 1974). Carbaryl is adsorbed on soil surfaces to a great extent. Laboratory measurements of absorption isotherms gave a Freundlich k value of 2.2. Of the 4 kg of carbaryl applied in a watershed, only 5.77 g (0.16%) were found in run-off water and sediment during the season; (75 and 25% of the seasonal loss, respectively). The distribution coefficient between sediment and water was 0.33. Carbaryl had completely disappeared from the sediment by day 70, but still remained in the water at low concentrations. Carbaryl could still be found in the soil, 1-2 years after application at 2 kg 85% WP/ha. It is carried by rainfall and cultivation from the surface layers of the soil into the deeper layers. During the first few days after application, it was found in the root zone. After 3 months, 90% of the carbaryl was present in the surface layers, and, after 300 days, it was found in the soil at a depth of 60-70 cm. Rainfall was not specified (Molozhanova, 1968). Carbaryl was applied to a sandy loam field plot at a dose of 25.4 kg/ha. A shallow water table was present at 1.1 m depth (LaFleur, 1976b). Rainfall during the following 16 months was 182 cm. After this length of time, the upper 1 m contained 6% of the applied carbaryl. No carbaryl was found in the 0-20 cm layer after the fourth month. The half-life of carbaryl in the upper 1 m was >1 month. Carbaryl appeared in the underlying groundwater within 2 months following treatment and could be detected through the eighth month. The maximum groundwater concentration occurred at the end of the second month (about 60 µg/litre). The dose rate was more than 10 times the usual one and the persistence was overestimated, as well as the residues that might be present in shallow groundwater. Norris (1991) reported a terrestrial field soil dissipation study conducted with carbaryl under actual use conditions. Carbaryl was applied to broccoli in California at 2.24 kg/ha, five times, on a weekly schedule. It was also applied once to sweet corn in North Carolina at a rate of 7.12 kg a.i./ha. The half-life in soil was estimated to be 6-12 days at the California site and about 4.8 days at the North Carolina site. Carbaryl residues were found mainly at a depth of 0-15 cm, which indicates that carbaryl has a low potential for leaching through the soil profile under typical agronomic practices. 4.3.2 Transformation Degradation of carbaryl in soil occurs as a result of the activity of microorganisms, and through physical and chemical effects. It undergoes hydrolysis, oxidation, and other chemical processes and, on the surface of the soil, is subjected to photolysis. When applied at the usual doses, carbaryl has a short persistence in soil. In the field, under temperate and warm climatic conditions, the half-life of carbaryl in the soil does not exceed one month (Table 7). 22.214.171.124 Photolysis in soil Studies have been conducted on the photolysis of carbaryl in soil. In one study, volatile substances were first identified and quantified and then soil TLC plates were used over a 30-day period to quantify degradation products (Chib, 1986b). The calculated half-life of carbaryl in sandy loam soil on TLC plates irradiated with a high-pressure mercury vapour lamp (200 watt, Hanovia) at 25 °C was 2.5 days. In controls kept in the dark, labelled carbaryl degraded with a half-life of >30 days. CO2 was the only major volatile degradation product from all treatments. The other soil degradation products identified were 5-hydroxy carbaryl, N-hydroxymethyl carbaryl and 1-naphthol. Only carbaryl and 1-naphthol were present in control soil extracts. The degradation products in the irradiated soil extracts suggest that hydrolysis and oxidation are the main mechanisms in the degradation of carbaryl under soil photolysis conditions. Das (1990b) also studied the photolysis of carbaryl in soil. Labelled carbaryl was applied to a sandy loam on plates (9.8±0.3 mg/kg) and maintained in artificial sunlight, intermittently (12 h irradiation + 12 h darkness) for 30 days. The measured irradiance of the artificial sunlight (502.6 watts/m2) was comparable to the natural sunlight (545.8 watts/m2). The evolution of volatile substances was also monitored. Controls were incubated in the dark. The calculated half-life of irradiated samples was 41 days (each day 12 h irradiation + 12 h darkness). No major metabolites were formed under irradiated conditions. 4.3.3 Biotransformation in soil The persistence and metabolism of 14C-carbaryl (200 mg/kg) was studied in 5 different soil types by Kazano et al. (1972). Persistence was influenced by soil type, and the production of 14CO2 varied from 2.2% (loamy sand) to 37.4% (clay loam) of the initial radioactivity during 32 days of incubation at 25 °C. The amount of radioactivity left in the soil after extraction was proportional to the soil organic matter content. A significant amount (17-57%) of the remaining radiolabel could not be extracted with organic solvents. Analysis of the extractable residues indicated the presence of 1-naphthyl N-hydroxymethylcarbamate, 4-hydroxy-1-naphthyl methylcarbamate and 5-hydroxy-1-naphthyl methylcarbamate, but these were not confirmed. The main pathway of degradation in soil was probably hydrolysis of the carbamate linkage, producing CO2 and the corresponding phenol, though it is possible that hydroxylation of the ring or the methyl carbon precedes hydrolysis. 1-Naphthol decomposed more rapidly in clay than in sandy loam. Most of the radioactivity (83-92%) was recovered in the soils after 60 days of incubation. 1-Naphthol was immobilized on humic substances in the soil, not by mechanical adsorption, but by chemical bonding. Four metabolites (coumarin, the others not identified) were also produced by a soil Pseudomonas. Gill & Yeoh (1980) studied the degradation of carbaryl in an extract of flooded paddy field soil (pH 3.7-4.8, organic matter content 0.5-3%, very high clay content) extract and in the paddy fish ( Trichogaster pectoralis). Under flooded conditions, the soil half-life of carbaryl was about 7 weeks. The major significant metabolite was 1-naphthol. Soil moisture plays an important role in the extent of degradation since, under flooded conditions and 100% field capacity, the degradation of carbaryl was more extensive than under 0 and 50% field capacity, providing more evidence that microorganisms play an important role in carbaryl degradation in paddy-field soil. Where sufficient moisture is available, oxidative mechanisms become important giving rise to ring and N-methyl hydroxylation in addition to cleavage of the carbonyl moiety, a common pathway of carbaryl metabolism. Carbaryl was also found to be more persistent in acidic soil than in alluvial soil. Rajagopal et al. (1983) measured the residues of carbaryl and of the 1-naphthol metabolite in 3 flooded soils (organic matter 0.54-1.61%, pH 6.2-9.5) following three applications of the test substance. Samples received either 1, 2, or 3 applications of carbaryl in aqueous solution. The concentration of carbaryl decreased with incubation time in all soils. The disappearance of the first 50% of the substance in all soils ranged from 10 to 15 days. Subsequent samplings provided evidence for the enrichment of carbaryl-degrading microorganisms in retreated soils. Thus, the disappearance time for 75% of the compound was 20-26 days for 3 applications, 28-30 days for 2 applications, and >30 days for 1 application. It appears that carbaryl degradation under flooded conditions does not follow a clear kinetic pattern (e.g., first order). However, the first order degradation rates were similar for the first, second, and third applications for 0-10 days. Degradation proceeded by hydrolysis with 1-naphthol as the major product, the amounts formed decreasing with incubation time in 2 out of the 3 soils. The disappearance of 1-naphthol was faster in retreated soils and may not be attributed only to degradation, because significant amounts of 1-naphthol may be bound to humic substances, especially in soils with a high organic matter content. The persistence of carbaryl was studied in 4 soils under flooded conditions by Venkateswarlu et al. (1980). They recovered a substantial portion of carbaryl from all soils 15 days after application. The recovery ranged from 37% in an alluvial soil to 73% in an acid sulfate soil. They concluded that flooded conditions enhance carbaryl degradation. The metabolism of 14C-carbaryl and 14C-1-naphthol in moist and flooded soils was studied by Murthy & Raghu (1989) in a continuous flow-through system, over a period of 28 days, permitting a 14C-mass balance. The percentage distribution of radiocarbon in organic volatile compounds, CO2, and extractable and non-extractable (bound) fractions of soils was determined. Organic volatile compounds could not be detected in either carbaryl- or 1-naphthol-treated soils. More 14CO2 (25.6%) was evolved from moist than from flooded soil (15.1%), treated with carbaryl. The mineralization of14C-1-naphthol was negligible. The level of extractable radiocarbon was higher (5.5%) in flooded soil treated with carbaryl. Less than 1% was the parent compound, and carbaryl was mainly metabolized to 5-hydroxycarbaryl in moist soil and to 4- and 5-hydroxycarbaryl in flooded soil. The extractable radiocarbon amounted to 18.2% and 24.3% in moist and flooded soils, respectively, and there was less than 1% of the parent compound with 1-naphthol treatment. Most of the 14C was found as soil-bound residues, levels being higher with 1-naphthol treatment than with carbaryl. The humus fraction of the soil organic matter contributed most to soil-bound residues of both carbaryl and 1-naphthol. Aerobic and anaerobic soil metabolism studies were conducted with carbaryl applied to sandy and clay loam soils in the dark (Wilkes et al., 1977; Khasawinah, 1978). Under aerobic conditions at room temperatures (23-25 °C), the half-life of carbaryl was approximately 9-17 days in sandy loam soil (Texas) and 21-27 days in clay loam (California). Lower temperature (15 °C) nearly doubled its half-life. The only solvent-extractable apolar material was parent carbaryl. A major proportion of the radioactivity initially applied to the soil was lost as CO2. It appears that, under aerobic conditions, carbaryl was degraded so that 1-naphthol carbon was lost as CO2, or it was incorporated into the organic matter of the soil (1-naphthol was not detected). After 112 days, carbaryl levels declined to 3.6% (average in Texas soil) and 15% (average in California soil) of the initial application. In an aerobic, soil study conducted by Miller (1990) with a sandy loam soil from North Carolina, soil was incubated with labelled carbaryl in the dark and maintained at 25±1 °C. The concentration of the parent compound rapidly decreased to a mean value of 11.9% of the applied dose by day 14. A significant amount of radioactivity was recovered as CO2. The average maximum value of CO2 was 59.8% by day 14. The only other major degradation product was 1-naphthol (maximum concentration of 34.9% of applied dose by day 1). Base hydrolysis released 41% of the unextractable residue. Carbaryl rapidly degraded under aerobic conditions with a half-life of 5.5 days. Murthy & Raghu (1991) studied the fate of carbaryl in the soil environment as a function of pH, with respect to the formation of extractable and non-extractable (soil-bound) residues. Soil samples (sandy clay, sandy loam, and clay) containing C14-carbaryl (10 mg/kg) were incubated at 28-30 °C for periods ranging from 7 to 56 days. More 14C-residues could be extracted from sandy clay and sandy loam than from clay soil, under both moist and flooded conditions. In general, flooding had no influence on the extractable 14C-residues. Thin-layer chromatography of chloroform extracts revealed the presence of carbaryl and 1-naphthol. At the end of 56 days, the percentage of carbaryl recovered was 32.1, 6.4, and 1% in sandy clay (pH 4.2), sandy loam (pH 6.8), and clay soils (pH 8.3), respectively. The authors considered that there appeared to be a correlation between soil pH and soil-bound residue formation as an increase in soil pH was reflected in increased bound residues. The humin portion of soil organic matter accounted for most of the 14C-residues. Low recoveries in sandy clay and in sandy loam soils may stem from the possible mineralization of carbaryl. Carbaryl persistence in the soil was studied under normal conditions of application (Caro et al., 1974) of 5.03 kg/ha in granules applied in corn seed furrows. About 135 days were required for 95% of the carbaryl to disappear (Fig. 1). The pesticide remained stable in the soil for 25 days (in some cases, more than 116 days) and then decayed rapidly. This decay is an indirect indication of microbiological degradation. This hypothesis is sustained by the finding that the stability of carbaryl in the soil is affected by the nature of the treatments applied during the period preceding its application. In soil that had been treated with carbaryl 6 months before sampling, more than 70% of the added radiolabelled carbaryl was degraded after 4 days, measured by the disappearance of radioactivity. Only 10% was lost in orchard soil that had been treated for 15 years with various pesticides, and only 3% was lost in soil that had never been treated with pesticides. It appeared that soil that was treated with carbaryl for 6 months increased the ability of the microorganisms to degrade carbaryl (Rodriguez & Dorough, 1977). Carbaryl was applied in soil at an agricultural rate and incorporated to a depth of 6 inches (15 cm) (Heywood, 1975). At 28 days after treatment, 63% of the applied dose appeared as CO2 and the identifiable residual carbaryl was about 6% of that applied. In India, degradation of carbaryl was studied in a greenhouse by Brahmaprakash & Sethunathan (1984, 1985). Since a soil planted with crops may be more dynamic and complex than unplanted soil, because of increased microbial activity, they studied carbaryl persistence in soil (pH 6.2, organic matter 1.6%) planted with rice in a greenhouse, under flooded and non-flooded conditions. Carbaryl disappeared more rapidly from soils planted with rice than from those without rice, under both flooded and non-flooded conditions. The amount of carbaryl decreased to between 30.2 and 32.1% of the original level within 30 days in unplanted soil under both flooded and non-flooded conditions. During this same period, the carbaryl concentration decreased to 17-18% of the original level in planted soil under both water regimes. Degradation occurred by hydrolysis, but there was no appreciable difference in the rates of degradation between flooded and non-flooded soils. The rate of degradation of carbaryl was little affected by moisture. Further degradation of 1-naphthol was slow in both planted and unplanted systems. A significant portion of the ring-14C accumulated in the soil as 1-naphthol and soil-bound residues. Evolution of 14CO2 from the labelled side-chain and ring was negligible, even in soil planted to rice. 4.3.4 Degradation by microorganisms Many studies have demonstrated the great ability of microorganisms to degrade carbaryl in the soil, and for some of them, to use it as a sole source of carbon. The most frequently identified organisms are bacteria ( Pseudomonas phaseolicola, P. cepaphia, Rhodococcus sp., Nocardia sp., Xanthomonas sp., Achromobacter sp.) and fungi ( Aspergillus niger, A. terreus, Fusarium solani, Gilocladium roseum, Rhizoctonia solani, R. practicola, Penicillium sp., Mucor sp., Rhizopus sp.). In many cases, it was demonstrated that the persistence of carbaryl in the soil decreased after a first application, which was interpreted as the selection and build-up of strains more capable of degrading the product. The same process may also explain that, in some studies, the rapidity of degradation suddenly rose after a lag period during which only minor amounts of carbaryl degraded. Tewfik & Hamdi (1970) mentioned that carbaryl was decomposed into 4 distinct compounds by a soil bacterium designated as S-1. They considered that this soil bacterium might utilize carbaryl in a similar way to Pseudomonas sp., which metabolize naphthalene via the salicylate pathway. Zuberi & Zubairi (1971) also reported that carbaryl is degraded by soil microflora. Pseudomonas phaseolicola and Aspergillus niger hydrolysed carbaryl to 1-naphthol. In the case of P. phaseolicola, an unidentified minor metabolite was also detected. Larkin & Day (1986) reported that 2 bacteria isolated from garden soil, Pseudomonas sp. (NCIB 12042) and Rhodococcus sp. (NCIB 12038), could grow on carbaryl as the sole source of carbon and nitrogen at pH 6.8, but failed to metabolize carbaryl rapidly. Both could use 1-naphthol as the sole source of carbon and metabolize it via salicylic acid. Strain NCIB 12038 produced also gentisic acid. Pseudomonas sp. (NCIB 12043), in a soil perfusion column enrichment at pH 5.2, metabolized carbaryl rapidly to 1-naphthol and methylamine. 1-Naphthol itself was metabolized via gentisic acid. A possible pathway for the catabolism of carbaryl and 1-naphthol was proposed. Walker et al. (1975a), working with Pseudomonas sp., observed degradation of 1-naphthol and suggested that carbaryl could serve as the sole source of nitrogen and carbon for bacteria. It was also noted that the presence of another nitrogen source in the media seemed to have a delaying effect on metabolism of carbaryl. Bacterial communities of at least 12 and 14 members were selected in continuous culture using carbaryl as the sole source of carbon and nitrogen at pH 6. These communities were supported by the slow formation of hydrolysis products and no carbaryl-degrading bacterium was selected after more than 83 days. When using equimolar 1-naphthol and methylamine as the sole source of carbon and nitrogen, a bacterial community of at least 8 members was selected. After a lag of 10-50 days, soil perfusion columns (pH 5.2) and continuous culture enrichments (pH 5) led to the selection of a Pseudomonas sp. that could utilize carbaryl as its sole carbon and nitrogen source (Larkin & Day, 1985). Sud et al. (1972) showed that Achromobacter sp. also utilized carbaryl as the sole source of carbon in a salt medium. Four degradation products of carbaryl were 1-naphthol, hydroquinone, catechole, and pyruvate. The organism also grew well in the first 3 degradation products. The rate of degradation of carbaryl after 1, 2, and 3 applications to 3 submerged soils was examined by Rajagopal et al. (1983). Soils that had been pretreated with carbaryl were able to degrade carbaryl more rapidly than those without pretreatment. The enrichment culture was inactivated upon autoclaving. The concentration of carbaryl decreased in the mineral medium inoculated with the enrichment cultures from the 3 soils, especially when it served as the sole source of both carbon and nitrogen. Rajagopal et al. (1984) studied the metabolism of side-chain and ring 14C-labelled carbaryl in a mineral salts medium by soil enrichment cultures. Hydrolysis was the major route of microbial degradation. During carbaryl degradation by enrichment cultures and Bacillus sp., 1-naphthol and 1,4-naphthoquinone accumulated in the medium. Rajagopal et al. (1986) also observed that carbaryl disappeared more rapidly from a laterite soil pretreated with 1-naphthol than from a control soil never exposed to 1-naphthol. The accumulation of 1-naphthol and bound residues formed from added14C-carbaryl was greater in soils pretreated with 1-naphthol than in untreated soils. In experiments with the soil fungus Rhizoctonia practicola, Bollag et al. (1976) found that it could transform 1-naphthol from an ether-extractable to a water-soluble product. It was also observed that, after removal of the fungal cells, the growth medium possessed the ability to transform 1-naphthol, indicating activity of an extracellular enzyme. Attempts to analyse the labelled material in the aqueous phase indicated that the radioactivity was associated with a compound of comparatively high relative molecular mass. Czaplicki & Bollag (1975) exposed 1-naphthol to Rhizoctonia solani, isolated from soil, and found that it was completely transformed to a compound not extractable with ether. Working with the soil fungus Aspergillus terreus, Liu & Bollag (1971b) investigated the metabolic transformation of carbaryl through 1-naphthyl- N-hydroxymethylcarbamate and tried to clarify the pathway of the side-chain. The next intermediate in biological transformation was 1-naphthyl carbamate, which was further degraded to 1-naphthol. Within one week, approximately half the amount of carbaryl was transformed to these other metabolic products. No attempt was made to clarify whether the formation of 1-naphthol was the result of the biological or the chemical degradation of 1-naphthylcarbamate, but there were clear indications that 1-naphthol was metabolized further in the presence of A. terreus. In vitro studies were carried out to investigate the degradation of carbaryl by soil microorganisms. Three isolates from soil, including Fusarium solani, a Gram-negative coccus, and a Gram-positive rod, accelerated the hydrolysis of carbaryl to 1-naphthol and other intermediates. Fusarium solani was the most effective in decomposing the 14C-labelled compound. Radioactivity decreased by 24% during the first 5 days and by 82% during 12 days in a growing culture after inoculation. A mixture of two or three of the microorganisms was more effective in decomposing carbaryl and 1-naphthol (Bollag & Liu, 1971). Bollag & Liu (1972a) studied the biological degradation of 14C-1-naphthol during growth, with replacement cultures and cell extracts of Fusarium solani. The radioactivity of 1-naphthol disappeared partially during growth, but it was completely dissipated by cell extract activity. The cell extract of F. solani degraded more than 80% of the 1-naphthol to form CO2 within 60 min of incubation, but it was not possible to identify intermediates. This implies rupture of the naphthol ring. No difference in activity could be observed between cell-free extracts from the spores or the mycelium of the fungus at pH 5.7 and 7.2. The enzyme system was relatively stable since there was no decrease in activity after the cell extract was stored at -10 °C during 4 months. The enzyme that participated in the degradation reaction was constitutive (always present) as shown by cell extracts prepared from cells grown in a medium with, or without, carbaryl as substrate. The fungus Gliocladium roseum, isolated from soil by Liu & Bollag (1971a), metabolized carbaryl to 3 metabolites which were identified as 1-naphthyl- N-hydroxy methylcarbamate, 4-hydroxy-1-naphthyl methylcarbamate, and 5-hydroxy-1-naphthyl methyl-carbamate. It was therefore considered that N-alkyl- and aromatic ring-hydroxylation of carbaryl were important detoxication reactions of G. roseum. About 70% of the radioactivity was recovered as carbaryl from an 11-day-old culture. The decrease in radioactivity from the growth medium containing side-chain labelled carbaryl indicated that a further degradation of the formed metabolites occurred, or that an additional pathway was involved in the degradation of carbaryl by G. roseum. Bollag & Liu (1972b) reported that most soil fungi ( Penicillium sp., Mucor sp., Rhizopus sp., and Aspergillus sp. except A. fumigatus), could hydroxylate carbaryl at different positions, but that the products differed qualitatively as well as quantitatively with the various fungi. In a different study, the enzyme or protein fraction (extracellular phenol oxidase) of the culture filtrate from Rhizoctonia practicola as chromatographed on a column of Sephadex G-200 and fractions were obtained that were able to transform 1-naphthol as a substrate (Sjoblad et al., 1976). The enzyme catalysed the polymerization of 1-naphthol to several products. Biological oxidation and coupling of phenols are key reactions in nature, that result in the formation of products such as lignins, melanins, tannins, alkaloids, and humus compounds. Thus, it can be assumed that enzymes interact with xenobiotic phenols that are incorporated into soil or sediment organic matter. Rhizoctonia practicola (and an extracellular enzyme) was able to polymerize 1-naphthol (dimers, trimer, tetramer) (Bollag et al., 1978). Rodriguez & Dorough (1977) studied the persistence of carbaryl in culture media in the presence of mixed and pure cultures of bacteria and fungi isolated from soil. All fungi ( Fusarium, Penicillium, Aspergillus, and one unidentified species), isolated from a soil treated with carbaryl six months before sampling, produced at least one metabolite from carbaryl. After 14 days of incubation, 54-79% of the carbaryl was recovered intact, and there was little formation of carbon dioxide by the fungi. In contrast, related experiments with bacterial isolates ( Arthrobacter, Nocardia, Pseudomonas, Xanthomonas, and Bacillus) from the same soil showed that only 1-9% of the added carbaryl remained unconverted ( Arthrobacter was the exception with 59.1% remaining carbaryl). Controls showed that non-biological degradation also occurred. Bacteria, like fungi, metabolized carbaryl qualitatively in the manner observed with natural soil populations. However, quantitative differences were so great that the use of isolates may be of little value in estimating the degradation rate of carbaryl and other pesticides in field soil. This is important to note when conducting microbial degradation studies in the laboratory. Bollag (1979) summarized the transformations of carbaryl by microbial activity. It was possible to isolate several microorganisms capable of hydrolising carbaryl to 1-naphthol. In addition, Gliocladium roseum showed the formation of: 4-hydroxy-1-naphthyl N-methylcarbamate 5-hydroxy-1-naphthyl N-methylcarbamate 1-naphthyl N-hydroxy-methylcarbamate. Other terrestrial fungi hydroxylated carbaryl at different positions, but the products differed quantitatively and qualitatively with the various fungi. Subsequent metabolic transformation of the hydroxylated products differed considerably. With A. terreus, the following products were obtained: 1-naphthyl N-hydroxy-methylcarbamate --> 1-naphthyl-carbamate --> 1-naphthol Several bacteria isolated from river water were capable of degrading 1-naphthol. At least two different pathways were involved. The first was a complete degradation with the release of CO2, the second produced principally 4-hydroxy-1-tetralone, which may involve hydroxylation of the naphthyl ring in the 4 position and conversion to an aliphatic cyclic compound. Such a pathway has also been described with a soil Pseudomonas by Walker et al. (1975a) and Davis & Evans (1964). 4.3.5 Persistence in soil Carbaryl is not usually applied as a soil treatment, therefore, the amounts of carbaryl that may reach the soil come principally from spray drift, or from washing off treated crops by rain. When applied at the usual doses, in the laboratory, carbaryl has a short persistence (half-life <40 days and usually from 6-20 days), but this may increase when the soil is flooded, or when the dose is increased. In field studies, the half-life of carbaryl, applied at the usual dose rates, in warm or temperate climatic conditions, did not exceed one month in the soil. The results of laboratory and field studies on the persistence of carbaryl in soil are summarized in Table 7. Table 7. Half-life of carbaryl in soil, calculated from experimental laboratory and field results Origin of soil Number Degradation Half-life References of days (%) (days) Laboratory 40 100 8 Johnson & Stansbury (1965) Laboratory 0.5 mg/kg 45 77 20 Molozhanova & Kanevskij (1971) Laboratory 1 mg/kg 45 53 40 Laboratory 10 mg/kg 45 13 Laboratory 30 55-63 < 20 Flores-Ruegg et al. (1980) Laboratory (flooded) 15 63-27 10-40 Venkatesvarlu et al. (1980) Laboratory 53 100 < 8 Odeyemi (1982) Orchard 184 99 30 Ivanova & Molozhanova (1973) Corn 135 95 30 Caro et al. (1974) Pretreated soil 4 70 < 3 Rodriguez & Dorough (1977) Grain cropa 30 53-31 27-55 Gangwar et al. (1978a) Corn soilb 5 42-56 5 Kavadia et al. (1978) Potatoes 50 < 99 < 7 Kovaleva & Talamov (1978b) - 30 96 6 Table 7 (continued) Origin of soil Number Degradation Half-life References of days (%) (days) Unspecified 90 Czaplicki (1979) Tropical 7 Rajukkannu et al. (1985) Sesamum 60 56 50 Yadav et al. (1985) Bare soil < 4 Davis (1986a) Bare soil 1-13 Forest 1.5 Sundaram & Szeto (1987) Forest 90 < 78 < 38 Springborn (1988b,c) Forest 90 < 90 < 24 Flooded rice 180 (56) Springborn (1988a) Flooded rice 180 (52) Broccoli 6-12 Norris (1991) Sweet corn 5 a20-60 kg/ha. b15-45 kg/ha. Johnson & Stansbury (1965) calculated a half-life of approxi-mately 8 days in an agricultural soil (sandy loam, pH 5.5) treated at 3 concentrations. Residues of carbaryl appeared to be completely degraded within 40 days. The considerable influence of the dose rate on the persistence of carbaryl in soil was demonstrated by Molozhanova & Kanevskij (1971). The percentages of the initial quantities that were degraded 45 days after treatment are shown in Table 8. Table 8. Degradation rate of carbaryl in relation to the dose Initial dose Degradation (%) (mg/kg) 0.5 77 1 53 1.5 37.3 2 30 2.5 27 3 23 4 20 6 16.7 8 15 10 13.2 The same authors also demonstrated the influence of the type of soil on the degradation of carbaryl. Forty-five days after treatment, about 50% degradation was observed in light grey forestry soil, and only about 20% in peat. The ability of soils to degrade carbaryl could be ranked as follows: light grey > grey forestry > turf podzol > southern chernozeme > ordinary chernozeme > meadow > peat. Ivanova & Molozhanova (1973) calculated that application of 1.36 kg carbaryl/ha in an orchard resulted in 19.7% being retained in the soil; 99% of the carbaryl present in soil disappeared within 184 days. Czaplicki (1979) conducted studies in Poland. The soil half-life of carbaryl was about 3 months and 90% degradation occurred within 18 months. From October to March, when the soil temperature was <5-8 °C, the disappearance of the insecticides in the soil almost stopped. Rajukkannu et al. (1985) studied the persistence of 4 products in the red and black soils of Tamil Nadu (India), where the tropical climate in conjunction with the soil properties shortened the persistence of insecticides. The half-life of carbaryl was 6.7-7 days and 95% degradation was achieved after 90 days. It was reported by Odeyemi (1982) that carbaryl disappeared from soil samples treated with 45 mg/kg after 53 days incubation in a greenhouse under tropical conditions (Nigeria). 4.3.6 Interaction with other physical, chemical, or biological factors Nitrogen fertilizers (ammonium sulfate and urea) increased the persistence of carbaryl in flooded laterite soil with a low native content of nitrogen (0.04%). In the alluvial soil with 0.11% total nitrogen, the persistence of the insecticides was little affected. The rates of degradation in the two soils treated with nitrogen fertilizers were almost identical. The authors speculated about the mechanism of this effect, which was possibly due to the preferential utilization of inorganic nitrogen by microorganisms, or to the inhibition of soil hydrolase activity (Rajagopal & Sethunathan, 1984). 4.3.7 Vegetation 126.96.36.199 Uptake and transformation in plants Carbaryl from soil treated at about 50 times the usual dose penetrated into apple trees and couch-grass (Molozhanova, 1968). Three months after application of carbaryl (85%) to soil at a dose of 100 mg/kg, residues of carbaryl were detected in the roots and stems in apples (39-13 mg/kg) and in couch-grass (50-38 mg/kg). Under the same conditions, no residues were detected in tomato fruits or in wheat grain, but 5.9 mg/kg were detected in potatoes (Table 9). Table 9. Migration of carbaryl from the soil into apple trees at different times following applicationa Experimental Sampling day Carbaryl content of samplesb conditions after (mg/kg) application Soil Foliage Fruits 85% WP carbaryl 5 9.3 0 - applied under each tree at a rate of 12 6.8 0 - 10 mg a.i./kg soil 19 6.0 5.0 - 56 4.4 0.04 4.4 138 2.1 0 - aFrom: Molozhanova (1968). bAverage data from 18 samples are given for each case. Huque (1972) reported that 14C-labelled carbaryl was readily taken up from granules by 2-week-old rice plants. After 3 days, a level of about 100 mg/kg was determined in the plant (excluding the roots). Residues then decreased to about 15 mg/kg after 18 days. Ferreira & Seiber (1981) studied the uptake and distribution of carbaryl in rice seedlings following exposure of the roots to the insecticide. Carbaryl was rapidly absorbed and transported upwards to the leaves and stems. After exposure was terminated, the major route of loss of carbaryl was through root exudation (22%) rather than volatilization from the leaves (4.2%). However, since most of the remaining carbaryl in the plant was present on the leaf surfaces, the authors surmised that volatilization might play a greater role in pesticide loss over longer periods of time. 14C-carbaryl and 14C-1-naphthol from soil-bound residues were partially released when barley was grown (Murthy & Raghu, 1988). 14C-residues could be detected in both shoots and roots in the case of carbaryl treatment, while only roots showed 14C-residues in the case of 1-naphthol. The rate of decomposition of carbaryl in plants depends on the climatic conditions. It is more rapidly decomposed in hot climates, at high temperatures, and by intensive ultraviolet radiation. Thus, residual levels in feed plants were lower in regions with a hot climate than in other regions (Atabaev, 1972). 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1 Environmental levels 5.1.1 Air Ambient air concentrations of carbaryl were measured before and after a treatment of a large area of forest in Maine, USA, to control spruce budworm. Concentrations in air ranged from less than 0.0035 to 0.107 µg/m3 several miles away (Shehata et al., 1984). Room concentrations of airborne carbaryl were measured following application for pest control as a 5% dust at an undetermined rate. The levels detected on the day of application, and 1, 2, or 3 days after application were 1.3, 0.2, 0.1, and 0.01 µg/m3, respectively (Wright et al., 1981). 5.1.2 Water Carbaryl concentrations in water from different sources in the USSR were studied by Molozhanova (1970). Data are presented in Table 10. Table 10. Concentrations of carbaryl in watera Type of sample Number of samples Mean Total Carbaryl-containing concentrations samples (%) (mg/litre) Well 114 87 0.13 Artesian water 64 70 0.16 Ground water 24 75 0.27 Dam 90 47 0.02 River 77 62 0.044 Lake 84 85 0.03 aFrom: Molozhanova (1970). According to an earlier report by Molozhanova (1968), carbaryl was present in trace amounts up to a maximum level of 1 mg/litre in well water and reservoirs during carbaryl application (6 kg/ha, 85% WP). Water irrigation channels, situated at a distance of 200-300 m from a carbaryl-treated cotton field, contained from 0.01 to 0.25 mg/litre up to 18 days after spraying at a dose of 2kg/ha (Guseynov, 1970). Residues of pesticides were monitored in the aquatic system of Ioannina basin (Greece) and its natural outlet, Kalamas River, for the period September 1984-October 1985 (Albanis et al., 1986). The carbaryl concentration in water was found to follow a seasonal fluctuation with maxima during summer and minima during winter months. Mean and range values are summarized in Table 11. Table 11. Mean and range values of carbaryl (ng/litre) on different sampling dates in the Ioannina basin Month of sampling River Canal Lake September 0.5 November NDa NDa March NDa May ND June NDa July 23.7 21.7 August 2.3 September 1.7 NDa April NDa July 8.8 aND = not detected. An oil-based, carbaryl formulation (Sevin-2-oil) was applied twice to a coniferous forest in New Brunswick (Canada). At a dose rate of 280 g/ha, the highest concentration in stream water was 0.314 mg/litre and only 0.123 mg/litre 0.5 h after spraying. More than 50% of the initial residues dissipated within 1 h (Sundaram & Szeto, 1987). Carbaryl was applied aerially as Sevin-4-oil (1.12 kg/ha) to aforest in Montana. Field samples of water were collected to study persistence. Residue levels in 4 streams sampled were variable (3-6, 3-175, 2-260, 4-108 µg/litre) 1.3-2.8 h after application (Pieper & Roberts, 1978; Pieper, 1979). Local accumulations of floating oil-based spray may have caused this variation. Grantham (1980) reported that carbaryl (Sevin-4-oil) levels were generally highest directly after spraying, but increased at some stream sites for 1-2 days after application. This was attributed to light rains, which washed the spray residue off the foliage and into the stream channel. However, Brown (1980) concluded that carbaryl enters streams in periodic doses and that rainfall dates are not related to the presence of residues in streams. Maximum residues of 26.32 µg/litre were found in water 12 h after spraying, with no residues detectable in samples taken hourly until 36 h. Residues were found again in samples taken 2.5-3 days and 5.5-6.5 days after spraying. In a study where carbaryl (Sevin-4-oil) was applied aerially (0.84 kg/ha) to a forest in Maine, in 1978, samples were collected in 9 streams (Stanley & Trial, 1980). Peak concentrations occurred shortly after spraying and maximum residue levels ranged from 0.93 to 7.8 µg/litre in brooks and from 0.44 to 2 µg/litre in rivers. In one stream, the maximum concentration was 16 µg/litre. Ott et al. (1981) reported that carbaryl levels were highest on the day of spraying (2.7 µg/litre), declined to about 0.7 µg/litre by the third day, but increased again to about 1.2 µg/litre on day 5. No residues were detected in stream water samples taken on the 17th day. Thomas et al. (1982) treated planted rice fields with carbaryl at rates of 0.63, 0.94, and 1.85 kg a.i./ha, either as a high volume spray (500 litre/ha) or as a low volume spray (150 litre/ha), and monitored the residues. In irrigation water sampled one hour after treatment, carbaryl levels amounted to 0.15-0.30 and 0.07-0.18 mg/litre, under high and low volume spraying, respectively. During the first 24 h after spraying, the concentration in the irrigation water remained fairly constant, but, by the fourth day, it had decreased significantly. By the tenth day after spraying the residues in water had decreased to 0.02-0.05 and 0.03-0.04 mg/litre, respectively. A rice field dissipation study was conducted in Arkansas (USA) by Springborn (1988). Carbaryl (Sevin XLR) was applied 3 times at 14-day intervals to an irrigated field plot in which rice was planted. In irrigation water, carbaryl residues disappeared with a half-life of <1 day after each treatment. The maximum concentration in water was 1466 µg/litre and, by day 3 following each treatment, the concentration was <121 µg/litre. In a similar study conducted in California, Springborn (1988a) found that carbaryl dissipated rapidly in irrigation water with a half-life of <1 day. The maximum concentration in water was 648 µg/litre. By day 3 following each treatment, the concentration was <27 µg/litre. Residues of 1-naphthol were found in the water collected from wells and ponds in and around Bhopal (India). The residues of 1-naphthol in well water (near a manufacturing plant) ranged from 0.002 to 0.024 mg/litre. In pond waters, the levels were found to be between 0.036-0.098 mg/litre. The level of 1-naphthol in soil samples was of the order of 0.153-0.656 mg/kg (Dikshith et al., 1990). 5.1.3 Soil Contamination of the soil occurs when carbaryl is used in agriculture. During the treatment period, levels of 1-3 mg carbaryl/kg were measured in the surface layer of the soil in the USSR (Molozhanova, 1968). Concentrations of 0.03-0.35 mg/kg were found in the surface layer of the soil up to 52 days after a single application of carbaryl at a rate of 2 kg/ha (Guseynov, 1970). Carbaryl levels in the soil varied depending on the type of soil (Table 12). Potatoes were sprayed with carbaryl at 4.25 kg/ha (5 kg Sevin WP, 85%/ha). One day, 10 days, and 2 months after treatment, residues in the soil under the potatoes were, respectively, 1.9, 9.55, and 0.02 mg/kg (Kovaleva & Talamov, 1978b). When the soil was sprayed before sowing, the residues in the soil 1 day, 1 month, 4 months, and 15 months after treatment were 1.5-2, 0.05-0.07, 0.02-0.08, and 0.06 mg/kg, respectively. Within 100 and 150 days following soil incorporation of carbaryl (Sevin, 10 kg/ha), residues in non-planted soil decreased to 0.05 and 0.02 mg/kg, respectively (Kovaleva & Talanov, 1980). Table 12. Carbaryl content in different types of soil during harvestinga Type of soil Number of Carbaryl concentration samples (mg/kg) Meadow-chernozem 200 2.22 ± 0.5 Chernozem southern 766 1.01 ± 0.23 Chernozem ordinary 288 1.09 ± 0.26 Chenozem podzol 100 0.15 ± 0.04 Turf-podzol 150 0.04 ± 0.01 Grey-woodland 246 0.0 aFrom: Molozhanova (1970). Yadav et al. (1985) detected 0.22 mg carbaryl/kg in the soil at harvest time, when sesamum ( Sesamum indicum) was sprayed twice with carbaryl (0.2%) on days 45 and 60 of crop growth (21 August and 5 September). As residues in the soil after the first spray were about 0.65 mg/kg, the rate of reduction of residues in soil was 56% within 2 months. Shilova et al. (1973) measured about 0.1 mg carbaryl/kg soil one month after treatment with carbaryl (5 kg/ha) against blood sucking insects in the subarctic. Gangwar et al. (1978b) applied a 4% granular formulation to sandy loam planted with a grain crop (bajra) at very exaggerated application rates of 20, 40, and 60 kg a.i./ha. Initial residues of 140-354 mg/kg decreased by a total of 31 to 53% within 30 days and 95 to 98% within 90 days. Residues at 90 days were 2.68-17.49 mg/kg. Application of carbaryl granules at very high doses of 15, 30, and 45 kg/ha resulted in deposits of 232, 397, and 525 mg/kg, respectively, in soils cultivated with corn, and in 104-109, 168-217, and 304-422 mg/kg, respectively, in clay loam soils cultivated with root crops (beet, radish, carrot). The dissipation rate of carbaryl residues from clay loam soil, in 5 days following the treatment, was 42-56% during autumn and 55-69% during spring. The highest residue levels sampled from all plots decreased to 229.7, 142.9, 73.8, 33.8, 6.7, and 1.3 mg/kg on days 5, 10, 15, 30, 60, and 100, respectively. Residues were below detectable levels in several plots by day 60 and in most plots by day 100 (Kavadia et al., 1978). According to Kuhr et al. (1974), the dissipation of carbaryl in the soil of apple orchards is rapid. Soil residues of carbaryl had almost completely disappeared from the top 2 inches of soil in 2 weeks. They were 13.8 mg/kg immediately after application and 3 mg/kg, 1-2 days later. An oil-based carbaryl formulation (Sevin-2-oil) was applied twice by a fixed-wing aircraft to a coniferous forest in New Brunswick (Canada). Initial residue levels 1 h post-spray in litter and soil for both applications were, respectively, 1.21 and 0.86 mg/kg and 0.78 and 0.48 mg/kg (Sundaram & Szeto, 1987). Relative to the amount sprayed, only a small amount of the chemical reached the litter and soil, probably because of canopy filtration. Within 1 day, an average of 40-45% of the initial residues was lost from litter and soil, respectively, indicating a rapid dissipation time of 1.5 days for the disappearance of 50% of the initial maximum concentration. Beyond 5 days, an average of 12% of the initial concentration remained in both the substrates. As already reported, two field dissipation studies were conducted by Springborn (1988b,c) with carbaryl (Sevin-4-oil) applied twice with a seven-day interval to forest. The first study was conducted in a coniferous Oregon forest. In soil from the treated site, carbaryl levels decreased from 0.196-3.877 mg/kg following the first application to 0.130-1.87 mg/kg, 3 days after treatment, and from 0.079-5.323 mg/kg following the second application to 0.242-1.187 mg/kg at 90 days. The second study was conducted in Pennsylvania. Carbaryl levels in soil decreased from 0.022-0.068 mg/kg following the first application to <0.012-0.075 mg/kg, 3 days after treatment, and from 0.11-0.932 mg/kg following the second application to 0.01-0.099 mg/kg at 90 days. A rice field dissipation study was conducted in Arkansas (USA) by Springborn (1988a). Carbaryl (Sevin XLR) was applied 3 times, at 14-day intervals, to an irrigated field plot in which rice was planted. In flooded soil, the concentration of carbaryl varied from <11 to 309 µg/kg throughout the study and was not directly related to application date. The carbaryl level was <11-309 µg/kg in soil following the first application and <11-129 µg/kg following the third application; 180 days later, carbaryl concentrations in the soil were 11-56 µg/kg. In a similar study conducted in California, under the same conditions, Springborn (1988) found that carbaryl dissipated rapidly in flooded soil. The concentration of carbaryl ranged from <11 to 198 µg/kg throughout the study. Carbaryl was <11-23 µg/kg in soil at sampling intervals following the first application, and increased to <11-180 µg/kg following the third application; 180 days after the third application, carbaryl concentrations in the soil were 11-88 µg/kg. A soil dissipation field study was conducted in 1985/1986 in California (sandy loam, pH 6.3) and Iowa (silt loam, pH 6.5) with carbaryl 80 S formulation (Davis, 1986a). Carbaryl was applied to strip plots of bare soil, 6 weeks later, 7 applications were made with intervals of about 5 days between applications. Soil sampling was performed before treatment, during treatment, and up to 6 months after the last application. No carbaryl residues were found in California pretreatment samples. After the last application, residues reached a maximum of 1.61 and 0.29 mg/kg in the 0-15 and 15-30 cm samples, respectively. Fourteen days after the last application, carbaryl residues below the 15-cm level were less than the limit of quantification. It took 28 days for residues to dissipate completely from the top 15 cm. The half-life in California soil was calculated to be 2.7-3.8 days. 5.1.4 Food and animal feed 188.8.131.52 Fruit, vegetables, and grain Contamination of vegetation by carbaryl occurs, either during spraying, or, by its migration through contaminated soil into the roots of plants. Residual levels of carbaryl after the spraying of plants depend on the type and species of the plants sprayed. Carbaryl levels of up to 30 mg/kg have been found in plants during the treatment period (Molozhanova, 1968). It has been found to be rather persistent in vegetables and fruits. Concentrations of 0.6-3.9 mg/kg were measured in lettuces 1 week after single or repeated sprayings, and levels up to 1 mg/kg were found in tomatoes treated according to requirements (Antonovich, 1970). In cabbage, initial residues after spraying ranged from 14.8 to 33.9 mg/kg, depending on the concen-tration used. After 7 days, the residues decreased to 2.5-5.13 mg/kg. In eggplant, the initial concentrations were 8.3-16.9, and 7 days later, 3.05-5.4 mg/kg. After washing, the concentrations decreased considerably to less than 3 mg/kg after day 7 (Mann & Chopra, 1969). Following application of carbaryl on cauliflower, in the form of dust (10%) at 1.5-2 kg a.i./ha or wettable powder (50%) at 0.75-1 kg a.i./ha, the residues of carbaryl declined to 3.64-9.59 mg/kg within 8 days of treatment. The carbaryl deposits on leaves were between 19.45 and 42.08 mg/kg. Washing of cauliflower with plenty of water reduced the residues of carbaryl by 36-95%. A waiting period of 8 days has been suggested for cauliflower (Singh et al., 1978). Persistence of carbaryl in brinjals and peas after spraying at 1 and 2 kg a.i./ha was found to be 3.42 and 5.03 mg/kg respectively. Washing of vegetables with water within one hour and after a day of spraying reduced the level of deposits of carbaryl to about 1.0 mg/kg level. In the case of pea pods, the levels of residues dropped to 0.20 mg/kg within 5 days. In pea seeds, the residue levels ranged between 0.03 and 0.09 mg/kg, after 8 days (Krishnaih et al., 1978). Measurements of carbaryl in different fruits were taken at intervals of 5-10 days after plants had been treated with a 0.2% suspension of carbaryl at 1000 litre/ha during the vegetation period (Bogomolova, 1968, 1970). The initial amount of carbaryl, 2 days after the treatment, varied in different fruits from 2.3 to 2.7 mg/kg. Ten days later, the concentrations were reduced by 50-70%, and, after 20 days, the levels were within the range of 0.2-0.8 mg/kg, depending on the species (Table 13). According to Mann & Chopra (1969), the dissipation of carbaryl from plants after the first day of spraying was about 40-45% of the initial residue. In apples, concentrations of 2.5-2.9 mg/kg, 0.4-2.4 mg/kg, and 0.15 mg/kg were found on days 7, 10, and 38, respectively, after spraying. One to two months after spraying apples with a 0.1% suspension at a dose of 2000 litre/ha, residues of 0.08-0.10 mg/kg were measured (Antonovich, 1970). Four months after spraying with a 0.06% suspension at a dose of 9 kg a.i./ha, levels in the range of 0.09-0.24 mg/kg were found in apples, depending on the spraying procedure. Fine-droplet spraying resulted in residue levels 2.5-3 times lower levels than those with coarse-droplet sprays (Atabaev, 1972). Table 13. Carbaryl levels in different fruits after spraying during vegetation (mg/kg)a Kind of fruit Day after spraying 2 10 20 Strawberries 2.3 1.25 Gooseberries 2.5 0.90 0.6 Blackcurrants 2.4 1.10 0.8 Cherries 2.7 1.90 0.2-0.4 Plums 2.4 1.40 0.2-0.4 aFrom: Bogomolova (1968). A survey of apples grown in Ontario, Canada, between 1978 and 1986 showed that out of 22% of carbaryl-treated apples (1.67 kg/ha) sold, 3.6% had detectable carbaryl residues (detection limit of 0.01 mg/kg), and that the average residue level was 0.03 mg/kg, with a maximum of 0.04 mg/kg (Frank et al., 1989). Prolonged storage of apples reduced the carbaryl residues only slightly (Bogomolova, 1968). The initial level (2.4-2.8 mg/kg) changed little during the first 3 months of storage at a temperature of +5 to +10 °C. A slight reduction was observed during the fourth month of storage. During the process of storage, carbaryl migrated inwards from the surface, so that the concentration in the skin was reduced, while that in the pulp was increased (Table 14). Residues on lemon foliage on day 0 of treatment were 4.6±0.3 µg/cm2. Five days after treatment, levels of 2.4 µg/cm2 and 5.6 µg/cm2 were found on lemon and orange foliage, respectively; on the 60th day, the residual values were 0.41 and 0.36 µg/cm2, respectively. Persistence half-lives were 22 days (lemons) and 14 days (oranges) (Iwata et al., 1979). Carbaryl is persistent, particularly in citrus fruits and grapes. Residues of 3-6 mg/kg were measured in lemons and oranges 2.5-3 months after treatment of trees with 3-10 kg carbaryl/ha, and amounts of 3.6, 1.9, and 0.4 mg/kg were found in grapes on days 7, 14, and 40, respectively, after repeated spraying of vineyards with a 0.12% suspension. The half-life of carbaryl in grapes was 29 days, compared with 2-9 days in cherries and cabbage (Antonovich, 1970). Washing and peeling reduced carbaryl levels in fruits and vegetables by 40%; thermal processing by 45-90%, and preparation of juices by 53% (Molozhanova, 1970). Canning also reduced carbaryl residues. Thus, canning of different fruits (strawberries, black currants, gooseberries) containing carbaryl in the range of 0.25-0.45 mg/kg, resulted in a reduction to 0.17-0.29 mg/kg; when the initial contents were higher (3-7 mg/kg) the levels after canning dropped to 1.4-2.2 mg/kg and 2.1-2.5 mg/kg, respectively, i.e., from 30 to 70%. Storage of canned fruit further reduced carbaryl residues by 2-3 times after 12 months (Bogomolova, 1968). Boiling vegetables (e.g., cabbage) reduced carbaryl residues by approximately 50%; however, the residues in pickled cabbage, 5 months after preparation, were only 25% less than the initial amount (Antonovich, 1970). Table 14. Carbaryl content in apples during storage (mg/kg)a Part of apple Day of storage 10 20 30 40 50 70 80 100 110 Skin 2.40 1.80 2.00 1.80 1.92 1.72 1.60 1.45 1.22 Pulp 0.35 1.00 1.02 1.10 1.00 1.10 1.34 1.42 1.30 Total 2.75 2.80 3.02 2.90 2.92 2.82 2.94 2.87 2.52 aFrom: Bogomolova (1968). Elkins (1989) reported that washing during the commercial processing of produce removed 97, 87, and 77% of the carbaryl residues from tomatoes, spinach, and broccoli, respectively. Blanching (short treatment with hot water) was stated to result in 68% removal of carbaryl from green beans, while blanching, in addition to washing, resulted in the removal of over 97% of carbaryl from both spinach and broccoli. After a single application of carbaryl (Sevin 50 at 2.5 kg/ha), residues in cauliflower decreased from 16.75 mg/kg on the day of application to 0.87 mg/kg, 15 days afterwards (Yadav & Jaglan, 1982). Washing further reduced the detectable residues at 15 days to 0.67 mg/kg, while no detectable residues were present after boiling. Carbaryl was found in vegetation adjacent to treated fields. Grass growing at a distance of 250 m from a carbaryl-treated cotton field contained 0.18-0.25 mg carbaryl/kg wet weight up to 45 days after spraying (Gusseynov, 1970). The results of the 1989 Pesticide Residue Monitoring Programme in California showed that carbaryl was detected in 2 samples of grape from 26 in quantities less than the accepted tolerance level. It was not found in oranges (Okumura et al., 1991). Summaries of residue data on different plants are given in Tables 15 and 16. Carbaryl residues were not detected in 44 samples of wheat in the United Kingdom analysed by the multiresidue method (Osborne et al., 1989). Table 15. Comparison of carbaryl residues with different formulationsa Formulation use Plant Mean residues Day after last (kg a.i./ha) value (mg/kg) application 44% SC 2x0.5 apple 0.25 7 pears 0.16 7 50% WP 2x0.5 apple 0.15 7 pears 0.09 7 44% SC 2x2 spinach 4.44 - 50% WP 2x2 2.38 14 44% SC 2x2 lettuce leaf 2.72 14 50% WP 2x2 1.55 44% SC 2x2 barley 7.87 14 50% WP 2x2 4.63 Table 15 (continued) 44% SC 2x2 wheat 0.96 14 50% WP 2x2 0.99 44% SC 2x2 oats 0.22 14 50% WP 2x2 0.26 aFrom: Davis (1987). Davis (1987) studied the impact of different types of formulation on food residues, including carbaryl (44% w/w) oil-based liquid formulation containing a "sticker", in order to prevent bees from carrying carbaryl particles back to the beehive. The 50 W formulation was a "standard" wettable powder formulation. The results are given in Table 15. This work showed that the addition of a "sticker" increased levels of food residues to some extent. Studies on the removal of carbaryl residues from tomatoes, green beans, spinach, and broccoli by commercial and home preparation procedures (Elkins et al., 1968; Farrow et al., 1968, 1969; Lamb et al., 1968) showed a considerable decrease in the residues with treatment (Table 17). For pre-harvest use on grain, the rate of application of carbaryl ranges from 2 to 9.5 kg/ha, depending on the degree of infestation, density of foliage, and the stage of the life cycle of the pest. Carbaryl, usually applied at a rate of 5 mg/kg, is also used to protect stored grain. Studies on stored wheat, barley, oats, and rice indicated that carbaryl residues on grain have a half-life of between 26 and 80 weeks, depending on the temperature (FAO/WHO, 1976). Residues of carbaryl in baked bread are then of the order of 1-1.5 mg/kg. 184.108.40.206 Animal products Technical carbaryl was fed to dairy cows of the Brown Swiss, Jersey, Holstein, and Ayrshire breeds at 50, 150, and 450 mg/kg of their average total daily roughage intake (dry weight) for a period of 2 weeks. Samples of milk were taken at regular intervals and the cream was analysed for carbaryl by means of the p-nitrobenzene-diazonium fluoborate coupling method. The concentration of carbaryl, if present, was below the sensitivity of the analytical method (0.01 mg/kg) (Gyrisco et al., 1960). Table 16. Carbaryl residues following different applications Formulation use Plant Mean value Day after Reference (kg a.i./ha) residues last (mg/kg) application 44% SC barley 5.4 14 Davis & Thomas (1987) 44% SC sugar beet 0.07-1.04 14 Thomas (1986) roots 44% SC pasture 59.7-183 3 Davis (1986b) aerial ground grass Carbaryl sweet 0.33 Frank et al. (1987a) cherries Carbaryl tomato 1.2 0 Frank et al. (1991) 0.5 3 0.03 6-8 tomato 0.47 0 juice 0.24 3 0.08 6-8 Table 17. Removal of carbaryl after preparation procedures Vegetable Initial Percentage removal References residues (mg/kg) Commercial procedure Home procedure Tomatoes 5.2 washing 82-99 washing 77 Farrow et al. (1968) canning 98-99 canning 92 juicing 98 juicing 77 Green beans 7.6 blanching 68-73 washing 52 Elkins et al. (1968) & canning blanching 81 freezing 94 canning 100 Spinach 20.8 washing 66-88 washing 70 Lamb et al. (1968) blanching 96-97 Broccoli 12.4 washing 77 washing & cooking 55 Farrow et al. (1968) blanching 82-97 washing, 90 blanching, & freezing Residues resulting from a single application and repeated applications of carbaryl spray on cattle were rapidly eliminated from body tissues. On days 1 and 3 after application, carbaryl was detected in the liver (0.05 mg/kg), muscles (0.04 mg/kg), and perirenal fat (0.16 mg/kg). Seventy-two hours after treatment, no residues were found in the tissues studied. The excretion in milk persisted for at least 69 h after spraying, the highest concentration being 0.075 mg/litre, 5 h after exposure (Hurwood, 1967). Data concerning the composition and levels of milk and tissue residues in cows after continuous feeding with 100 mg 14C-carbaryl/kg diet are shown in Tables 18 and 19. Table 18. Metabolites in tissues after continuous 28-day feeding of 100 mg 14C- carbaryl/kg dieta Metabolite (mg/kg) Kidney Liver Muscle carbaryl 0.03 0.04 0.02 5,6-dihydrodihydroxycarbaryl 0.05 0.01 0.04 5,6-dihydrodihydroxynaphthol 0.02 0.02 0.0 naphthyl sulfate 0.29 0.02 0.0 water-soluble unknowns 0.43 0.13 0.03 unextractable unknowns 0.18 0.19 0.01 aFrom: Dorough (1971). Table 19. Composition of milk residues after continuous 28-day feeding with 100 mg 14C-carbaryl/kg dieta Percentage mg/kg Organic phase 5,6-dihydrodihydroxycarbaryl 38.5 0.11 carbaryl 8.4 0.02 3,4-dihydrodihydroxycarbaryl 4.0 0.01 Table 19 (continued) Percentage mg/kg 5,6-dihydrodihydroxynaphthol 1.0 0.01 Water soluble 5-methoxy-6-hydroxycarbaryl 25.6 0.07 unknowns 10.6 0.03 1-naphthol 5.5 0.02 5-hydroxycarbaryl 3.1 0.01 carbaryl 1.2 0.01 aFrom: Dorough (1970a). Effective horn fly control for a period of 15 days was obtained by spreading carbaryl down a cow's back. Results indicated that, if applications of a 0.5% spray or a 50% dust were made immediately after the morning or evening milking, no residues of carbaryl would result in subsequent milkings (Eheart et al., 1962). The results obtained by Petrovski (1970) were similar. After treating cows with a 0.85-1% suspension of carbaryl, residues of from 0.1 to 0.3 mg/litre were found in milk, 20 h after application. On the third day, only trace amounts of carbaryl were found. After treatment with a 0.5% suspension of carbaryl, no traces were found in the milk. 220.127.116.11 Animal feed crops Generally, animal feed crops contain less carbaryl than fruit and vegetables. Mean levels of 0.82 mg/kg were found in 80 samples of animal feed plants (Molozhanova, 1970). Levels of 0.3 mg/kg were found in maize stems at harvest, 70 days after spraying (Antonovich, 1970). Studies on the carbaryl content of animal feed plants in hot climates showed that carbaryl can be detected 3-3.5 months after treatment, depending on the dose and the number of applications. Thus, after repeated applications of 50% carbaryl dust at a rate of 0.5 g/m2, the carbaryl content in animal feed plants was in the range of 1-1.8 mg/kg wet weight, but, after a single application of 0.7 g/m2, concentrations of 0.10-0.31 mg/kg were found (Atabaev, 1972). 5.1.5 Other products Measurement of carbaryl residues in tobacco plants showed that, 15 days after field spraying with 950 g/ha, the content of carbaryl in green tobacco leaves was 10.1 mg/kg. Drying leaves by hot air decreased carbaryl residues by only 10% (Antonovich, 1970). Application of carbaryl at a rate of 2.2 kg/ha resulted in carbaryl residues on cotton foliage of 7.2 µg/cm2 on the day of spraying (Estesen et al., 1982). Levels decreased to 5.9, 0.58, and 0.26 µg/cm2 after 3, 6, and 8 days, respectively. Carbaryl was found in the foliage of cotton plants in concentrations of 0.03-1 mg/kg from 10 days to 2.5 months after treatment. Carbaryl penetrated the plant through foliage and roots and contaminated the cotton and the seeds. Cotton harvested from treated fields 2 weeks to 1 month after treatment contained 0.015 mg/kg. Trace amounts of carbaryl were found in cotton oil (Guseynov, 1970). After treatment with a 2% suspension of carbaryl against mountain pine beetle, residues in bark disks after 1 year were 359 mg/kg. In another case, residues in bark disks were 890±146 mg/kg, after spraying, and 531±178 mg/kg 16 months later (Page et al., 1985). 5.1.6 Terrestrial organisms The quantities of carbaryl that were absorbed by songbirds, either by contact or by eating food from a sprayed area, were studied by Kurtz & Studholme (1974). Towhees (American finches) were collected 3 days after the forest had been sprayed for gypsy moths at a rate of 1.1 kg/ha. The amounts of carbaryl found in the towhees were small, even though the samples were taken close to the spraying time. Trace amounts of carbaryl were found in three samples out of five, compared with two samples out of five control birds. The minimum level of detection was 0.1 µg/g. The low levels of carbaryl found in "sprayed" birds were explained by the fact that towheeds are groundfeeders and that only a small amount of carbaryl might have passed through the trees to the ground. 5.2 General population exposure 5.2.1 Exposure through the food The daily intake of carbaryl was studied for several years in the USA (see Table 20). Table 20. Carbaryl daily intake in the USA Year Positive samples Daily Intake References (%) (mg/day per person) 1964-65 7.4 0.15 Duggan et al. (1971); 1965-66 2.7 0.026 Duggan & Corneliussen (1972) 1966-67 1.1 0.007 1968-69 0.8 0.003 1969-70 0 - Infant Toddler 1978 - 0.088 0.05 Gartrell et al. (1986) 1979 - - 0.049 1980 - 0.06 0.035 Reed et al. (1987) 1981-82 - 0.129 0.127 1982 - - 0.012 1982-84 - 0.117 0.017- Gunderson (1988) 0.0315 Carbaryl residues in total diet samples in the USA are relatively low (Table 21). Carbaryl is found in potatoes, leguminous vegetables, root vegetables, and fruit. In foods processed in the usual manner, i.e., by peeling, stripping outer leaves, and cooking, when appropriate, carbaryl residues usually decreased to undetectable levels or traces (Manske & Corneliussen, 1974). Table 21. Carbaryl residues in food in the USA Number of composites Number of Number of Range in References analysed composites composites mg/kg with residues with traces 270, from 30 grocery 20 15 trace to Manske & Corneliussen stores in 27 cities 0.5 (1974) June 1970-April 1971 420, from 35 market 6 5 0.02 Manske & Johnson baskets, 6 June 1971- (1975) July 1972 360, 12 August 1972- 12 10 0.05-1.10 Johnson & Manske (1976) July 1973 360, August 1973- 8 2 0.001-0.284 Manske & Johnson (1977) July 1974 1044, 1978-82 11 (1%) a a Yess et al. (1991a) 3744, 16 market 135 (4%) a a Yess et al. (1991b) basket collections 1982-86 aNo figures given. During a 5-year period, from 1982 to 1986, the Los Angeles District Laboratory analysed 19 851 samples of domestic and imported food and feed commodities for pesticide residues. A single, rapid, multiresidue method was used. Carbaryl was detected in 164 samples from the total 19 851 samples analysed. Not one sample exceeded the US Federal tolerance levels (Luke et al., 1988). In another publication, the same authors reported comparative studies on US and imported food. Carbaryl was found in 21 samples from a total 6391 US samples in quantities ranging from 0.1 to >2.0 mg/kg, but less than 10 mg/kg. From 12 044 imported agricultural commodities 132 were contaminated by carbaryl, and levels in 14 of them exceeded the tolerance levels (Hundley et al., 1988). 5.2.2 Exposure during insect control Exposure to carbaryl, used to control the gypsy moth in camping and picnic areas, was monitored on the day of application and then weekly for 3 weeks. Carbaryl was applied from the air at a rate of 1.12 kg/ha. Exposure was measured using dermal pads. Extrapolating from the most contaminated group of pads (they contained an average of 279 µg/pad), the authors calculated a "worst case" exposure of 0.54 mg/kg on the day of spraying. The authors concluded that the risk to those who use public areas during, and after, carbaryl application to trees is negligible (Cameron et al., 1985). A 5% spray of carbaryl 85% wettable powder (WP) was applied for insect control in homes (Vandekar, 1965), as a surface application, at the rate of 2 g/m2. Urine samples taken from villagers 1 week after spraying showed a significant increase in 1-naphthol levels from 30.5 µg/ml baseline to 50.3 µg/ml. Inhibition of plasma ChEA was found in 48 out of 63 subjects, 1 week after spraying. During a monitoring study carried out from 1976 to 1980 in the USA, 1-naphthol was found in 1.4% of the urine samples from persons between 12 and 74 years old. The source was thought to be carbaryl and naphthalene (Carey & Kutz, 1985). 5.3 Occupational exposure during manufacture, formulation, or use Harry (1977) estimated that about 13 million people in the USA were potentially exposed to carbaryl during its manufacture, formulation, packing, transportation, storage, and during and after application, and while working with treated crops or during harvest. Best & Murray (1962) published a survey on the exposure of plant workers during the production of carbaryl. Air concentrations varied from 0.23 to 31 mg/m3. Urine samples contained an excess of 1 mg total 1-naphthol per 100 ml of urine. During air blast spraying of orchards, Jegier (1964) found air concentrations of 0.6 mg/m3 (0.18-0.81 mg/m3). The mean respiratory exposure, measured by the respirator pad technique was 0.29 mg/h (0.24-0.53 mg/h) and mean dermal exposure, measured by skin pads, was 25.3 mg/h (18.5-30.3 mg/h). The maximum total exposure was 31 mg/person per h, or 0.025% of the toxic dose. Simpson (1965) estimated that the amount of dermal exposure was less than 0.1% of the toxic dose in orchard sprayers. During cotton spraying by aircraft, Yakim (1965) found 4 mg carbaryl dust/m3 in the breathing zone of flagmen, 1 mg/m3 during preparation of the solution, and 0.7 mg/m3 in the pilot's cabin. Adylov (1966) reported the following air concentrations found during the aerial application of a water solution: flagmen's breathing zone 1.92 (0.64-2.84) mg/m3; pilot cabin, trace amounts, workers on the ground (preparation of solution, etc.,) 6.28 (0.48-19.2) mg/m3. In the urine samples of carbaryl formulators, 1-naphthol levels varied from 6.2 to 78.8 mg/litre. Agricultural workers who used carbaryl for pest control excreted from 0.07 to 1.7 mg/litre in their urine (Shafik et al., 1971). Exposure studies were completed for pesticide application and formulating plant workers by the biological monitoring of 1-naphthol in the urine, dermal pads, and respirator filter pads (total 480 samples of dermal pads and 73 respirator pads). Workers operating tractor-drawn airblast equipment applied carbaryl at 0.045-0.06% as spray (Comer et al., 1975). An estimate of the amount of dermal and respiratory exposure that could occur was made using the procedures described by Durham & Wolfe (1962). The results are presented in Table 22. The concentration of 1-naphthol in the urine varied from 0.2 to 65 mg/litre with a mean of 9 mg/litre. The rate of excretion per hour varied from 0.004 to 3.4 mg with a mean of 0.5 mg/h. The maximum level during the working day was reached by late afternoon. An approximate calculation of the excreted carbaryl (0.5 mg 1-naphthol/h = 0.7 mg/h excretion of carbaryl), and potential exposure, 75 mg/h, shows great differences. About 1/100 part of possible absorption occurs. Exposure to carbaryl during agricultural application was studied by Leavitt et al. (1982). WP 80 carbaryl was used (0.45 kg in 200 litre water) to spray trees, lawns, and gardens. The mean dermal exposure was 128 mg/h and the mean inhalation exposure was 0.1 mg/h. The maximum percentage of the toxic dose that the applicators received was 0.12%/h. No symptoms or inhibition of ChE were reported. In another group of applicators, the mean dermal exposure was 59.4 mg/h and inhalation exposure 0.1 mg/h. Table 22. Potential dermal and respiratory exposure of formulating plant workers and field spraymen to carbaryla,b Subject Route of Exposure Exposure (mg/h) exposure situations studied Range Mean Formulating dermal 48 0.80-1209.30 73.90 plant workersc respiratory 48 0.03-4.10 1.90 Field dermal 32 1.70-211.80 59.00 spraymend respiratory 25 0.01-1.08 0.09 aFrom: Comer et al. (1975). bCalculated on the basis of the worker wearing a short-sleeved, open-necked shirt, no gloves or hat, with his clothing protecting the areas covered. cWorkers on mixing and bagging operations (4 and 5% dust). dOperating power air-blast spray machines in fruit orchards (0.045-0.06% solution spray). Dermal exposure is influenced by the type of spray equipment. The dermal deposit and respiratory intake of carbaryl in humans was monitored during home garden spray operations using compressed air or garden hose-end sprayers (Puech, undated). The greatest mean dermal deposit in cm2/min was received on the feet. When the spray target was above shoulder height the next highest dermal spray was received on the forearm (Table 23). Exposure through inhalation was insignificant. A study of the urban application of carbaryl was performed by Gold et al. (1982). The maximum dermal exposure recorded in this study was 2.86 mg/kg per h. The maximum air concentration was 0.28 µg/litre. An insignificant decrease in ChEA in serum and erythrocytes was found in some of the applicators. The mean dermal carbaryl exposure of the applicator, expressed as a percentage of toxic dose per h, was 0.01%, with a maximum of 0.08%. This exposure rate is below the risk rate for applicators. No symptoms of intoxication were reported by the authors. During the urban application of carbaryl to trees and ornamental shrubs using hand-held equipment, air concentrations measured at the breathing zone in full-shift samples, of 0.010-0.070 mg/m3, were detected in only 30% of the samples (Leonard & Yearly, 1990). Table 23. Human exposure during spray operations Type of spray equipment Mean deposit Total exposure (µg/cm2 per min) (µg/kg per min) Compressed air sprayer below waist 0.030 4.3 above shoulder height 0.096 5.6 Hose-end sprayer below waist 0.154 18.3 above shoulder height 0.353 24.4 Dermal exposure to carbaryl in harvesters of strawberries was studied by Zweig et al. (1984, 1985). They observed dermal exposure on the hands and forearms, and to a much lesser degree, on the lower legs. The first day, 3 mg carbaryl/h was found on cotton gloves, 0.66 mg/h on pads on the forearms, and 0.07 on the lower legs. The following day, the values were 1.23, 0.41, and 0.07 mg/h for gloves, forearms, and lower legs, respectively. The ratio of dermal exposure to dislodgeable foliar residues (DFR) was 4.34, 2.82, and 6.17, for 3 consecutive days. The half-life of carbaryl on strawberry leaves was 4.1 days. Dislodgeable carbaryl residues on cotton foliage, expressed as µg/cm2 of cotton leaf (one surface only), following application by man-pulled ground rig were 7.2, 5.9, 0.58, and 0.26 at 0, 72, 144, and 192 h after application, respectively (Estesen et al., 1982). Approximate dermal exposure rates may be calculated using the following expression, proposed by Zweig et al. (1985). Dermal exposure rate (mg/h) = 5x103xDFR (in µg/cm2). This method is suggested to obtain the exposure rate of fruit harvesters, in order to establish safe re-entry periods without human studies. Factors affecting the levels of exposure during the agricultural application of pesticides were analysed by Wolfe et al. (1967). Wind is the most important environmental condition. The type of activity, equipment used, the duration of exposure, formulation, individual protection, including attitude, are also discussed. Carbaryl deposits from air jet application on orchards were found at 500 m downwind in the presence of inversion, and at 300 m, in its absence. Ground application results gave deposits at 150 and 50 m, respectively (MacCollom et al., 1985, 1986). Air concentrations were 17.88 µg/m3 during spraying above the orchard downwind edge, 9.5 µg/m3, 30 min after spraying, and 8.17 µg/m3, 1 h after spraying. Airborne and deposit levels of carbaryl were measured after three applications by air to an apple orchard (Currier et al., 1982). Samples of air were taken at distances of from 12.2 to 3994 m from the target orchard. At a distance of 12.2 m, the concentration in air was between 3.3 and 76.2 µg/m3. One hour later it was between 2.5 and 11.3 µg/m3. At a distance of 3.2-3.3 km, the initial concentration was between 9 and 28.8 µg/m3 during spraying and 0.9-14.4 µg/m3, one hour after treatment. Because the area of the study was one of concentrated agriculture, it is possible that carbaryl could have been used on other orchards and gardens near to the sampling point. 6. KINETICS AND METABOLISM 6.1 Absorption When only 0.1 ml of carbaryl solution (0.025-0.05 mmol/litre) was administered into the lungs of anaesthetized rats, it was rapidly absorbed. About 50% of the dose was absorbed in 2.6 min. The amount of carbaryl absorbed per unit time was directly proportional to the administered dose (Hwang & Shanker, 1974). Blase & Loomis (1976) demonstrated that carbaryl could be taken up and metabolized by the isolated perfused rabbit lung. Retention by rats of 14C-labelled carbaryl, inhaled as vapours during a 1-h exposure period, was 75.4% of the total dose inhaled, which did not exceed 50 µg (Dorough, 1982). When a dose of 7.5 µmol radiolabelled carbaryl/kg body weight was administered intragastrically to fasted, anaesthetized, female rats, 22 and 67 min after dosing, the proportions absorbed were 52.6±14.1% (n=3) and 81.7±15.7% (n=3), respectively; 89.3% of the radiolabelled material in the collected portal blood was [1-naphthyl-1-14C] N-methyl carbarmate (Casper et al., 1973). Absorption from the small intestine was studied in anaesthetized rats with a ligature around the pylorus and an ileocecal junction with major blood vessels not occluded. A concentration of carbaryl of 0.005-0.1 mmol/litre was used. The absorption half-time was 6.4 min (Hwang & Schanker, 1974). On the basis of ligation studies on mice, Ahdaya & Guthrie (1982) determined that the absorption of carbaryl from the stomach was relatively low compared with that from the entire gastrointestinal tract (29%), but was relatively high in comparison with that of other pesticides. Variation in the digestive absorption kinetics, according to the vehicle used, was reported in studies on female Wistar rats (Cambon et al., 1981). The results indicated that carbaryl was absorbed more rapidly in the intestine when either DMSO or tragacanth was used as a vehicle. It seems that milk does not facilitate carbaryl absorption. Inhibition of ChEA was closely related to the absorption rate. The rate of dermal penetration of carbaryl (in acetone solution) into mammals, birds, amphibia, and insects was studied by Shah et al. (1983). The half-time of penetration of 14C-labelled carbaryl was 317 min in Japanese quail, 12.8 min in mice, 6.4 min in grass frogs, 4576 min in American roaches, and 791 min in tobacco horn worms. In an in vitro study, only about 1% of an applied dose of carbaryl penetrated through the skin of the rat over an 8-h period (MacPherson et al., 1991). Using two different methods, Shah & Guthrie (1983) measured the half-time for penetration of carbaryl, applied dermally at a rate of 4 µg/cm3, and found 10.34 h and 4.75 h, respectively. Shah et al. (1987) compared the rate of dermal penetration of carbaryl in young, versus adult, Fischer 344, female rats and did not find any consistent age-related differences. Percutaneous absorption of carbaryl in rats was also studied by Knaak et al. (1984). Carbaryl dissolved in acetone was applied to back skin (not occluded) at 43.4-48 µg/cm2. Recovery studies indicated that 57.7% of the applied carbaryl was absorbed. Approximately 5.8% of carbaryl penetrated the skin within 1 h, the rate of absorption being 0.18 µg/h per cm2. The half-life of absorption by blood was 1.26 h, and that for elimination, 67 h. Carbaryl labelled with radioactive carbon (14C), dissolved in acetone, was applied to the skin of six volunteers, in order to study percutaneous penetration (Maibach et al., 1971; Feldmann & Maibach, 1974). The results showed almost complete penetration of carbaryl on the forearm and jaw angle. After a 24-h application, the cumulative urinary excretion over 5 days was 74%. According to other authors using the same data, the estimated cumulative absorption over 5 days, as a percentage of the applied dose, was 63% (Fisher et al., 1985), 45% of this occurring 8 h after the onset of penetration, which had a lag of 3.5 h. Comparing the different studies, it is clear that some solvents can facilitate the dermal penetration of carbaryl. 6.2 Distribution Plasma levels of carbaryl in rats were compared after iv, intraduodenal, or hepatic portal administration of 0.5 mg/kg (Houston et al., 1974). In Fig. 2, it is shown that plasma concentrations were lower after intraduodenal application, when carbaryl was subjected to the liver's first pass metabolizing effect. Plasma concentrations following portal application were also lower (approximately 80% of the concentration after the systemic route of application). The distribution of carbaryl in rat tissues after a single oral administration of 144.2 mg/kg body weight (0.2 LD50) was studied (Klisenko & Yakim, 1966) at 5 and 30 min, and 1, 2, 4, 24, 48, and 72 h. Carbaryl and 1-naphthol were identified by thin-layer chromatography with a sensitivity of 0.5 µg/g. At 5 min, carbaryl was found in all organs. Thirty min after administration of carbaryl, peak concentrations found were: muscle, 35 µg/g, brain, 16 µg/g, spleen, 25 µg/g, and erythrocytes, 120 µg/g; at 60 min, a level of 45 µg/g was found in the liver. After 24 h, levels of only 1-4 µg carbaryl/g were detected in the liver, kidney, muscles, and skin. At 48 h, no residues were found. The authors suggested that carbaryl is rapidly distributed and excreted. 1-Naphthol was found in the liver, stomach, intestines, kidneys, and lungs. Other non-identified metabolites were present in the liver and lungs. Levels of carbaryl in the tissues of rats poisoned with oral doses of 800 and 1200 mg/kg, respectively, were as follows: liver, 7-58 and 52-80 µg/g; heart, 3.5-31.3 and 40.6-45.9 µg/g; brain, 3-26.8 and 25.9-30.9 µg/g. Higher concentrations were found in animals that died from poisoning than in animals killed, even though they were treated with the same doses (Mount et al., 1981). Yakim (1970) studied the distribution of carbaryl and 1-naphthol after 6-month oral administration of 0.2, 0.1, or 0.05 LD50 carbaryl in rats. Carbaryl was found in the intestines (20-40 µg/g), liver (4-20 µg/g), and kidneys and lungs (in trace amounts); 1-naphthol was found in the kidneys (20-50 µg/g), and in the liver (5-10 µg/g) in groups treated with 0.2 LD50. The carbaryl level was <1 µg/g in the organs studied in the group treated with 0.1 LD50; no traces of carbaryl were found in the group treated with 0.05 LD50. The author suggested that a higher quantity of carbaryl is found 30 min after a single oral application, which corresponds to the highest percentage of ChE inhibition. Two to 6 h after a single dermal application of 500 mg carbaryl/kg to cats, the compound was found in plasma at 15 µg/ml and in erythrocytes at 25 µg/ml. Inhalation studies were also performed on cats (Table 24). The author stressed that carbaryl was present in smaller amounts in plasma than in erythrocytes because of the more active metabolism by blood proteins. More 1-naphthol and other unidentified metabolites were present in plasma. Elimination of carbaryl and normalization of ChE activities occurred in 48-70 h with all routes of administration of carbaryl. Carbaryl has a low cumulation capacity. In a study in which the abilities of several pesticides to bind to potential carriers isolated from human blood were compared, carbaryl was much more effectively bound by albumin than by either high- or low-density lipoproteins (Maliwal & Guthrie, 1981). Table 24. Relation between carbaryl inhalation and resulting cholinesterase (ChE) inhibitiona Experimental Carbaryl and 1-naphthol ChE inhibition in % conditions Plasma Erythrocytes Plasma Erythrocytes Single 6 h exposure 2.5-5 µg/ml after 5-10 µg/ml 39-80 (after 4 h) 52-84 (after 4 h) to 80 mg/m3 exposure; no traces (after exposure) 8 cats 4 h later 10-15 µg/ml (4 h later) 2-2.5 µg/ml (24 h later) Single 6 h exposure 0-10 µg/ml 28-44 in blood to 20 mg/m3 in single animal (after 4 h); 8 cats 0 after 48 h 0 (after 48 h) 4-Month exposure to traces in single animal 16 mg/m3 in blood 4 cats 1-Month exposure to 5-15 µg/ml, 0 on the 5-15 µg/ml, 0 on the 44 on the day of 62 on the 7th day of 63 mg/m3 6th day after the end 6th day after the end exposure exposure 4 cats of exposure of exposure 4-Month exposure to 5 µg/ml on the third 10 µg/ml on the third - - 38 mg/m3 day of exposure day of exposure 4 cats aFrom: Yakin (1979) Levels of carbaryl in some tissues of male, albino, Fisher Strain 344 rats, after single and multiple oral administration (Table 25) were reported by Hassan (1971). Table 25. Tissue levels of carbaryla Treatment Time after Concentration of carbaryl administration Whole blood Heart Brain (µg/ml) (µg/g) (µg/g) Single dose 80 mg/kg 2 h 16.2 2.6 3.45 Rats fed 700 mg/kg diet 90 days 4.8 0.85 0.68 Rats fed 100 mg/kg diet 90 days 2.7 < 0.5 < 0.5 aFrom: Hassan (1971). Andrawes et al. (1972) studied the 14C residues in hen tissues following the feeding of 1-naphthyl-14C-carbaryl at 70 mg/kg for 4 days (Table 26). Radiolabelled carbaryl naphthyl 14C (6600 dpm/µg) and carbaryl carbonyl 14C (4000 dpm/µg) were administered intratracheally to rats as aerosols for 15 seconds. The maximum concentration in blood occurred after 2-5 min. Distribution of the residues in the organs, 1 h after inhalation, was highest in the lung (9.2-10.5%), liver (4.7-9.2%), bladder (2.7-5.9%), and kidney (2.5-3.7%) (Nye & Dorough, 1976). Distribution of 14carbon after intraperitoneal and oral administration of 7.4 µmol carbaryl/kg body weight to rats was studied by Krishna & Casida (1966) (Table 27). Carbaryl was found in all tissues analysed. No marked differences with sex and administration route were noted. On the basis of the rate of excretion of radioactivity following an intraperitoneal injection of labelled carbaryl, Shah & Guthrie (1983) calculated a half-time for clearance of label of 6.46 h. Table 26. Concentration of14C residues in hen tissues after feeding 1-naphthyl-14C carbaryl at the level of 70 mg/kg for 4 daysa Sample µg/kg at indicated times after last treatment 16 h 7 days Brain 17.3 6.8 Heart 47.5 20.9 Kidney 405.5 80.7 Pancreas 69.6 15.6 Skin 86.6 22.4 Fat 25.2 5.2 Gizzard 43.0 13.9 Thigh 28.6 10.1 Breast 25.9 12.7 Leg muscle 29.1 9.0 Blood 197.2 152.2 Lung 138.5 122.5 Liver 332.6 33.4 Spleen 108.7 74.2 Intestine and contents 300.1 < 5.0 Intestinal wall 44.0 11.1 Oviduct 50.5 6.7 Developing egg (small) 534.3 7.4 Developing egg (large) 508.1 35.5 Remaining carcass 35.5 < 5.0 aFrom: Andrawes et al. (1972). Distribution of carbaryl was studied in 7 Sprague-Dawley rats and Swiss mice on day 18 of pregnancy. Whole-body auto-radiography was performed after oral application of 13.5 µCi carbaryl methyl 14C/kg. The transfer to the placenta began in the first hour. Distribution of the carbaryl occurred in the excretory organs, fetus, digestive tract, and the bone marrow and brain. The major portion was quickly eliminated. A more stable localization occurred in highly active protein-building organs, such as the fetus, digestive tract, and bone marrow. The 14carbon concentration in the eye, liver, and brain of the fetus was relatively constant from 8 to 96 h (Declume & Derache, 1976, 1977; Declume & Benard, 1977a,b, 1978). Table 27. Distribution of 14carbon in various tissues in rats, 48 h after ip administration of carbaryl and its hydrolysis product, in µmol equivalent/kg of fresh tissue, based on total 14Ca Labelled position Blood Bone Brain Fat Heart Kidney Liver Lung Muscle Spleen Testes Corpuscle Plasma Carbonyl 14C 0.38 0.16 0.26 0.33 0.16 0.39 0.47 0.63 0.32 0.18 0.33 Methyl 14C 0.51 0.22 0.47 0.67 0.19 1.23 1.37 1.78 0.97 0.47 0.97 1-Naphthyl-1-14C 0.02 0.19 0.19 0.03 0.18 0.06 0.17 0.09 0.05 0.03 0.26 0.09 1-Naphthol-1-14C < 0.01 0.06 0.03 0.01 0.04 0.01 0.05 0.14 0.02 1.16 0.13 (hydrolysis product) aAdapted from: Krishna & Casida (1966). Fernandez et al. (1982) determined that the elimination of label following intravenous administration of 20 mg 14C carbaryl/kg to rats could be best described by a three compartment model, with 73% of the label excreted within 24 h. Strother & Wheeler (1976, 1980) reported that 14C-carbaryl rapidly crossed the rat placenta and was distributed in all fetal tissues. Fetal brain, heart, and lung contained more 14C on a weight basis than the maternal counterpart. 6.3 Metabolism The metabolism of carbaryl has been studied extensively, and its complexity and the need for additional studies are recognized. As with other carbamates (WHO, 1986), the principal metabolic pathways are hydroxylation, hydrolysis, and epoxidation, resulting in numerous metabolites subjected to conjugation, forming water-soluble sulfates, glucoronides, and mercapturates (Carpenter et al., 1961; Dorough et al., 1963; Dorough & Casida, 1964; Knaak et al., 1965; Menzie, 1969; Bend et al., 1971). Hydrolysis of carbaryl results in the formation of 1-naphthol, carbon dioxide, and methylamine (Fig. 3) (Carpenter et al., 1961; Sakai & Matsumura, 1971). The first evidence for carbaryl hydroxylation was reported by Hodgson & Casida (1961). Carbaryl is metabolized by a rat liver microsome system, requiring NADPH2 and oxygen, to form a formaldehyde-yielding derivative. The use of 14carbon-labelled carbaryl (Fig. 4) and thin-layer chromatography contributed to further studies on carbaryl metabolism (Skraba & Young, 1959; Krishna et al., 1962). Chin et al. (1974) used in vitro techniques on tissues from animals and human beings. Reviews on carbamate and carbaryl metabolism have been published (Lykken & Casida, 1969; Kuhr, 1970; Knaak, 1971; Kuhr, 1971; Ryan, 1971; Fukuto, 1973; Dorough, 1973; Kuhr & Dorough, 1976). 6.3.1 In vitro studies on animal tissues An investigation of the individual metabolism of carbaryl in the liver, lung, and the kidney of rat was conducted using the tissue explant maintenance technique. Hepatic tissue of the rat, incubated with carbaryl, actively performed demethylation, hydrolysis, hydroxylation, and oxidation, followed by sulfate and glucuronide conjugations (Chin et al., 1979b). Rat liver microsomes fortified with reduced nicotineamide- adenine dinucleotide phosphate were used to study the hydroxylated products of carbonyl 14C, methyl 14C, and naphthyl 14C carbaryl (Dorough & Casida, 1964). The metabolites were identified as N-hydroxymethylcarbaryl, 4-hydroxycarbaryl, and 5-hydroxycarbaryl. At least 2 unidentified metabolites had the C-O-C(O)-N-C structure intact. 1-Naphthol and at least two unidentified metabolites without the carbamyl groups were formed as a product of hydrolysis. The nature of carbaryl metabolites in liver microsomes in mice, rats, and rabbits was studied by Leeling & Casida (1966) and in guinea-pigs and rats by Knaak et al. (1965). Two more metabolites were identified (Table 28): 5,6-dihydro-5,6- dihydroxycarbaryl, and 1 hydroxy-5,6-dihydro-5,6-dihydroxy- naphthalene. It was suggested that hydrolysed metabolites are probably conjugated as glucoronides and sulfates (Knaak et al., 1965; Hassan et al., 1966; Leeling & Casida, 1966). A study on rat liver microsomes and small intestine later showed (Mehendale & Dorough, 1971) that about 90% of 1-naphthol and N-hydroxymethylcarbaryl and about 40% of 5-hydroxycarbaryl and 4-hydroxycarbaryl were conjugated as glucuronides. The presence of thioether conjugates in incubation mixtures of mouse liver homogenates with carbaryl has been confirmed (Bend et al., 1971; Ryan, 1971). The in vitro technique for metabolic studies using liver tissues qualitatively reflects the in vivo metabolic processes of carbaryl in animals and human beings (Sullivan et al., 1972) and their similarity (Matsumura & Ward, 1966). Methylmercury hydroxide pretreatment in rats (10 mg/kg daily for 2 days) decreased the hepatic microsomal cytochrome P-450 content and aminopyrine demethylase by 50%, as well as the microsomal hydroxylation reaction in vitro of carbaryl to form 4-hydroxycarbaryl and N-hydroxymethylcarbaryl. Chlordane pretreatment increased both cytochrome P450 and hydroxylation (Lucier et al., 1972). However, there were no quantitative changes in the metabolite pattern. When the metabolism of carbaryl in rat intestine was studied in vitro, hydrolysis and the synthesis of 1-naphthyl glucuronide were reported to occur mainly in the first third of the intestine (Pekas & Paulson, 1970; Pekas, 1972). MacPherson et al. (1991) studied the metabolism of carbaryl using a rat skin preparation in vitro. A post-mitochondrial fraction was able to catalyse hydrolysis and sulfation and glucuronidation conjugation reactions, but not ring hydroxylation. The activities were very small in comparison with activities in liver microsomes. The degradation of carbaryl by an esterase of the American cockroach ( Periplaneta americana) was reported by Matsumura & Sakai (1968). Table 28. Carbaryl naphthyl-1-14C metabolism by liver microsomes from mice, rabbits, and rats, in the presence of NADPH2a Substance determined Total radiocarbon (%) using liver microsomes from: Mice Rabbits Rats Ether extract Carbaryl 32.1 19.4 46.9 Hydroxylated metabolites 1-naphtyl N-hydroxymethylcarbamate 11.9 6.3 11.7 4-hydroxy-1-naphthyl methylcarbamate 6.7 8.1 6.1 5-hydroxy-1-naphthyl methylcarbamate 2.4 1.7 1.3 5,6-dihydro-5,6-dihydroxy-1-naphthyl methylcarbamate 5.3 9.1 3.8 1-hydroxy-5,6-dihydro-5,6-dihydroxy-naphthalene 1.9 2.7 1.5 1-Naphthol 7.2 6.3 5.8 Unidentified metabolites Metabolite A 4.0 4.5 3.5 Metabolite C 0.6 2.0 0.8 Aqueous fraction 28.5 39.6 19.4 aFrom: Leeling & Casida (1966). 6.3.2 In vivo studies on animals The metabolism of carbaryl has been studied in a variety of mammals including rat, rabbit, guinea-pig, monkey, sheep, cow, pig, and dog. Although many organs have been shown to be able to metabolize carbaryl, the most important one is the liver. The metabolism of 1-naphthyl-14C carbaryl was studied in male and female Beagle dogs following a single, oral administration of 2.5 or 25 mg/kg. The metabolic pathways identified involved hydrolysis, N-methyl oxidation, ring hydroxylation, and conjugation. No significant qualitative differences were found between male and female dogs or between high and low dosage levels. Faecal elimination accounted for 30-66% of the applied dose and was found to be primarily the result of incomplete absorption from the intestinal tract of the solid material and subsequent elimination of unchanged carbaryl. The metabolic pathway defined is illustrated in Fig. 5 (Andrawes & Bailey, 1978c). The metabolism of 1-naphthyl-14C carbaryl was studied in male and female Sprague-Dawley rats following a single, oral administration of 2.5 mg/kg. The metabolic pathways identified in the rat involved hydrolysis, N-methyl oxidation, ring hydroxylation, and conjugation. New metabolites identified, previously unknown in the rat, were: 1,5-naphthalenediol, 1,6-naphthalenediol, 3,4-dihydro-3,4-dihydroxy-1-naphthol, and 3-hydroxy-1-naphthyl methylcarbamate. These new metabolites had been previously identified in the dog. The metabolic pathway defined is illustrated in Fig. 5. Faecal elimination accounted for only 2-7% of faecal 14C as carbaryl, indicating more complete absorption of the test material than in the dog (Andrawes & Bailey (1978a). Conjugated metabolites of 1-naphthyl-14C carbaryl excreted in rat and dog urine, after similar single oral treatments of 2.5 mg/kg, were separated and compared as intact conjugates using gel permeation and thin layer chromatography. The metabolic products were found to be qualitatively similar in the two animal species, with evidence of glutathione conjugation in the dog. Urinary metabolites only differed quantitatively between species. The rat appeared to be considerably more active in hydrolysing carbaryl to 1-naphthol followed by conjugation, whereas, in the dog, the principle urinary metabolites were formed through direct conjugation of carbaryl itself. A significant amount of urinary radioactivity in both species remained unidentified, i.e., 27 and 34% in the rat and dog, respectively (Andrawes & Bailey, 1978b). Tables 29 and 30 illustrate the quantitative urinary metabolic differences in rats and dogs. Note that "Free" refers to unconjugated metabolites whereas "Acid" and "Enzyme" refer to acid- and enzyme-hydrolysed conjugates. Table 29. Metabolic products present in a 24-h sample of urine of a rat treated orally with 2.5 mg 1-naphthyl-14C carbaryl/kg in corn oil Products Percentage of total radioactivity in urine Free Enzyme Acid Total 1-Naphthol 0.26 15.86 0.49 16.61 Carbaryl 0.14 0.45 4.22 4.81 Methylol NDa NDa 0.64b 0.64 1,5-Naphthalenediol 0.17 1.12 0.08 1.37 1,6-Naphthalenediol 0.03 0.42 NDa 0.45 5-Hydroxy carbaryl 7.76 3.07 0.24 11.07 5,6-Dihydrodihydroxy naphthol 1.16 4.99 b 6.15 5,6-Dihydrodihydroxy carbaryl 5.66 7.75 b 13.41 1,4-Naphthoquinonec 0.03 1.12 0.10 1.25 4-Hydroxy carbaryl 2.30 2.76 0.19 5.25 3-Hydroxy carbaryl NDa NDa 0.06 0.06 3,4-Dihydrodihydroxy naphthol 0.71 1.53 b 2.24 Unknown 1 0.25 2.95 0.06 3.26 Unknown 2 NDa 3.46 NDa 3.46 Other unknowns 0.37 1.92 0.38 2.67 Highly polar materials NDa 6.99 20.31 27.30 aND = none detected. bAcid hydrolysis degrades the methylol to desmethyl carbaryl and the dihydrodihydroxy derivatives to phenols. cA decomposition product of 1,4-naphthalenediol during work-up. Table 30. Metabolic products present in a 24-h sample of urine of a female dog treated orally with 25 mg 1-naphthyl-14C carbaryl/kg Products Percentage of total radioactivity in urine Free Enzyme Acid Total 1-Naphthol 0.38 2.74 1.47 4.59 Carbaryl 1.71 0.16 9.90 11.77 Methylol NDa NDa 0.22 0.22 1,5-Naphthalenediol 0.21 1.83 1.14 3.18 1,6-Naphthalenediol NDa 1.62 NDa 1.62 5-Hydroxy carbaryl 1.61 1.67 0.20 3.48 5,6-Dihydrodihydroxy naphthol 1.12 9.79 NDa 10.89 5,6-Dihydrodihydroxy carbaryl 3.80 3.09 NDa 6.89 1,4-Naphthoquinoneb NDa 1.24 1.36 2.60 4-Hydroxy carbaryl 0.64 5.30 0.44 6.38 3-Hydroxy carbaryl NDa 0.04 0.01 0.05 3,4-Dihydrodihydroxy naphthol NDa 0.50 NDa 0.50 Unknown 1 NDa 0.20 0.62 0.82 Unknown 2 NDa 1.30 NDa 1.30 Other unknowns NDa 1.26 0.43 1.69 Highly polar materials NDa 9.06 34.96 44.02 aND = none detected. bA decomposition product of 1,4-naphthalenediol during work-up. The above work supersedes the work by Knaak & Sullivan (1967), who mistakenly concluded that the Beagle dog metabolic pathway was qualitatively different from that of the rat, because of poor absorption of solid/slurry test material. The marked emphasis on differences in metabolic pathways between species, together with the analytical techniques available at the time of the study contributed to their conclusions. Studies on the nature of the biliary, water-soluble metabolites of carbaryl were conducted by Bend et al. (1971) on 19 male Wistar rats, using a technique with the bile duct cannulated. Water-soluble conjugates of carbaryl with sulfur-containing amino acid were found in the urine as well as the bile of treated rats: S-(4-hydroxy-1- naphthyl) cysteine and S(5-hydroxy-1-naphthyl)-cysteine. Biliary secretion of 5,6-dihydro-5,6-dihydroxycarbaryl glucuronide following an infused dose was found to be greatly reduced, from 10% to less than 1%, by pretreatment with antibiotics (Struble et al., 1983b). This indicates that bacteria play a role in the enterohepatic circulation and bilary secretion of this metabolite of carbaryl. The functional activity of the reticulo-endothelial system (RES) can influence carbaryl metabolism (Pipy et al., 1980). The elimination of carbaryl from the blood decreased significantly in rats (24 male Sprague-Dawley) with RES inhibited by colloidal carbon, and increased in those in which the RES was activated with glyceryl trioleate. Correlation of the activity of RES with the enzyme activity of monoxygenases of the hepatic microsomal fraction could possibly explain this effect of the RES in the toxicokinetics of carbaryl. An intravenous injection of colloidal carbon, which inhibits liver microsomal metabolism, reduced biliary excretion of an iv dose of carbaryl given 18 h later (Pipy et al., 1981). Pretreatment of rats with 75 mg phenobarbital/kg per day, ip, for five days resulted in an increase in the rate of sulfate conjugation of carbaryl, following a high dose of carbaryl (16.4 mg/kg), but not a low dose (1.64 mg/kg) (Knight et al., 1987). 6.3.3 Metabolic transformation in plants The formation of carbaryl metabolites in plants is primarily dependent on the hydrolytic, oxidative, and conjugative potential of the plant tissues, which are similar to the tissues of insects and mammals. The metabolism in plants is of a shorter duration than that in insects and mammals, and the tendency to accumulate metabolites is more pronounced. The carbamate ester bond appears to be quite stable in plants, which explains the small amount of recovery of 1-naphthol from treated plants. The extent of oxidation is generally higher in plants and insects than in mammals. Several hydroxymetabolites are formed by oxidation of the N-methyl group and naphthalene ring. They are conjugated as glycosides. Non-enzymatic factors, such as light, heat, and humidity may contribute to the degradation of carbaryl in plants as well (Kuhr, 1971). The metabolism of carbaryl has been defined in a wide variety of plant species (Table 31). Metabolites were isolated in microgram quantities for mass and ultraviolet spectroscopic analyses (Mumma et al., 1971). 6.3.4 In vitro studies with human tissues The metabolism of carbaryl in selected human tissues was studied in vitro by Chin et al. (1974). On the basis of the total anionic characteristics of the metabolites derived from each organ, metabolic activity occurred in the following organs in descending order: liver, lung, kidney, placenta, vaginal mucosa, uterus. The metabolic profiles of carbaryl in human postembryonic fetal autopsy tissue were determined using 1-naphthyl-14C or N-methyl-14C-carbaryl. The anionics from fetal liver amounted to 20% of those found with the adult liver. Naphthyl glucuronide and naphthyl sulfate were produced in the kidney, whereas the lung produced naphthyl sulfate from carbaryl (Chin et al., 1979a). Carbaryl was metabolized oxidatively by primary human embryonic cells in culture (Lin et al., 1975). Complete degradation occurred after 72 h of incubation. Unconjugated metabolites were identified as 1-naphthol, 5-hydroxycarbaryl, 4-hydroxycarbaryl, and 5,6-dihydro-5,6-dihydroxycarbaryl. The water-soluble components were identified as 4-hydroxycarbaryl, 1,4-naphthalenediol, and 5,6-dihydro-5,6-dihydroxycarbaryl. The primary human embryonic lung cells did not convert carbaryl to carbon dioxide. They may not possess the enzyme system that is necessary to break down the naphthalene ring of carbaryl to form carbon dioxide. Sakai & Matsumura (1971) studied the degradation of carbaryl by brain esterases. Carbaryl was degraded by band E4 and E6, whereas, in the mouse brain preparation, the compound was degraded by band E8, E9, and E6. Cell culture techniques were used to examine the products of carbaryl degradation by cultures of an L-132 cell line derived from normal human embryonic lung. The data indicated that detoxification was similar to that observed in animals and in in vitro enzyme systems (Baron & Locke, 1970). Table 31. The composition of water-soluble metabolites of carbaryl in plants. (% distribution of released aglycones 21 days after treatment (for apples, 53 days)) Metabolite Beana Beana Wheatb Potatoc Cornd Riced Tomatoc Applese (mature (shell (seedlings) (mature (seedlings) (seedlings) (mature foliage) only) foliage) foliage) 5,6-Dihydrodihydroxycarbaryl 2.5 12.7 2.1 NDd 1.3 2.9 ND 2.1 Methylol carbaryle 29.9 47.9 10.5 16.2 13.3 7.8 25.4 2.8 7-Hydroxycarbaryl 24.2 trace ND ND ND ND ND 0.8 4-Hydroxycarbaryl 15.0 4.1 23.6 4.1 8.9 3.7 13.8 13.7 5-Hydroxycarbaryl 7.3 9.4 21.3 4.1 12.9 5.6 3.4 9.2 1-Naphthol 13.8 21.0 1.1 5.8 3.2 3.5 8.1 11.3 5,6-Dihydrodihydroxynaphthol ND ND ND ND 0.5 1.0 ND Carbaryl 0.3 0.8 20.8 27.0 24.1 42.8 16.8 33.3 Unknown(s) ND ND 3.2 ND 2.9 2.2 5.2 3.0 Unhydrolysed conjugate 7.1 4.1 17.4 42.8 33.0 30.4 27.3 12.6 References: aWiggins & Weiden (1969); bAndrawes & Chancey (1970); cChancey & Andrawes (1971b); dChancey & Andrawes (1971a); eChancey (1974). fND = not detected. Studies on the in vitro metabolism of carbaryl by a human liver fraction indicated that there was a difference in the metabolic pattern compared with that in the rat liver. The human liver produces at least two more metabolites (Strother, 1970). 6.3.5 In vivo studies on humans The metabolism of carbaryl in humans appears to be qualitatively similar to that previously reported in other mammals. Metabolic reactions include: hydroxylation, hydrolysis, and conjugation. Metabolites of carbaryl were identified in the urine of human volunteers after ingestion of a 2 mg/kg dose (Andrawes & Myers, 1976). Only traces of the unchanged carbaryl could be detected in the urine indicating rapid metabolism. From the spectrum of the metabolites identified, a metabolic pathway of carbaryl in humans is proposed and shown in Fig. 6. The only detectable metabolites in urine samples taken from workers exposed to carbaryl dust were 1-naphthylglucoronide and sulfate (Knaak et al., 1965). Later 1-naphthyl and 4-hydroxycarbaryl, as conjugates of glucuronic and sulfuric acid, were found in a study on volunteers. Human exposure to carbaryl during aerial application for Gypsy moth control was assessed by Schulze et al. (1979). The results of this study indicated a strong positive correlation between carbaryl exposure and the excretion of urinary 1-naphthol within 24 h of known exposure and a total lack of 1-naphthol in pre-exposure samples. The presence of these metabolites has been confirmed in six urine samples of workers with high exposures to carbaryl (Andrawes & Myers, 1976). One major and three minor interfering chemicals were detected in no-exposure samples as well as the maximum exposure samples. These interfering materials developed a colour similar to that of carbaryl metabolites upon spraying the chromatograms with p-nitrobenzene diazonium fluoborate. It is estimated that the major interfering pigment accounted for as much as one-third of the total colour observed on thin-layer chromatograms. These interfering chemicals may also be the cause of high blank values often encountered during the routine analysis of urine samples (Andrawes & Bailey, 1979). 6.4 Elimination and excretion in expired air, faeces, urine, milk, and eggs The elimination of metabolized carbaryl is rapid, and accumulation in animals seems unlikely, under normal exposure conditions. Carbaryl is generally excreted entirely within 24-96 h of intake. Elimination takes place mainly through the urine, faeces, and respiration, and, to a lesser extent, through the milk of dairy animals and the eggs of poultry. Carbaryl is mainly excreted as its product of hydrolysis, 1-naphthol, which is detoxified to water-soluble glucuronide (Carpenter et al., 1961), and sulfate (Whitehurst et al., 1963), and only in trace amounts as 1-naphthol or unchanged. The average percentage of metabolites recovered after orally administered doses of 1-naphthyl 14C, methyl 14C, and carbonyl 14C carbaryl to rats was 94% over a 7-day period (Knaak et al., 1965). Only residues from carbaryl methyl 14C were detected after that, since the methyl moiety is incorporated in tissue (2-3%). Liberated naphthol is conjugated and excreted, while the liberated carbonyl groups are disposed of as respiratory14CO2 (Knaak et al., 1965). Forty-seven to 57% of the metabolites excreted retain the intact C-O-C(O)N-C structure, indicating a nonhydrolytic pathway for carbaryl. Monkeys and pigs (2 young females) excreted carbaryl as a conjugate of intact carbaryl and 4-hydroxycarbaryl, mainly glucuronides. Sheep also excreted 1-naphthyl glucuronide and sulfate and 4 (methylcarbamoyloxy)1-naphthylsulfate (Knaak et al., 1968). Myers (1977) studied the correlation between the amount of carbaryl ingested and urinary metabolites levels. Metabolites were measured as three groups: I. naphthyl sulfate; II. free and sulfated 5,6-dihydrodihydroxycarbaryl and dihydrodihydroxynaphthol; III. other conjugated (glucuronides) of naphthol, 5,6-dihydrodihydroxycarbaryl, and 5,6 dihydrodihydroxynaphthol. The amounts in groups I and II were 25.5-36.5% of the dose. When all three groups of metabolites were analysed, the amounts of metabolites for the same period represented 41.5-52.7% of the dose. Naphthol was the major metabolite found in Group III (mean 75% of all metabolites of the group). A good correlation was found between the amount ingested and urinary metabolites. Rats treated orally with naphthyl14C carbaryl (2.3 µci) excreted 90% of the administered radioactivity in the urine within 3 days. During the 72-h sampling period, the faeces contained only 2-5% of the radioactivity (Lucier et al., 1972). Eighty per cent of 5 mg of 14C naphthyl carbaryl, given intraperitoneally to rats, (see section 6.3.2) was eliminated in the 24-h urine and 10% in the faeces (Bend et al., 1971). Enzymatic hydrolysis and reverse isotope dilution showed that 10% of the dose was excreted as 1-naphthyl-glucoronide, 5%, as 1-naphthylsulfate, 3%, as conjugates of 4-hydroxycarbaryl, 5%, as 1-naphthylsulfate, 3%, as conjugates of 4-hydroxycarbaryl, and 5% of 5-hydroxycarbaryl. About 2% of the carbaryl appeared unchanged in urine. Other radioactive metabolites were not identified. Following a single, ip injection in albino rats of both sexes, of methyl 14C and carbonyl 14C carbaryl at 30 mg/kg, 75-80% was recovered after 48 h in the expired air and urine. Liberated by hydrolysis, N-methyl carbamic acid was spontaneously decomposed to methylamine and carbon dioxide (43.5%). The methylamine moiety was later demethylated to 14CO2, which was eliminated in the expired air and 14C formate (9.2%), which was excreted in the urine (Hassan et al., 1966). In the study of Nye & Dorough (1976), 2.5% of the endotracheally administered carbaryl dose was exhaled as 14C-carbon dioxide. More than 90% of the dose was eliminated in the urine during 3 days. The faeces contained 2-5%. A comparative study on the excretion of carbaryl after a 7.5 µmol/kg, ip dose in rats was reported by Krishna & Casida (1966) who used three types of labelled carbaryl (Table 32). Table 32. Fate of 14carbon in male Sprague-Dawley rats after ip administration of carbaryla Labelled position Administered 14carbon recovered (%) Expired 14CO2 Urine Faeces 0-24 h 24-48 h Carbonyl 14C 24.5 62.1 2.4 2.1 Methyl 14C 12.3 54.6 3.4 3.9 Naphthyl 14C 0.2 74.2 2.3 8.9 Hydrolysis product < 0.1 86.4 3.3 1.4 naphthol 14C aFrom: Krishna & Casida (1966). Biliary excretion of carbaryl was studied in bile-duct cannulated rats. Naphthyl 14C and methyl 14C carbaryl (50 µg) were administered intravenously to rats (see section 6.3.2). During the first 2 h of collection, 90% of the total biliary radioactivity was eliminated (Bend et al., 1971). After oral application of 0.01 mg/kg naphthyl-14C carbaryl to rats, 37.5% was excreted via the bile in the first 3 h and 45.4% (cumulative percentage) of the dose was eliminated within 48 h. Faecal elimination was 1.4% and urine elimination was 42.3% (Marshall et al., 1979). The results of Struble et al. (1983a), who used higher doses of carbaryl (1.5, 31, and 300 mg/kg) on male Sprague-Dawley rats were similar. Twelve hours after administration, 15-46% of the 14C was excreted in the bile, 10-40% in the urine, and less than 1% was eliminated in faeces. Three metabolites were identified from the bile, the major one being 5-6-dihydro-5-6-dihydroxycarbaryl glucoronide (12-18% of the biliary 14C). On the basis of a study on 50, male, Sprague-Dawley rats, Borzelleca & Skalsky (1980) reported that carbaryl metabolites in the blood were also present in the saliva, at similar concentrations. Leeling & Casida (1966) studied carbaryl metabolites in the urine of one male and one female New Zealand White rabbit. The ether-extractable metabolites found in the urine were: N-hydroxymethyl carbaryl, 4-hydroxycarbaryl, 5-hydroxycarbaryl, 5,6-dihydro-5,6-dihydroxycarbaryl, 5,6-dihydro-5,6-trihydroxy-1- naphthol, and 1-naphthol. Thirty-five, adult, female American cockroaches ( Periplaneta americana) were injected through the central abdominal wall with 5 µg of carbaryl-carbonyl 14C, carbaryl methyl 14C, or 2 µg carbaryl naphthyl 14C. Metabolites similar to those appearing in rat liver microsomes were formed. About 19% of the radioactivity of carbaryl carbonyl 14C was eliminated as 14C-carbon dioxide in 24 h (Dorough & Casida, 1964). Excretion of carbaryl in milk was studied by Baron (1968). Application of a total dose of 2 g carbonyl carbaryl 14C to a lactating cow resulted in approximately 1% of radioactive residues in the milk. In skim milk, 87% of these residues were water-soluble and 13% chloroform-soluble. About 90-95% of the14C radioactivity in the soluble component was removed after crystallization of lactose. The metabolism of carbaryl naphthyl 14C was studied in one lactating Jersey cow (Dorough, 1967). Two consecutive single treatments at 6-day intervals with 0.25 mg/kg body weight (total 125 mg) and one single dose of 3.05 mg carbaryl naphthyl 14C/kg body weight added to the feed, resulted in radiolabelled residues in the milk for as long as 60 h after treatment. The first sample of milk, taken after 6 h, showed maximum concentrations of chloroform extractables, water-soluble, and unextractable, residues in whole milk in the range of 0.063-0.95 mg/litre. Residues of only a slightly lower magnitude were detected in the 12-h milk samples and rapidly declined in 24-h samples. Thirty per cent of the residues in the 6-h sample was a chloroform-extractable metabolite, tentatively identified as 5,6-dihydro-5,6-dihydroxy-1-naphthyl- N-methylcarbamate. About 0.35% of the total dose was detected in the milk in both treatments, 70% in the urine, and 11% in the faeces in 0.25 mg/kg treatment, and 58% in the urine and 15% in the faeces in the 3.05 mg/kg treatment. The highest levels in the tissues of the cow, killed on an unspecified day after treatment, were found in the liver, kidney, and ovaries. A single lactating cow was treated orally with a daily total dose of 518 mg 14C-carbaryl (0.77 mg/kg body weight), given in two doses, 12 h apart. Results are shown in Table 33 (Dorough, (1969). Fifty-nine per cent of the radioactivity from a14C-labelled oral dose of carbaryl administered to a cow was recovered from the urine within the first 24 h (Saini & Dorough, 1978). The passage of14C into the milk and its presence in the suckling neonates has been studied by whole-body autoradiography in rats fed 14C-methyl carbaryl. The highest concentrations in newborn offspring, after 48 h, were found in the stomach contents, the liver, the hair, and the bone marrow (Benard et al., 1979). Table 33. Distribution and excretion of total-14C equivalents in milk, urine, faeces, and tissues, 24 h after one-day oral application of carbaryl in a lactating cow Sample mg/kg Dose recovered (%) Milk 0.04 0.09 Urine 22.4 58.64 Faeces 0.47 1.65 Tissues: - liver 0.098 0.21 - kidney 0.382 0.17 - leg muscle 0.027 nil - fat 0.014 nil A Saanen goat was treated orally with 1.34 mg carbaryl carbonyl 14C/kg. The excretion of radioactivity in the urine was 7.4% at 2 h, 24% at 4 h, and 45% at 24 h. In total, 47% of the radioactivity was excreted the urine. In the milk, the peak was 0.9 mg/litre at 8 h, dropping to 0.008 mg/litre 60 h after the administration of carbaryl (Dorough & Casida, 1964). The excretion of carbaryl was studied in hens. Carbaryl- carbonyl14C (10 mg/kg body weight) was given orally to Leghorn hens. Fifty per cent of the 14C was expired within 48 h. When carbarylnaphthyl 14C was applied at the same dose, no radioactivity was detected in the expired gases. The urine was the primary route of excretion. 14Carbon remaining in the carcass, 48 h after dosing, accounted for 1.4 and 7.1% of the dose given as the ring-labelled and carbonyl-labelled compound, respectively (Paulson & Feil, 1969). The main metabolites found in the urine of hens were: 1-naphthol, 1-naphthyl glucoronide, and sulfate esters of 1-naphthol, 4-hydroxycarbaryl, and 5-hydroxycarbaryl. Conjugates with other metabolites were also found in small quantities (Paulson et al., 1970). Mature hens were given a single oral dose of carbaryl naphthyl 14C at 10 mg/kg body weight. Eggs collected for 12 days contained a total dose of 0.33% of the 14C administered (Paulson & Feil, 1969). The fate of naphthyl-1-14C carbaryl in laying chickens was studied by Andrawes et al. (1972). Chickens (3 White Leghorn in each group) were fed 7, 12, or 70 mg/kg carbaryl in the feed twice daily for 14 days. They had been pretreated for 17 days with the same doses of nonlabelled carbaryl in feed. Residues reached maximum levels within 1 day in excrement, 2 days in egg white, and 6-8 days in egg yolk. After the end of dosing, the half-life of 14C residues was <1 day in the excrement and egg white, 2-3 days in the egg yolk, and 5 days in the carcass. Metabolites and carbaryl found in the eggs of hens, fed 70 mg carbaryl/kg averaged in total 19.7 µg of 14C carbaryl equivalent per egg (16.3 µg in yolk and 3.4 µg in white), collected between the 9th and the 14th day of dosing. Naphthyl-l-sulfate was the largest single component of egg residues accounting for the higher concentration of residues in the egg yolk. 7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT 7.1 Microorganisms 7.1.1 Soil microorganisms Studies of the effects of insecticides on soil microorganisms are complex and subject to many sources of variation. It is therefore difficult to assess the practical significance of the results of such studies. Varsheny & Gaur (1972) studied the impact of carbaryl (as Sevin) on soil fungi. They determined the size of the fungal population and the species composition of the fungal community after plating out extracts of soil samples on various media. The authors suggested that, at very high rates of addition to the soil (up to 5000 mg/kg), there was evidence of a change in species composition and an increase in the total fungal population. In laboratory cultures of saltmarsh protozoans (dominated by Euplotes sp.), Weber et al. (1982) found that both carbaryl and 1-naphthol caused dose-related mortality. For both compounds, mortality at 1 mg/litre was about 50%. There was almost 100% mortality at 10 mg/litre. Carbaryl inhibited the growth of cultures of Bacillus subtilis (DeGiovanni & Donnelly, 1968). 7.1.2 Aquatic microorganisms Edmiston et al. (1985) conducted a detailed study of the effects of carbaryl on Paramecium multimicronucleatum. The 24-h LC50 was 28 µg/litre in a static plate test. Effects on the oxygen uptake of cultures and effects on the cell surface were also reported, but these results were mostly obtained using concentrations in excess of the 24-h LC50, and must be of limited value. Murray & Guthrie (1980) studied the responses of bacterial cultures originating from Lake Houston. The respiratory rate of bacterial cultures was increased at water temperatures of between 23-33 °C. Plate counts of bacterial colonies suggested that carbaryl treated cultures contained more bacteria than control cultures on most days in a 26-day study. 7.2 Aquatic organisms Aquatic toxicity studies have been conducted in the laboratory and field to define the biological effects associated with acute and long-term exposure to carbaryl and the degradation product, 1-naphthol, of aquatic vertebrates and invertebrates. More data are available for freshwater organisms than for saltwater organisms. In addition, actual and simulated field studies have been conducted to assess the impact of carbaryl exposure on freshwater invertebrates. 7.2.1 Aquatic invertebrates Studies on the toxicity of carbaryl for non-target aquatic invertebrates have included investigations on molluscs, crustaceans, and insects, concentrating on organisms of commercial importance, organisms most likely to be exposed during a typical application of carbaryl, or on standardized test species typically used in aquatic toxicity tests. The acute and long-term laboratory studies under various environmental conditions (pH, temperature, water hardness, etc.) are briefly reviewed in Table 34. Most of the studies on the toxicity of carbaryl for aquatic invertebrates have been conducted under laboratory conditions and, consequently, the results only yield data on the relative toxicity of the substance. They do not reflect realistic exposure in the environment. Most of the laboratory studies evaluate constant, short-term (less than 1 week) exposure to carbaryl under static or flow-through conditions. There are few long-term studies on the effects of carbaryl on aquatic invertebrates. Exposures in the environment are typically localized, at much lower concentrations than in aquatic toxicity tests and are not constant. The toxicity of carbaryl on the early development of the sea urchin was studied by Hernandez et al. (1990). Developmental stages with active cleavage and cellular mobilization (blastula and gastrula) turned out to be more sensitive. The next two stages (prism and pluteus) were less sensitive and EC50 values were 8-26 times higher. This effect may be connected with increased detoxication processes by the cytochrome oxidase system. Table 34. Acute toxicity of carbaryl for invertebrates Organism Test Size/age Stat/ Temperature Hardness pH Parameterc Concentration Reference material Flowa (°C) (mg/litre)b (µg/litre) Crustacea (Freshwater) Water flea Daphnia magna unknown stat 16 42 7.4 48-h LC50 5.6 Sanders et al. (1983) Daphnia magna technical 96-h LC50 3280 Lejczak (1977a) Daphnia magna unknown 48-h EC50 0.26 Rawash et al. (1975) Daphnia magna technical 21-day MATC 1.5-3.3 Surprenant (1985c) 21-day NOEC 6.0 (survival) Daphnia pulex technical stat 16 44 7.1 48-h LC50 6.4 Mayer & Ellersieck (1986) Simocephalus unknown stat 10 44 7.1 48-h LC50 11 Johnson & Finley (1980) serrulatus stat 16 44 7.1 48-h LC50 7.6 stat 21 44 7.1 48-h LC50 8.1 Ostracod Cypridopsis vidua unknown adult stat 21 272 7.4 48-h LC50 115 Mayer & Ellersieck (1986) Sow bug Asellus brevicaudus technical adult stat 18 44 7.1 96-h LC50 280 Johnson & Finley (1980) Table 34 (continued) Organism Test Size/age Stat/ Temperature Hardness pH Parameterc Concentration Reference material Flowa (°C) (mg/litre)b (µg/litre) Crustacea (Freshwater) Amphipod Gammarus fasciatus unknown adult stat 21 44 7.1 96-h LC50 26 Johnson & Finley (1980) Gammarus lacustris unknown adult stat 21 44 7.1 96-h LC50 22 Johnson & Finley (1980) Gammarus unknown adult stat 12 40 6.5 48-h LC50 8.13 Mayer & Ellersieck (1986) pseudolimnaeus adult stat 12 40 7.5 48-h LC50 11.5 adult stat 12 40 8.5 48-h LC50 7.8 Gammarus pulex technical 48-h LC50 29 Bluzat & Seuge (1979) Gammarus technical 96-h LC50 16 Sanders et al. (1983) pseudolimnaeus Gammarus technical 96-h LC50 13 Woodward & Mauck (1980) pseudolimnaeus Glass shrimp Palaemonetes unknown adult stat 21 272 7.4 96-h LC50 5.6 Johnson & Finley (1980) kadiakensis 25-31 mm stat 96-h LC50 120 Chaiyarach et al. (1975) Table 34 (continued) Organism Test Size/age Stat/ Temperature Hardness pH Parameterc Concentration Reference material Flowa (°C) (mg/litre)b (µg/litre) Crayfish Procambarus sp. technical immature stat 12 42 7.5 96-h LC50 1900 Mayer & Ellersieck (1986 Procambarus unknown 60-70 mm stat 96-hLC50 2430 Chaiyarach et al. (1975) simulans Procambarus technical 96-h LC50 500 Cheah et al. (1978) acutus Crustacea (Freshwater) Prawn Macrobrachium technical 96-h LC50 19 Omkar & Shukla (1985) lamarrei Upogebia wettable powderd 48-h EC50 90 Stewart et al. (1967) pugettensis 1-naphthol 48-h EC50 4400 Callianassa wettable powderd 48-h EC50 80 californiensis 1-naphthol 48-h EC50 3500 Hemigrapsus wettable powderd 24-h EC50 710 oregonensis 1-naphthol 24-h EC50 74 200 Table 34 (continued) Organism Test Size/age Stat/ Temperature Hardness pH Parameterc Concentration Reference material Flowa (°C) (mg/litre)b (µg/litre) Crustacea (estuarine & marine) Blue crab Callinectes unknown juvenile flow 29 27s 48-h LC50 320 Mayer (1987) sapidus Brown shrimp Penaeus aztecus unknown juvenile flow 30 28s 48-h LC50 1.5 Pink shrimp Penaeus duorarum unknown juvenile flow 23 29s 48-h LC50 32 Grass shrimp Palaemonetes unknown juvenile flow 23 29s 48-h LC50 28 pugio 3-4 days stat 24 28-30s 96-h LC50 22 Thursby & Champhon (1991) Crustacea (estuarine & marine) Table 34 (continued) Organism Test Size/age Stat/ Temperature Hardness pH Parameterc Concentration Reference material Flowa (°C) (mg/litre)b (µg/litre) Mysid shrimp Mysidopsis bahia unknown flow 96-h LC50 > 7.7 Nimmo et al. (1981) Mysidopsis bahia technical 96-h LC50 6.7 Surprenant (1985b) NOEC 5.1 Mysidopsis bahia 24 h flow 25 31s 96-h LC50 8.46 Thursby & Champhon (1991) 24 h stat 24 30s 96-h LC50 19 24 h flow 26 31-32s 28-day LC50 9.9 Ostracod Cypretta kawatai technical 72-h LC50 1800 Hansen & Kawatski (1976) Insects Stonefly Isogenus sp. unknown larva stat 7 35 7 96-h LC50 2.8 Mayer & Ellersieck (1986) Pteronarcys technical 96-h LC50 4.8 Johnson & Finley (1980) californica Mosquito Culex pipiens technical 24-h LC50 75 Rawash et al. (1975) Table 34 (continued) Organism Test Size/age Stat/ Temperature Hardness pH Parameterc Concentration Reference material Flowa (°C) (mg/litre)b (µg/litre) Insects Midge Chironomus technical 24-h LC50 7 Karnak & Collins (1974) tentans Chironomus technical 72-h LC50 5900 Hansen & Kawatski (1976) tentans Chironomus technical 48-h EC50 10 Sanders et al. (1983) plumosus Chironomus unknown larva stat 20 4 24-h LC50 106 Fisher & Lohner (1986) riparius larva stat 20 6 24-h LC50 133 larva stat 20 8 24-h LC50 127 Back swimmer Notonecta technical 96-h LC50 200 Federle & Collins (1976) undulata Water stick Ranatra elongata wettable 96-h LC50 624 Shukla et al. (1982) powder Table 34 (continued) Organism Test Size/age Stat/ Temperature Hardness pH Parameterc Concentration Reference material Flowa (°C) (mg/litre)b (µg/litre) Diptera Chaoborus technical 48-h LC50 296 Bluzat & Seuge (1979) Ephemeroptera (Mayfly) Cloeon technical 48-h LC50 480 Crawling water beetle Peltodytes sp. technical 96-h LC50 3300 Federle & Collins (1976) Insects Stone fly Pteronarcella unknown larva stat 16 44 7.1 96-h LC50 1.7 Johnson & Finley (1980) badia larva stat 12 38 6.5 96-h LC50 11 Woodward & Mauck (1980) larva stat 12 38 7.5 96-h LC50 13 Mayer & Ellersieck (1986) larva stat 12 38 8.5 96-h LC50 29 Claasenia unknown larva stat 16 44 7.1 96-h LC50 5.6 Johnson & Finley (1980) sabulosa Table 34 (continued) Organism Test Size/age Stat/ Temperature Hardness pH Parameterc Concentration Reference material Flowa (°C) (mg/litre)b (µg/litre) Molluscs Common mussel Mytilus edulis unknown 48-h EC50 > 30 000 Roberts (1975) for byssal attachment Mytilus edulis wettable larva 48-h EC50 2300 Stewart et al. (1967) powder 48-h EC50 1300 1-naphthol Giant oyster Crassostrea gigas wettable larva 48-h EC50 2200 powder 48-h EC50 800 1-naphthol Clinocardium wettable adults 24-h EC50 7300 nuttallii powder 24-h EC50 6400 1-naphthol Molluscs Eastern Oyster Crassostrea juvenile flow 29 27s 96-h EC50 > 2000 Mayer (1987) virginica Table 34 (continued) Organism Test Size/age Stat/ Temperature Hardness pH Parameterc Concentration Reference material Flowa (°C) (mg/litre)b (µg/litre) Crassostrea technical 48-h EC50 2700 Surprenant (1985a); virginica Dionne et al. (1985) Mactrid clam Rangia cuneata 35-50 mm stat 5s 96-h LC50 125 000 Chaiyarach et al. (1975) Lymnaea technical 48-h LC50 21 000 Bluzat & Seuge (1979) stagnalis aStat = static conditions (water unchanged for the duration of the test). Flow = flow-through conditions (carbaryl concentration in water continuously maintained). bHardness = expressed as mg CaCO3/litre. s = salinity (%). cEC50s for oyster based on shell deposition. d50-80% wettable powder. Carbaryl is slightly toxic for freshwater and marine molluscs (clams, mussels, oysters). Acute toxicity typically occurs in adult molluscs at concentrations ranging from 1 mg carbaryl/litre to over 100 mg/litre. The major hydrolytic product of carbaryl, 1-naphthol, is slightly more acutely toxic for molluscs (mussel, pacific oyster) than the parent compound (Stewart et al., 1967). In addition, larvae and young juveniles are more sensitive to carbaryl exposure than older juveniles and adults. There have been few studies on the effects of carbaryl on the growth of molluscs. Davis (1961) and Butler (1962) found that carbaryl (80% WP) concentrations of 1.0-2 mg/litre reduced the larval development and growth of oysters ( Crassostrea virginica) and clams ( Venus mercenaria). The effects of a 1-h exposure to carbaryl (80% WP) and its hydrolytic product, 1-naphthol, were studied on six developmental stages of the mussel ( Mytilus edulis) (Armstrong & Millemann, 1974c). The six stages included unfertilized eggs to the early veliger stage, 32 h after fertilization. Unfertilized eggs and the first polar body stage were exposed to carbaryl and 1-naphthol (separately). The acute toxicity values of carbaryl and 1-naphthol were similar for the first two stages, ranging from approximately 5 to 25 mg/litre. The stage of development most sensitive to carbaryl was the first polar body stage. Thereafter, susceptibility decreased as age of the larvae increased. LC50 values for crustacea varied from 5 to 9 µg/litre (water fleas, mysid shrimps), 8 to 25 µg/litre (scud), and 500 to 2500 µg/litre for crayfish. The hydrolytic metabolite 1-naphthol was less toxic for daphnids and shrimps. The effects of carbaryl (80% WP) on the life stages of crustacea have been studied in the Dungeness crab ( Cancer magister) (Buchanan et al., 1970). Early larvae were more sensitive to carbaryl than juveniles and adults. Carbaryl (1.0 mg/litre) did not affect hatching of eggs but prevented moulting of all prezoeae to zoeae. The concentration that killed 50% of the first zoeae during a 96-h exposure was estimated to be 1 mg/litre. Young juvenile crabs were more sensitive than older juveniles or adults. The 24-h EC50s (death or irreversible paralysis) were estimated to be 0.076 and 0.35-0.62 mg/litre for second and ninth stage juveniles, respectively. At 0.032 mg/litre, juvenile crabs were not affected when held in uncontaminated water after exposure. In adult crabs, the 24-h and 96-h EC50s (death or paralysis) were 0.49 and 0.26 mg/litre, respectively. Post-larval Dungeness crabs had about the same sensitivity to carbaryl as other crabs. The 24-h EC50 (death or paralysis) of small stone crabs was 1.0 mg/litre (Butler, 1962), that of juvenile blue crabs, 0.55 mg/litre (Butler, 1963), and that of adult crabs, 0.06-1.05 mg/litre (Stewart et al., 1967). As part of fresh water biota, daphnids have been used as assay organisms to determine toxic concentrations of a variety of pesticides. Laboratory bioassays were conducted with carbaryl to determine its toxicity and immobilization values for two species of daphnids, Daphnia pulex and Simocephalus serrulatus. The EC50s for overt effects were 0.0064 mg/litre for Daphnia pulex and 0.0076 mg/litre for Simocephalus serrulatus (Sanders & Cope, 1966). In a long-term study, Daphnia magna was exposed to technical carbaryl for 21 days; reproductive performance was the most sensitive indicator; the MATC was 1.5-3.3 µg/litre (Surprenant, 1985c). Aquatic insects in the orders Plecoptera (stoneflies) and Ephemeroptera (mayflies) are generally highly sensitive to low levels of carbaryl. The effects of 0.6-50.0 mg carbaryl/litre were studied on selected aquatic organisms, including: Scanedesmus quadricauda, Lemna minor, Lebistes reticulatus and Daphnia magna (Bogacka & Groba, 1980). Carbaryl depressed reproduction, biomass, and chlorophyll content in Lemna minor (vascular plant) after 24 h exposure at a concentration >6.6 mg/litre. An almost 100% decrease occurred at a concentration of 50 mg/litre. Inhibition of photosynthesis intensity in the alga Scenedesmus quadricauda was about 50% at a concentration of carbaryl in the water of 32 mg/litre, and 65% at a concentration of 56 mg/litre, after 24 h exposure. Less than 10% inhibition occurred at a concentration of 1.8 mg/litre. A decrease in the production of chlorophyll was demonstrated at 4.4 mg/litre. Carbaryl applications of 5.7 or 11.4 kg/ha were effective in controlling ghost shrimp ( Callianassa californiensis) as an oyster pest. The numbers of various clams in the mud were reduced by 22 and 38%, respectively. Clam species differed in susceptibility. For example, gaper clams (Tresus capax) were reduced by 58 and 69%. Polychaetes and nemertean worms were not affected during 30 days of observation (Armstrong & Millemann, 1974a,b). 7.2.2 Fish 18.104.22.168 Acute toxicity Acute toxicity studies have been conducted with technical carbaryl, several formulations, and 1-naphthol to determine LC50 values for several freshwater and marine fish (Table 35). The results indicated that short-term exposure (< 4 days) to carbaryl and its formulations showed some toxicity for fish (96-h LC50s = 1-30 mg/litre). There were no differences in the toxicity of two formulations (4-oil-sevin and XLR) for rainbow trout and bluegill. The LC50s for sheepshead minnow (exposed to technical material) and rainbow trout (exposed to 4-oil and XLR) were similar and both were generally more sensitive than bluegill. The toxicity of 1-naphthol for rainbow trout, bluegill, and sheepshead minnow was determined by Springborn (1988a); 96-h LC50 values for these species were 0.76, 1.4, and 1.2 mg/litre, respectively. The corresponding NOECs were < 0.43, 0.55, and 0.47 mg/litre. The metabolite, 1-naphthol, was more acutely toxic than the formulations for bluegill. However, the toxicities of 1-naphthol and the formulations were similar for rainbow trout and sheepshead minnows. Few studies report acute toxicity values for fish that are lower than 1 mg/litre. Cold water fish (Salmonidae), such as Coho Salmon and trout, seem to be susceptible to carbaryl, while the catfish (Ictaluridae) is tolerant (Macek & McAllister, 1970; Post & Schroeder, 1971). Irritability, sluggishness, and loss of equilibrium were classical signs of acute intoxication. Rainbow trout (Oncorhynchus mykiss), exposed to 1 mg carbaryl/litre for 96 h, exhibited decreased swimming capacity and swimming activity; the capacity to capture and consume prey was also diminished (Little et al., 1990). Other end-point concentrations have been established for carbaryl for various species. Reference should be made to the original documentation to obtain a fuller understanding of these measures of toxicity. The lowest no-observed-effect concentration (NOEC) was found for rainbow trout at 0.065 mg/litre. Other NOECs were at least an order of magnitude greater. Maximum acceptable toxic concentrations (MATCS) have also been calculated. Bansal et al. (1980) estimated 30-day MATCs for 4 species of carp exposed to carbaryl (Sevin as a 50% wettable powder). For all species, the MATC was between 0.052 and 0.078 mg/litre. Verma et al. (1984) reported a 60-day MATC of between 0.09 and 0.11 mg/litre for another carp species, exposed to the same type of formulation of Sevin. Table 35. Acute toxicity of carbaryl for fish Organism Size/age Stat/ Temperature Hardness pH Formulationc Parameter Concentration Reference flowa (°C) (mg/litre)b (mg/litre) Freshwater Coho salmon 1 g stat 13 44 7.1 96-h LC50 4.3 Mayer & Ellersieck (1986) (Oncorhynchus stat 13 42 7.1 96-h LC50 2.4 kisutch) 4.6 g stat 13 42 7.1 96-h LC50 1.8 5.1 g stat 13 42 7.1 96-h LC50 2.7 10.6 g stat 13 42 7.1 96-h LC50 1.2 19.1 g CT 96-h LC50 0.8 Macek & McAllister (1970) CT 96-h LC50 1.3 Post & Schroeder (1971) Chinook salmon fingerling flow 13 314 7.5 96-h LC50 2.4 Mayer & Ellersieck (1986) (Oncorhynchus tshawytscha) Cutthroat trout 0.5 g stat 12 40 7.5 96-h LC50 7.1 (Salmo clarkii) 0.5 g stat 12 330 7.8 96-h LC50 4.0 0.6 g stat 7 42 7.5 96-h LC50 6.0 0.7 g stat 12 40 6.5 96-h LC50 5.0 0.6 g stat 12 40 8.5 96-h LC50 1.0 CT 96-h LC50 6.0 Woodward & Mauck C49 96-h LC50 6.7 (1980) Table 35 (continued) Organism Size/age Stat/ Temperature Hardness pH Formulationc Parameter Concentration Reference flowa (°C) (mg/litre)b (mg/litre) Rainbow trout 1.5 g stat 12 42 7.1 96-h LC50 2.0 Mayer & Ellersieck (1986) (Oncorynchus 1.5 g stat 12 272 7.4 96-h LC50 1.2 mykiss) 1.2 g stat 12 40 7.4 96-h LC50 3.5 1.2 g stat 12 320 7.4 96-h LC50 3.0 1 g stat 12 40 7.4 96-h LC50 1.6 1 g stat 17 40 7.4 96-h LC50 1.1 1 g stat 12 40 6.5 96-h LC50 1.2 1 g stat 12 40 7.5 96-h LC50 0.8 1 g stat 12 40 8.5 96-h LC50 1.5 0.5 g flow 17 314 7.5 96-h LC50 1.3 CT 96-h LC50 4.3 Macek & McAllister (1970) XLR 96-h LC50 1.4 Springborn (1985) S4 96-h LC50 1.3 CT 96-h LC50 2.2 Sanders et al. (1983) CT 96-h LC50 1.5 Post & Schroeder (1971) Atlantic salmon 0.2 g stat 7 42 7.5 96-h LC50 0.3 Mayer & Ellersieck (1986) (Salmo salar) 0.2 g stat 12 42 7.5 96-h LC50 0.9 0.2 g stat 17 42 7.5 96-h LC50 1.0 0.4 g stat 12 42 6.5 96-h LC50 1.3 0.4 g stat 12 42 8.5 96-h LC50 0.9 Brown trout 0.6 g stat 12 42 7.5 96-h LC50 6.3 (Salmo trutta) fingerling flow 12 314 7.5 96-h LC50 2.0 Table 35 (continued) Organism Size/age Stat/ Temperature Hardness pH Formulationc Parameter Concentration Reference flowa (°C) (mg/litre)b (mg/litre) Brook trout 0.8 g stat 12 42 7.5 96-h LC50 2.1 Mayer & Ellersieck (1986) (Salvelinus 0.8 g stat 7 42 7.5 96-h LC50 3.0 fontinalis) 1 g stat 17 42 7.5 96-h LC50 0.7 0.7 g stat 12 42 6.5 96-h LC50 4.6 0.7 g stat 12 42 8.5 96-h LC50 2.1 0.7 g stat 12 42 9 96-h LC50 1.1 0.8 g stat 12 42 7.5 96-h LC50 1.2 0.8 g stat 12 300 7.5 96-h LC50 1.3 Lake trout 1.7 g stat 12 40 7.5 96-h LC50 0.7 (Salvelinus 1.7 g stat 12 40 6 96-h LC50 0.7 namaycush) 1.7 g stat 12 40 9 96-h LC50 0.9 0.5 g stat 12 162 7.4 96-h LC50 0.9 2.6 g flow 12 162 7.4 96-h LC50 2.3 Gold fish 0.9 g stat 18 40 7.1 96-h LC50 13.2 (Carassius 0.9 g stat 18 272 7.4 96-h LC50 12.8 auratus) Common carp 0.6 g stat 18 40 7.1 96-h LC50 5.3 (Cyprinus carpio) fry CT 96-h LC50 1.7 Chin & Sudderuddin (1979) Table 35 (continued) Organism Size/age Stat/ Temperature Hardness pH Formulationc Parameter Concentration Reference flowa (°C) (mg/litre)b (mg/litre) Carp C50 WP 72-h LC50 10.4 Toor & Kaur (1974) (Cyprinus carpio communis) (Catla catla) S 96-h LC50 6.4 Tilak et al. (1981) (Cirrhina S50 WP 96-h LC50 2.0 Verma et al. (1984) mrigala) Fathead 0.5 g stat 12 42 7.5 96-h LC50 14.0 Mayer & Ellersieck (1986) minnow 0.8 g stat 18 40 7.1 96-h LC50 14.6 (Pimephales 0.8 g stat 18 272 7.4 96-h LC50 7.7 promelas) Sheepshead CT 96-h LC50 2.2 Springborn (1985) minnow (Cyprindon variegatus) Black bullhead 1.2 g stat 18 40 7.1 96-h LC50 20.0 Mayer & Ellersieck (1986) (Ictalurus malas) Channel catfish 1.5 g stat 18 40 7.1 96-h LC50 15.8 (Ictalurus 1.5 g stat 18 272 7.4 96-h LC50 7.8 punctatus) fingerling flow 12 314 7.5 96-h LC50 17.31 Table 35 (continued) Organism Size/age Stat/ Temperature Hardness pH Formulationc Parameter Concentration Reference flowa (°C) (mg/litre)b (mg/litre) Freshwater 1.1 g stat 28 S50 WP 72-h LC50 17.5 Arunachalam et al. (1980) catfish (Mystus vittatus) S 96-h LC50 2.4 Tilak et al. (1981) (Mystus S 96-h LC50 4.6 cavasius) Freshwater CT 96-h LC50 46.9 Tripathi & Shukla (1988) catfish (Clarias batrachus) CT 96-h LC50 107.7 Catfish CT 96-h LC50 9.7 Lejczak (1977a) (Lebistes reticulatus) Green sunfish 1.1 g stat 18 40 7.1 96-h LC50 11.2 Mayer & Ellersieck (1986) (Lepomis 1.1 g stat 18 272 7.4 96-h LC50 9.5 cyanellus) Table 35 (continued) Organism Size/age Stat/ Temperature Hardness pH Formulationc Parameter Concentration Reference flowa (°C) (mg/litre)b (mg/litre) Bluegill 1.2 g stat 18 40 7.1 96-h LC50 6.8 (Lepomis 1.2 g stat 18 272 7.4 96-h LC50 5.2 macrochirus) 0.4 g stat 12 44 7.4 96-h LC50 7.4 0.4 g stat 22 44 7.4 96-h LC50 5.2 0.8 g stat 12 40 7.4 96-h LC50 16.0 0.8 g stat 17 40 7.4 96-h LC50 7.0 0.8 g stat 22 40 7.4 96-h LC50 8.2 0.4 g stat 17 320 7.4 96-h LC50 6.2 0.7 g stat 17 40 6.5 96-h LC50 5.4 0.7 g stat 17 40 7.5 96-h LC50 5.2 0.7 g stat 17 40 8.5 96-h LC50 1.8 0.7 g stat 17 40 9.5 96-h LC50 2.6 0.6 g flow 12 314 7.5 96-h LC50 5.1 XLR 96-h LC50 9.8 Springborn (1985) S4 96-h LC50 10 Mosquito fish 30-40 mm stat 96-h LC50 31.8 Chaiyarach et al. (1975) (Gambusia affinis) Largemouth 0.9 g stat 18 40 7.1 96-h LC50 6.4 Mayer & Ellersieck (1986) bass (Micropterus salmoides) Black crapple 1 g stat 18 40 7.1 96-h LC50 2.6 (Pomoxis nigromaculatus) Table 35 (continued) Organism Size/age Stat/ Temperature Hardness pH Formulationc Parameter Concentration Reference flowa (°C) (mg/litre)b (mg/litre) Yellow perch 1.4 g stat 18 40 7.1 96-h LC50 0.7 Mayer & Ellersieck (1986) (Perca 0.6 g stat 12 42 7.5 96-h LC50 5.1 flavescens) 1 g stat 7 42 7.5 96-h LC50 13.9 1 g stat 12 42 7.5 96-h LC50 5.4 1 g stat 17 42 7.5 96-h LC50 3.4 1 g stat 22 42 7.5 96-h LC50 1.2 0.9 g stat 12 42 6.5 96-h LC50 4.0 0.9 g stat 12 42 7.5 96-h LC50 4.2 0.9 g stat 12 42 8.5 96-h LC50 0.5 0.9 g stat 12 42 9 96-h LC50 0.4 1 g stat 12 42 8 96-h LC50 3.8 1 g stat 12 170 8 96-h LC50 5.0 1 g stat 12 300 8 96-h LC50 3.8 fingerling flow 12 314 7.5 96-h LC50 1.4 Snakehead fish CT 48-h LC50 8.7 Rao et al. (1985a) (Channa punctatus) C50 WP 96-h LC50 19.5 Singh et al. (1984) C50 WP 48-h LC50 8.1 Rao et al. (1985b) (Anabas S 96-h LC50 5.5 Tilak et al. (1981) testulus) Table 35 (continued) Organism Size/age Stat/ Temperature Hardness pH Formulationc Parameter Concentration Reference flowa (°C) (mg/litre)b (mg/litre) Tilapid fish S50 WP 72-h LC50 8.0 Koudinya & Ramamurthi (Sarotherodon (1980) mosambica) (Tilapia sp.) C 48-h LC50 5.5 Basha et al. (1983) Estuarine C 96-h LC50 2.2 Lingaraja & Venugopalan teleost (1978) (Therapon jarbus) (Hetero-pneustes C50 WP 96-h LC50 20.1 Singh et al. (1984) fossilis) (Macropodus) CT 96-h LC50 3.5 Arunachalam & Palanichamy (1982) (Cypanus) CT 96-h LC50 4.0 Estuarine & marine Longnose juvenile stat 28 19s 48-h LC50 1.6 Mayer (1987) killifish (Fundulus similis) Table 35 (continued) Organism Size/age Stat/ Temperature Hardness pH Formulationc Parameter Concentration Reference flowa (°C) (mg/litre)b (mg/litre) Striped mullet juvenile stat 24 17s 48-h LC50 2.4 (Mugil cephalus) aStat = static conditions (water unchanged for the duration of the test); Flow = flow-through conditions (carbaryl concentration in water continuously maintained). bHardness = expressed as mg CaCO3/litre; s = salinity (%). cFormulation: C = carbaryl; CT = Carbaryl technical (> 95%); C50 WP = Carbaryl 50% wettable powder; S = Sevin; S4 = Sevin-4-oil; S50 WP = Sevin 50% wettable powder; XLR = Sevin (44% carbaryl); C49 = Carbaryl (49%). Where information on formulation is not given the carbaryl formulation used was mostly carbaryl technical. Exposure of C. punctatus to carbaryl at a concentration of 3 mg/litre for 48 h resulted in an increase in free fatty acids, cholesterol, and lipase activity in the liver (Rao et al., 1985b). However, the total lipid content was reduced. The literature indicates that water temperature, hardness, and pH may influence the toxicity of carbaryl, as well as the size of the fish (Table 35). In a study by Post & Schroeder (1971), water was supplied from a well, classified as very hard and highly alkaline at temperatures of 13.6-14.6 °C. Carbaryl was more toxic for cutthroat trout weighing 0.37 g than for those weighing 1-2 g (96-h LC50 1.5 and 2.2 mg/litre, respectively) and more toxic for brook trout weighing 1.15 g than for those weighing 2.04 g (96-h LC50 1.2 and 2.1 mg/litre, respectively). A comparative toxicity study on carbaryl and 1-naphthol, under laboratory conditions showed that 1-naphthol was approximately 5 times more toxic than carbaryl for goldfish ( Carassius auratus) and 2 times more toxic for killifish ( Fundulus heteroclitus) (Shea & Berry, 1983). Synergism of the effects of carbaryl and phenthoate on Channa punctatus was found (Rao et al., 1985a,b). Carbaryl produced potentiation of the effects of 2,4-D, n-butyl ester, dieldrin, rotenone, pentachlorophenol, and arecoline on trout (Statham & Lech, 1975). Application of carbaryl to a forest resulted in a significant (15-34%) decrease in brain acetylcholinesterase activity in brook trout from a nearby stream (Haines, 1981). No other effects were observed. 22.214.171.124 Short-term and long-term toxicity There is a limited data base on the effects of long-term carbaryl exposure on fish. In one study, juvenile spot Leiostomus xanthurus were exposed for five months to carbaryl (technical, 98%) at a level of 0.1 mg/litre in a flow-through system (Lowe, 1967). No carbaryl-related mortality was observed and there was no effect on growth. Carlson (1972) conducted the only full life-cycle study on fish with carbaryl (80%), in which fathead minnows ( Pimephales promelas) were exposed to five concentrations (0.008-0.68 mg/litre) for 9 months, beginning with the larvae. Survival of fatheads after 6 months at 0.68 mg/litre was lower than that in the controls. After 9 months at 0.68 mg/litre, the mean number of eggs produced per female was reduced, the mean number eggs per spawning was also affected and no hatching occurred. No other demonstrable effects were noted at 0.017, 0.062, and 0.21 mg/litre concentrations; thus, the maximum acceptable toxicant concentration (MATC) for fathead minnows, exposed to carbaryl in water, was between 0.21 and 0.68 mg/litre. The lethal threshold concentration for 2-month-old minnows was 9 mg/litre. Kaur & Toor (1977) exposed different stages of the embryo of carp ( Cyprinus carpio) to carbaryl through the hatching stage. There was 100% mortality of carp eggs and embryos at 2.5 mg/litre. There appeared to be no effect of carbaryl on hatching at 0.01-0.75 mg/litre. However, there was decreased hatching at 1.0 mg/litre and deformed larvae (3.3%) with enlargement of the pericardial sac and coiling of the posterior region of the embryo. Thirty days exposure of Channa striatus to 10 or 20 mg carbaryl/litre retarded oocyte production, with an increase in the number of immature oocytes and a decrease in the number of mature oocytes (Kulshrestha & Arora, 1984). Statistically significant reductions in gonadotropic hormone in the pituitary gland and plasma in Channa punctatus were observed following exposure to carbaryl at a concentration of 1.66 mg/litre (Ghosh et al., 1990). Gonadotropic hormone levels continued to decrease with continued exposure, with a 30% decrease in the pituitary gland and a 50% decrease in serum after 30 days of exposure. Exposure of the freshwater fish Puntius conchonius to carbaryl at a concentration of 0.194 mg/litre for 15 days resulted in an increase in the incidence of lesions in the gill and liver (Gill et al., 1988). A higher level of exposure also resulted in lesions in the kidney. A 27-day exposure to 12.5 mg/litre led to a decrease in the feeding and growth rate of the catfish ( Mystus rittatus) (Arunachalam et al., 1980). Long-term toxicity of carbaryl for fish in most natural surface waters will not occur, since exposure would not be high enough or constant, because of the substance's degradation. 7.2.3 Amphibians The LC50s for bullfrog ( Rana tigrina) 0.1 g tadpoles with 24, 48, 72, or 96 h of exposure were determined to be 12.8, 8.2, 6.7, and 6.3 mg/litre, respectively (Marian et al., 1983). Growth and feeding were decreased in a dose-dependent manner by doses ranging from 0.5 to 5 mg/litre. 7.3 Terrestrial organisms 7.3.1 Worms Carbaryl was classified as extremely toxic for earthworms (LC50 = 9.1 µg/m2) by Roberts & Dorough (1984). The LC50 value for the worm Eisenia fetida-Savigny was 14 µg/cm2 (Neuhauser et al., 1985). The number of earthworms was reduced by 60% when carbaryl was applied at 0.5 kg (1.12 lb) a.i. per hectare (Thompson, 1971). Exposure of earthworms to carbaryl (1-8 mg/kg) significantly increased the burrowing time, which was directly proportional to the dose and time of exposure, because of the AChE inhibition in the neural tissue (Gupta & Sundararaman, 1991). 7.3.2 Insects Administration of carbaryl in sublethal doses produced death in the early embryonal stage of the silkworm (Kuribayashi, 1981). The alfalfa leaf-cutting bee ( Megachile pacifica), which is very important in the pollination of alfalfa, has been reported to be particularly tolerant to carbaryl (Lee & Brindley, 1974). Waller (1969) also classified carbaryl as relatively non-toxic for the alfalfa leaf-cutting bee. The carbaryl tolerance was related to sex and age. The LD50 was similar for 1-day-old males and 1-and 4-day-old females (240, 245, and 262 µg/g, respectively). However, four-day-old males were much more susceptible to carbaryl, and had an LD50 of 51 µg/g. Guirgius & Brindley (1975, 1976) showed that carbaryl toxicity in alfalfa leaf-cutting bees was controlled by the activity of mixed function oxidase or microsomal enzymes. This detoxification system varies with the age and sex of the bees and results in significantly different carbaryl persistence which, in turn, leads to differences in carbaryl toxicity. In the more tolerant groups, carbaryl metabolites were rapidly conjugated and moved to an aqueous fraction of the bee. Less tolerant insects (4-day-old males) accumulated these metabolites with time, indicating that the conjugation mechanisms had deteriorated with age. Carbaryl is known to be highly toxic for honey-bees. When ingested, the LD50 was 0.18 µg/bee (Alvarez et al., 1970). The contact LD50 (approximately 10-15 mg/kg) for adult bees is approximately 1.3 µg (Stevenson et al., 1977; Stevenson, 1978). The carbaryl residue content of bee bread was correlated with the amount of residue found in the bees and occurred at higher levels than in honey throughout the 56-day period (Winterlin & Walker, 1973). 7.3.3 Birds The toxicity of carbaryl for birds appears to be low (Table 36). LD50s for six species of waterfowl and game birds were all greater than 1000 mg/kg (Bart, 1979). There were some exceptions; thus the red-winged blackbird has an LD50 of 56 mg/kg (Schafer, 1972). In this study, 180 compounds were found to be toxic for the red-winged blackbirds and the LD50s ranged between 0.24 and 100 mg/kg. Food intake, body weight, and locomotor activity were monitored in adult male bobwhites ( Colinus virginianus), given diets that contained levels of carbaryl typical of normal exposure under agricultural conditions in Kansas. No changes were observed in diets containing 127 or 1235 mg carbaryl/kg (Robel et al., 1982). Technical carbaryl fed to young Mallard ducks at dietary levels of 10, 100, 1000, or 3000 mg/kg, revealed dose-correlated signs of toxicity (reduced intake and/or body weight depression) at the 100, 1000, and 3000 mg/kg level (Fletcher & Leonard, 1986). There was some tentative evidence that low dosages of carbaryl may increase the susceptibility of bobwhites ( Colinus virginianus) to the protozoan parasite Histomonas meleagridis, to which they are usually resistant (Zeakes et al., 1981). A study of the effects of carbaryl on forest birds was conducted in southern New York; plots had been treated 3-4 weeks previously with carbaryl at the normal rate of 1.1 kg/ha, and at 6 times the normal rate (6.6 kg/ha). In this study, carbaryl had little, if any, effect on birds. Young birds gained weight normally, adults continued nesting in the area, no changes were detected in song frequency, and there was no evidence of birds leaving the area to forage. This lack of detectable effects, despite the heavy dose (6.6 kg/ha), indicates that carbaryl applied at the normal rate (1.1 kg/ha) would have little if any adverse effect on birds (Bart, 1979). The LC50 of carbaryl for mallard embryos, following field applications, was determined by Hoffman & Albers (1984) to be greater than 26.4 kg/ha or 118 µg/g egg. Carbaryl was estimated to be relatively nontoxic compared with other pesticides. Inhibition of the brain ChE activity of birds from forests sprayed with carbaryl 1.13 (kg/ha) was found in 3 out of 12 species studied (Zinkl et al., 1977) up to 5 days after application. Table 36. Acute toxicity of carbaryl for birds Species Age Parametera Concentration Reference (mg/kg) Japanese quail 7 days LD50 2290 Hudson et al. Coturnix coturnix (1984) japonica 2 months 5 day-LC50 > 10 000 Hill & Camardese (1986) Bobwhite quail 23 days 5 day-LC50 > 5000 Hill et al. (1975) Colinus virginianus California quail 10 months LD50 > 2000 Hudson et al. Callipepla californica (1984) Chukar Alectoris chukar 4 months LD50 1888 Sharp-tailed grouse LD50 < 1000 Tympanuchus phasianellus Pheasant 3-4 months LD50 707-> 2000 Phasianus colchicus 23 days LD50 > 5000 Hill et al. (1975) Mallard 3 months LD50 > 2564 Hudson et al. (1984) Anas platyrhynchos 24 days 5 day-LC50 > 5000 Hill et al. (1975) Rock dove LD50 1000-3000 Hudson et al. (1984) Columbia livia Canada goose LD50 1790 Branta canadensis Red-winged blackbird LD50 56 Schafer (1972) Agelaius phoeniceus a LD50 = single oral dose expressed as mg/kg body weight. 5 day-LC50 = 5-day dietary exposure (expressed as mg/kg feed) followed by 3 days on a "clean" diet. 7.3.4 Mammals The effect of carbaryl on wild mice ( Clethrionomys glareolus and Apodemus sylvaticus) in their natural environment was studied by Krylov (1970). Carbaryl-containing bait, each consisting of 2 kg grain treated with 50 g carbaryl in oil suspension, were distributed twice (in March and June) over 10 ha of woods, at a rate of 1 bait/ha. An adjacent territory of 10 ha was used as a control. Effects on mice, assessed 1´ months after the last application, were: reduction of the population by 31.5% compared with the control; changes in the reproduction system (e.g., decreased number of embryos in utero, and of corpora lutea, reduced weight of testicles), and increased weight of adrenal glands. 7.4 Effects on the population and ecosystem The effects of carbaryl on terrestrial ecosystems have been studied by Stegeman (1964), Barrett (1968), and Spain (1974). A large-scale study was performed by Barrett in 1968. It was designed to determine the effects of carbaryl on an intact ecosystem in a field that was planted with millet ( Panicum ramosum). The area was sprayed with a single application of 2.24 kg carbaryl/ha. There was a highly significant decrease in litter decomposition in the treated area, 3 weeks after spraying, which was probably because of a reduction in microarthropods and other decomposers. After 5 weeks, there was a more than 95% reduction in the number of arthropods and in the total biomass. Phytophagous insects were severely affected, and predatory insects and spiders were less affected. The number of species was also reduced, but all species returned to control levels within 1-2 weeks, except Hemiptera and Hymenoptera. Reproduction of cotton rats was delayed by 4 weeks. However, the total mammal population was not affected, because of a compensating increase in the population of house mice. Old field mice did not seem to be affected. The effects of carbaryl on forest soil mites and Collembola, the two most numerous soil arthropods, were studied by Stegeman (1964). Mites and Collembola are an important link in the decomposition process of dead plants and animal matter. They also are a natural means of decomposing the accumulating litter, and they provide nutrients for already existing or future crops and fungi. Application of carbaryl to a test plot in a red-pine plantation, at doses of 11.2 and 56 kg/ha, reduced the arthropod population proportional to the severity of the treatment. Neither mites nor Collembola were totally exterminated by any treatment. The rate of population increase of the mites, 4-5 months after treatment, was directly proportional to the dose applied. Collembola were more vulnerable to treatment than mites and did not recover as rapidly. The effect of carbaryl at two application rates (0.11 and 1.13 g a.i. per m2) on mixed species population of the litter fauna of a Corsican pine forest was studied by Spain (1974). He took a quantitative sampling of the fauna at intervals of 11, 106, 209, and 315 days after application to record the pattern and the time of the recovery process (Table 37). The effect of carbaryl varied in different populations. For Collembola, there was a marked reduction at the two treatment levels. For Coccoidea and Symphyla, there was a slight effect at the lower level, and a marked decrease at the higher level. For Coleoptera, Diptera, Cryptostigmata, Mesostigmata, and Prostigmata, the effect was small or insignificant. The recovery process during the period of 315 days was not sufficient to reach the status of the untreated population. Tagatz et al. (1979) studied the effects of carbaryl on animal communities that develop from planktonic larvae in aquaria containing sand and estuary water. Samples collected after 10 weeks of exposure were analysed. The numbers of animals and species were significantly less at 11.1 and 103 µg carbaryl/litre (Table 38) being decreased to nearly half (from 21 to 12). A carbaryl concentration of 1.1 µg/litre did not produce significant effects, except a decrease in the particularly sensitive amphiopod, Coraphium acerusicum. Carbaryl might have caused changes in biological interactions that affect the relative abundance of species. The annelid Polydora ligni increased at a concentration of 103 µg/litre, and there was a marked decrease in the number of other annelids and nemerteas. There have been a number of field studies evaluating the impact of carbaryl applications on macroinvertebrate populations. In one study in New York State, carbaryl was applied aerially at a rate of 1.3 kg/ha, in fuel oil with a paraffin oil sticker, and its effects upon the aquatic fauna of two streams were studied (Burdick et al., 1960). The data indicated that carbaryl (in oil suspension) was toxic for mayflies (Ephemeroptera), stoneflies (Plecoptera) and caddisflies (Trichoptera). Other groups of insects were less affected and the application did not affect fish. Square-foot samples, collected before, and shortly after, spraying, showed reductions of from 50 to 97.2% in the weight of invertebrate (standing crop) fish food. A progressive effect from upstream to lower sections was correlated with increased 44-151 exposure time. No exposure concentrations in water were documented. Owen (1965) also reported a reduction of 54.2 to 62.8% in the standing crop of aquatic insects following aerial spraying with carbaryl (80 WP) (1.3 kg/ha). A control stream showed a 5.9% increase in the same period. Table 37. Geometric means for assessed taxa (individuals/m2), post-application sampling of carbaryl-treated plotsa Sampling day (after treatment) Taxon Control plot High carbaryl Low carbaryl Coccoidea 272.9 90.8 129.4 Coleoptera (imagines) 425.6 137.7 153.1 Coleoptera (immature) 54.0 47.3 41.4 Collembola 42 771.5 5033.4 9819.7 Diptera 350.9 244.2 243.7 11 Cryptostigmata 5707.3 3933.2 5863.5 Mesostigmata 7239.5 3688.9 5038.2 Prostigmata 1335.6 305.3 564.8 Aranese 238.7 222.7 155.0 Chilopoda 71.1 102.4 37.4 Symphyla 872.1 359.0 388.9 Coccoidea 847.8 157.0 162.9 Coleoptera (imagines) 41.9 379.1 170.7 Coleoptera (immature) 41.9 31.7b 0.0b Collembola 18 185.1 1621.3 5205.9 Diptera 264.0 173.8 160.5 106 Cryptostigmata 5855.3 6237.9 6623.7 Mesostigmata 8610.3 846.4 595.2 Prostigmata 631.5 846.4 595.2 Aranese 360.6 224.9 253.2 Chilopoda 114.0 157.8 94.3 Symphyla 317.5 47.0b 223.4 Table 37 (continued) Sampling day (after treatment) Taxon Control plot High carbaryl Low carbaryl Coccoidea 358.3 181.0 141.0 Coleoptera (imagines) 520.0 86.6 232.6 Coleoptera (immature) 289.6 125.9 74.1 Collembola 41 198.7 3469.4 19 238.9 Diptera 449.9 28.2b 209.5 209 Cryptostigmata 16 298.4 11 184.8 12 014.8 Mesostigmata 16 150.0 7293.9 11 709.0 Prostigmata 2721.6 2407.2 3152.4 Aranese 264.7 108.3 220.2 Chilopoda 241.9 28.2b 150.5 Symphyla 357.3 78.3 174.4 Coccoidea 142.0 207.5 51.5 Coleoptera (imagines) 261.6 64.4 132.5 Coleoptera (immature) 37.0 88.8 23.1b Collembola 40 139.5 4121.3 10 294.6 Diptera 533.9 291.2 173.1 315 Cryptostigmata 10 889.3 12 743.6 10 506.6 Mesostigmata 11 290.3 7762.7 14 912.0 Prostigmata 2995.0 1844.7 4765.7 Aranese 217.4 157.4 117.8 Chilopoda 159.5 83.4 88.8 Symphyla 349.6 85.4 158.8 aSource:Spain (1974). b95% confidence limits for the parametric mean include zero. Table 38. Animals and species, by phylum, collected from control aquaria and aquaria exposed to carbaryla Control Carbaryl 1.1 µg/litre 11.1 µg/litre 103 µg/litre Phylum Number Species Number Species Number Species Number Species Mollusca 1691 3 1563 3 1340 3 1321 5 Arthropoda 380 7 339 8 336 2 269 2 Annelida 102 8 94 7 79 4 200 5 Nemerica 16 1 25 1 20 1 0 0 Coelenterata 3 1 5 1 0 0 0 0 Platyhelmintes 0 0 2 1 0 0 0 0 Echinodermata 0 0 0 0 1 1 0 0 All phyla 2192 20 2086 21 1776 11 1790 12 aFrom:Tagatz et al. (1979). The effects on stream invertebrates of carbaryl, applied at a rate of 840 g a.i./ha for spruce budworm suppression, was studied. Benthos samples showed significant declines among Plecoptera, Ephemeroptera, and Trichoptera. Plecoptera had not repopulated any treated stream, 60 days after treatment (Courtemanch & Gibbs, 1980). Gibbs et al. (1984) conducted a 42-month (1980-83) study on the occurrence/persistence of carbaryl residues in pond water and sediment as a result of an application of Sevin-4-oil (840 g a.i./ha). The immediate and long-term effects on pond macroinvertebrates and emerging aquatic insects were also evaluated. A preliminary study by these investigators in 1977 and others (Coutant, 1964) had shown that there were large increases in the number of drift organisms, several days after aerial spraying with carbaryl. Most drift was accompanied by dead Amphipoda, Ephemeroptera, Plecoptera, and Trichoptera, and carbaryl residues persisted longer than 30 days in pond sediment. The increase in the number of dead organisms was accompanied by a reduction in the standing crop of benthic macroinvertebrates. The most severe and persistent impact was on Amphipoda with Hyallela azteca and Crangonyx richmondensis reduced to almost 0/m2; C. richmondensis failed to recolonize in one of the two treatment ponds, 42 months after treatment. The numbers of immature Ephemeroptera and Trichoptera were reduced immediately following spray application, but this effect did not persist throughout the season or into the following year. Immediate reduction in numbers of adult Ephemeroptera and Trichoptera emerging from the ponds was also found, but recovery of populations was observed. Numbers of immature Odonata were also reduced following treatment and remained low the following year. The Chironomidae populations did not appear to be affected, either as immatures or emerging adults. The effects of two consecutive years of spraying with Sevin-4-oil on other aquatic systems appear similar to those observed in areas treated once (Courtemanch & Gibbs, 1978; Trial, 1978,1979). In simulated aquatic field studies, 1 mg carbaryl (WP)/litre was applied to one outdoor concrete pond (4 x 5 x 1 m deep) (Hanazato & Yasuno, 1987). All zooplankton (Cladocera included) and Chaoborus larvae were killed. The zooplankton community recovered rapidly and Cladocera reappeared only two days after application. Since Chaoborus populations are predators of crustacean zooplankton, their suppression may also have added to the recovery of increased populations of Cladocerans. Carbaryl exposure also changed the community from a rotifer-predominating zooplankton community to that of Cladocera-predominating. Rotifers in this study were also highly sensitive to carbaryl. The same phenomenon was observed again after the second application of carbaryl. Subsequent to this study, Hanazato & Yasuno (1989) applied carbaryl to simulated ponds in the same way as in the previous study, but in the spring when the water temperature was approximately 10 °C lower. Cladocerans never recovered to the density level of the pre-treatment period. The rapid recovery of Chaoborus seemed to interfere with the recovery of Cladoceran populations after treatment. The authors suggested that the different recovery patterns of the zooplankton community resulted from different temperatures in the ponds. In another study, Hanazato & Yasuno (1990a) examined Chaoborus density in relation to the effects of carbaryl (0.1 and 0.5 mg/litre) on zooplankton communities in ponds, where the abundance of Chaoborus larvae was controlled. They concluded that Chaoborus density and/or temperature may influence the recovery of the zooplankton community following the effects of carbaryl. The recovery of a zooplankton community may differ in different aquatic ecosystems (with different community structures) and under different temperatures, even when the same treatment is applied. Hanazato & Yasuno (1990b) also studied the effect of the time of application of carbaryl (0.5 mg/litre) on recovery patterns of zooplankton communities in simulated ponds. The loss of carbaryl from pond water was rapid. The concentration decreased to less than 1% of its initial value, three days after the first or second application, and six days after the third application. This study also showed that applications of carbaryl at different times induced different zooplankton structures (and different recovery patterns), and, various factors other than toxicity of carbaryl, such as temperature, competitive interactions between zooplankters, and trends of zooplankton populations may play important roles in determining zooplankton community structure after chemical application. Furthermore, the significance of predators in the recovery process after chemical treatment was re-emphasized. 8. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS Reviews of toxicological aspects of carbaryl were prepared by NIOSH (1976); US EPA (1977, 1980, 1982, 1984); USDHHS/USDL (1978); US EPA (1980b); Mount & Oehme (1981a); Weston (1982); and Cranmer (1986). Studies from the Soviet Union were reviewed by IRPTC (1982, 1989). 8.1 Single exposures 8.1.1 Oral toxicity The oral LD50s for the rat are given in Table 39 and for other mammals in Table 40. The values varied by about a factor of 4 depending on formulation, route of production, vehicle, and strain of rat. Interspecies differences were found. Cats are the most sensitive, with guinea-pigs, rats, mice, and rabbits showing more resistance in that order. Pigs and monkeys seem to be less sensitive (Carpenter et al., 1961; Gladenko & Malinin, 1970; Smalley, 1970). Cattle are more sensitive than pigs. After a single oral application of carbaryl in cattle, symptoms appeared at a dose level of 25 mg/kg; 100 mg/kg was the minimum effective dose in pigs (Gladenko & Malinin, 1970). Mount & Oehme (1981b) observed that the lethality of carbaryl, administered to sheep at doses ranging from 300 to 1000 mg/kg, was highly correlated with concentrations in the brain (>1 mg/kg) and liver (>3 mg/kg), and with inhibition of acetylcholinesterase (greater than 50% inhibition). 8.1.2 Acute inhalation toxicity Acute toxic effects following inhalation at different concentrations are presented in Table 41. 8.1.3 Dermal toxicity A dose of 2500 mg/kg, applied as a 40% aqueous suspension of 50% wettable powder, killed 1 out of 4 rabbits (Carpenter et al., 1961). When 99% technical carbaryl was applied to male and female rabbits, the LD50 was >2000 mg/kg (Bushy Run, 1983b). In rats, the LD50 is thought to be >4000 mg/kg (Yakim, 1965; Gaines, 1969). Table 39. Acute oral LD50s for the rat Strain (sex) Weight Formulation of Vehicle LD50(mg/kg Symptoms References (g) carbaryl body weight) CF-N technical 0.25% agar (male) 90-120 510 (360-650) - Carpenter et al. (female) 90-120 610 (490-750) - (1961) Sherman 850 (600 LD min) Gaines (1969) (male) (female) 500 (100 LD min) Inbred 721 (653-789) Yakim (1965) Inbred 120-200 sunflower oil 515 Rybakova (1966) Sprague Dawley 203-246 technical carboxymethyl-cellulose 300 Hamada (1990) (male, female) in water Sprague Dawley 112-173 technical 0.25% methyl-cellulose 685 (612-767) death, 4-24 h Field (1980b) (male, female) Sprague Dawley 100-160 95% technical 0.25% methyl-cellulose 225 (202-321) death, 2-24 h Field (1980a) (male, female) Table 39 (continued) Strain (sex) Weight Formulation of Vehicle LD50(mg/kg Symptoms References (g) carbaryl body weight) Sprague Dawley 200-264 40.38% water tremor, prostration, Hazleton Lab. (male) 750 (467-1202) laboured respiration, American Inc. salivation 10 February 1982 (female) 527 (257-977) death, 4-3 days Harlem Sevin XLR not diluted death, 0.5 h-3 days Kuhn (1991a) Sprague Dawley plus 44% (male) 229-286 867 (562-1336) (female) 182-219 575 (459-721) Harlem Sevin-4-oil not diluted 658 (456-979) 3 h-3 days Kuhn (1991b) Sprague Dawley 47% (w/w) (male) 229-286 963 (802-1160) (female) 182-219 473 (364-620) Hilltop Wistar 200-250 98% technical 0.25% methyl-cellulose Bushy Run (male) 283 death, 1.5-24 h (1983b) (female) 246 Hilltop Wistar 204-237 80% sprayable water (male) 406 Bushy Run (female) 203 death, 1 min-24 h (1983c) Table 40. Acute oral toxicity for mammals other than rats Species Number of animals Toxicitya Reference (mg/kg body weight) Mouse 6 per dose 363 (294-431) Yakim (1965) Mouse (female) 80 white 437 ± 70 Rybakova (1966) Mouse 1310 white 206 (175-480) Bukin (1965) Guinea-pig 5 per dose 280 Carpenter et al. (1961) Rabbit 4 per dose 710 Rabbit 56 700 (LD100) Bukin (1965) Cat (female) 3 250 (LD100) Carpenter et al. (1961) Cat no data 150 Yakim (1965) Swine 3 per dose 800-1000 Gladenko & Malinin (1970) Swine 1 per dose 1500-2000 Smalley (1970) Monkey no data > 1000 FAO/WHO (1970) Duckling 106 500 (LD15) Bukin & Filatov (1965) 6000 (LD100) Table 40 (continued) Species Number of animals Toxicitya Reference (mg/kg body weight) Chick 48 250 (LD33) 1000 (LD100) Hen 12 > 1000 (no death) Hen 12 > 3000 Bukin (1965) aLD50, unless otherwise stated. Table 41. Inhalation toxicity single exposure Species Concentration Effects observed References Guinea-pig 6 390 mg 50% wettable powder/m3 nasal and ocular irritation after 14 days - Carpenter et al. (average particle size 15 µm) 4 h haemorrhage areas in the lungs (1961) Guinea-pig 6 230 mg carbaryl 85S/m3 slight weight decrease; recovered by day 14 (average particle size 5 µm) 4 h Dog 75 mg carbaryl 85S/m3 5 h typical symptoms for ChE inhibition Cat 3 groups 82 mg/m3 dust 6 h tremor salivation, muscle fibrillation decreased Yakim (1967, 1968) of 4 animals ChEA by 39-55% serum; 53-71% red blood cells; normalization after 72 h 37 mg/m3 dust 6 h decreased ChEA by 23% in serum and 41% in red blood cells; recovery after 48 h 20 mg/m3 dust 6 h decreased ChEA by 11-27% in serum and 15.28% in red blood cells; recovery after 24 h Rat 20-23 mg/m3 dust no effect Weil & Carpenter 98% particles (1974) less than 1.0 µm diameter Rat 1800 mg/m3 water aerosols lacrimation, tremor with 1.5 h of exposure Myers et al. (1975) Rat Wistar Albino Aerosols Sevin XLR 44% 792 mg 1/5 females died, tremor, ataxia, increased Faït (1984) male 5 a.i./m3 per 4 h (the highest respiratory rate; recovery period 6 days female 5 attainable concentration) particle size 3.6 ± 2.64 µm 8.1.4 Other routes of exposure Other routes of exposure are presented in Table 42. Table 42. Toxicity following parenteral application Species Weight Vehicle Route of LD50 References (g) administration mg/kg body weight) Rat 10-107 propylene intravenous 18 Mellon Institute female glycol (1958) Rat 92-126 polyethylene intravenous 24 glycol 400 intravenous (17-33) Rat 90-120 95% ethyl intravenous 33 alcohol (26-41) Rabbit 1686-3544 0.25% agar intraperitoneal 223 (122-407) Rat 90-120 in lard subcutaneous 1410 Leghorn 10-11 days in alantoic 3.44 mg Tós-Luty et al. chick old cavity per embryo (1973) embryos (250) 8.2 Skin and eye irritation, sensitization 8.2.1 Skin and eye irritation The results of several studies on skin irritation from carbaryl were negative (Carpenter et al., 1961; Yakim, 1965). Transient erythema was noted after an application of 0.5 ml 43.4% carbaryl on occluded rabbit skin (Bushy Run, 1983a). Carbaryl is a weak eye irritant (Table 43). Table 43. Eye irritation Species Number Formulation Effects observed Reference Rabbit 5 technical grade mild injury in one Carpenter et al. 10% suspension of five eyes (1961) in propylene glycol 25% aqueous no injury suspension 50 mg dust spots of corneal-necrosis Rabbit 6 0.1 ml (90 mg) conjunctival irritation Bushy Run (1983b) New 99% technical after 2 days recovery Zealand product White 0.1 ml transient iritis in Bushy Run 43.4% carbaryl 2 of 6, conjunctival (1983a) irritation in 6 of 6. Recovery in 3 days 8.2.2 Sensitization The sensitization response following topical application of carbaryl was studied by Myers & Christopher (1987). The induction which consisted of an application of carbaryl to covered skin, once a week during 3 weeks, was followed by a 2-week incubation period. A single challenge dose of 50% (w/v) technical carbaryl in 0.25% aqueous methyl cellulose solution (0.1-0.3 ml) was administered. Carbaryl did not produce a positive response in any of the guinea-pigs tested. Four out of 16, male, albino guinea-pigs were treated with 8 intravenous injections (3 per week) of 0.1 ml of a 0.1% dispersion of carbaryl in 3.3% propylene glycol. After a 3-week incubation period, a challenge dose was given, but did not cause sensitization (Carpenter et al., 1961). In a more recent study, a similar procedure, but with 9 doses and a 2-week incubation period, was used. Although there was some skin irritation, it was considered that carbaryl had little or no sensitizing potential (Bushy Run, 1983a). 8.3 Short- and long-term oral exposure Several short- and long-term feeding studies with carbaryl have been reported (Carpenter et al., 1961; Rybakova, 1967; Orlova & Zhalbe, 1968; Gladenko & Malinin, 1970; Dikshith et al., 1976; Hamada, 1991a). Results are shown in Table 44. Doses that do not show any effects are 200 mg/kg diet equal to 7.9 mg/kg body weight in rats, and 100 mg/kg diet for mice, and 1.8 mg/kg body weight for dogs (approximately 100 mg/kg diet). The cumulation coefficient (LD50 for 3-month exposure/LD50 single application) was 18 (Kassin, 1968), demonstrating a very low cumulative potential for carbaryl. 8.4 Short- and long-term inhalation toxicity Short-term and long-term inhalation toxicity data are given in Table 45. 8.5 Reproduction and developmental toxicity The reproduction and developmental toxicity of carbaryl has been studied in many vertebrate species using a wide variety of study designs. The data have shown that carbaryl can affect reproduction (Table 46) and embryo/fetal development (Table 47 and 48) in a number of species. The relevance of these studies for risk assessment is influenced by several factors related to experimental design, dose levels, and the types of effects noted. Shortcomings of the studies included small sample size; inappropriate dose selection; the variable degree of maternal toxicity (ranging from no effect to lethality); and a lack of historical data for some species. The following sections have been arranged according to end-point (reproduction and developmental toxicity) and species of animals studied (mammalian, non-mammalian). Studies that are of dubious value for risk assessment are listed at the end of the table sections, and are not discussed in the text. The reasons for such an evaluation are given below the references to the individual papers. Table 44. Short- and long-term feeding and oral studies Species Sex Number Dosage (mg/kg diet Period of Effects observed References of or mg/kg body weight) exposure animals Mouse male 48 100, 400 mg/kg diet 80 weeks no changes in survival rate, pathology, and tumour FAO/WHO (1965) and incidence female Mouse male 10/sex 100, 1000, 7000 mg/kg 53 weeks at 7000 mg/kg diet: decreased survival, body Hamada (1991b) and per group diet weight gain and erythrocyte count; increased liver female weight and decreased ovary weight; increased frequency and severity of chronic nephropathy in females; cholinesterase (plasma, RBC, and brain) depressed at 100 and 7000 mg/kg; NOEL 100 mg/kg diet Rat male 2x10 1500 (58.5 mg/kg 96 days no changes Carpenter et al. body weight) (1961) female 2x10 1500 (58.5 mg/kg 96 days kidney weights significantly increased body weight) male 2x10 2250 (87.5 mg/kg 96 days increase in liver weight as a % of body weight, body weight) diffuse cloudy swelling in the kidney tubules in 4 animals (male and female) female 2x10 2250 (87.5 mg/kg 96 days decrease of body weight, increase in kidney weight body weight) Table 44 (continued) Species Sex Number Dosage (mg/kg diet Period of Effects observed References of or mg/kg body weight) exposure animals Rat male 4x40 50, 100, 200 (2, 4, 2 years no changes Carpenter et al. CF-N and 7.9 mg/kg body weight) (1961) female Rat male 2x40 400 (15.6 mg/kg 2 years weight depression in male, cloudy swelling of the Carpenter et al. CF-N and body weight) hepatic cords, principally located around the (1961) female central veins in both sexes; transitory diffuse cloudy swelling of the epithelial lining of the primarily convoluted proximal, and loop tubules Rat 75, 150, 300 mg/kg 3 months cytoplasmatic vacuolization in the proximal tubules FAO/WHO (1970) body weight Rat male 4x48 7, 14, 70 mg/kg weight depression at all dose levels; at 70 mg/kg, Rybakova (1967) and body weight slight morphological liver changes; cloudy swelling female in convoluted tubules; decreased motility of spermatocytes at all dose levels, more pronounced at 70 mg/kg; oedema in the interstitial tissue; desquamation of spermatogenic epithelium; destruction of parenchyma; decreased production of spermatocytes was found at 14 and 70 mg/kg dose levels; increased estral cycle; increased hypophysis gonadotropic function; decreased ascorbic acid contents; cell proliferation, hypertrophy, increased lipid content in suprarenal glands; decreased thyroid function; augmentation of follicles, colloid retention, thickness of follicular epithelium Table 44 (continued) Species Sex Number Dosage (mg/kg diet Period of Effects observed References of or mg/kg body weight) exposure animals Rat male 80-90/ 250, 1500, 7500 mg/kg 52 weeks body weight and food consumption lower at middle Hamada (1991c) Sprague female group diet dose; significantly increased total cholesterol; Dawley increase in liver and kidney weight; NOEL 250 mg/kg diet Rat male 876 2, 5, 15 mg/kg 1 year changes in sex glands function at 15 and 5 mg/kg Orlova & Zhalbe and body weight dose levels; enzymatic activity, spermatozoids and (1968) female the estral cycle; reduced fecundity; no effect at 2 mg/kg dose level Rat male total 200 mg/kg 90 days decreased AChE in blood with 33.8%; no Dikshith et al. local 28 body weight, histological changes (1976) strain 3 x per week in peanut oil Dog male total 0.45, 1.8, 1 year diffuse cloudy swelling of proximal convoluted and Carpenter et al. Basenji- and 14 7.2 mg/kg body loop tubules of kidney; local sudanophilic granules (1961) cocker female weight in capsules, in the glomeruli at 400 mg/kg level; the same in hybrids 5 days/week, to control dogs to a lesser extent; transient hind leg approximate levels weakness in 1 female after 189th dose at of 24, 95, 414 mg/kg 0.45 mg/kg body weight dry diet Beagle male 24+ 125, 400, 1250 mg/kg 1 year at 400 and 1250 mg/kg diet, decreased AChE in Hamada (1987) dogs and 24 diet plasma, red blood cells, and in brain; increase in female leucocyte count and segmented nitrophil count; increase in liver weight in male; no effect level 125 mg/kg diet Table 44 (continued) Species Sex Number Dosage (mg/kg diet Period of Effects observed References of or mg/kg body weight) exposure animals Beagle male 24 0, 20, 45, 125 mg/kg 5 weeks significant inhibition of cholinesterase activity Hamada (1991a) dogs female 24 diet in plasma at week 2 for males dosed 20 and 125 mg/kg Monkey 150, 300, 600 mg/kg 38 weeks kidney alterations similar to those found in rats FAO/WHO (1970) body weight Swine male 3 150 mg/kg body weight 72-83 progressive myasthenia, incoordination ataxia, Smalley et al. female 3 in diet days intentional tremor, chronic muscular contraction, (1969) until terminal paraplegia and prostration; death myodegeneration Swine 5 and 10 mg/kg 147-176 no changes Gladenko & Malinin body weight days (1970) Cattle 1 and 4 mg/kg 148 days decreased Hb with 20%, and erythrocytes with (young) body weight 30% occasionally Table 45. Short- and long-term inhalation toxicity Species Number of Concentration of carbaryl Days of Effects References animals exposure Cat 4 0.06 mg/litre 30 typical cholinergic symptoms, Yakim (1968) inhibition of ChE plasma 31-40%, erythrocytes 40-59% Cat 4 0.03-0.04 mg/litre 30 reaction time increased Cat 4 0.016 mg/litre 120 no symptoms, fluctuation in plasma ChE inhibition around 18% Rat no data 10 mg/m3 dust (85% 90 no mortality no gross visible injury Carpenter et al. suspension) 7 h/day inhalation (1961) 5 days per week periods Table 46. Reproduction studies Species Treatment End-point(s) Reference (number) Mouse Swiss Webster males 30-40 g 0, 8.5, 17, 34 mg/kg body weight weight and uptake of testosterone: Thomas et al. (1974) (10 + animals/group) orally (5 days) testes and accessory glands Swiss Webster males 30-40 g 0, 34, 68 mg/kg body weight biotransformation of Dieringer & Thomas (5 + animals/group) orally (5 days) testosterone-1,2-3H (1974) C57Bl/6 x C3H 6-8 weeks of 0, 12, 25, 50...800 mg/kg body sperm morphology, testes weight Osterloh et al. (1983) age (4 animals/group) weight per day 5 days i.p. on day 35 [route of administration] Rat Osborne-Mendel 0, 2000, 5000, 10 000 mg/kg diet fertility, litter size, and viability Collins et al. (1971) (20 females and 1 male per (3 generations) group) Wistar 0, 7, 25, 100, 200 mg/kg body fertility, litter size, and viability Weil et al. (1973) (13-21 animals/group) weight per day in the diet 0, 3, 7, 25, 100 mg/kg body weight per day orally (3 generations) Male and female 0, 1, 5, 10, 20, 40, 50 mg/kg general toxicity, serum protein, Vashakidze (1975) body weight per day orally numbers of male reproductive (1 month) cells, sperm viability, testicular histology, litter viability Table 46 (continued) Species Treatment End-point(s) Reference (number) Wistar males 0, 12.5, 25.0, 250.0 mg/kg body sperm morphology Luca & Balan (1987) (36 animals per group) weight per day Rats Male and female 0, 50, 100, 300 mg/kg body fertility indices Vashakidze (1966) weight per day orally 2 weeks to [insufficient data] 3 months Male and female 0, 2, 5, 15 mg/kg body weight per fertility indices Orlova & Zhalbe (total of 876 animals) day orally 12 months for F° (1968); Zhalbe et al. and 6 months for the F1 (1968) [insufficient data] Male and female 0, 2, 5 mg/kg body weight per day fertility indices Shtenberg & Ozhovan for 6 months (follow-up to Orlova (1971) [insufficient & Zhable) 2 through 5 generations data] Male and female 0, 2, 5, 15 mg/kg body weight per fertility indices Shtenberg et al. day for 12 months (1973) [insufficient data] Gerbil (32-80 animals per group) 0, 2000, 4000, 6000, and fertility, litter size, and viability Collins et al. (1971) 10 000 mg/kg body weight diet (3 generations) Table 47. Developmental toxicity studies, mammalian Species Treatment End-point(s) Reference (number) Mouse CF-1 0, 100, 150 mg/kg body weight fetal examination Murray et al. (1979) (23-44 in a group) per day orally or 5660 mg/kg diet. Gestation day 6-15 Swiss albino 0, 100, 150, or 200 mg/kg body fetal examination Mathur & Bhatnagar (10 per group) weight per day orally. Gestation (1991) days 8, 12, or 6-15 Charles River 0, 10, 20 mg/kg body weight. fetal examination Benson et al. (1967) (8 per group) Gestation day 6-parturition [doses too low] CD-1 100 mg/kg body weight per day postnatal viability Chernoff & Kavlock (23 animals) orally. Gestation day 8-12 (1982) [teratology screen test] CD-1 200 mg/kg body weight per day postnatal viability Kavlock et al. (1987) (30 animals) orally. Gestation day 8-12 [teratology screen test] Rat Harlan Wistar 0, 20, 100, 500 mg/kg diet. fetal examination Weil & Carpenter (6 per group) Gestation days 1-7, 5-15, 1-21 (1965); Weil et al. (1972) Wistar 0, 200, 350 mg/kg body weight orally fetal examination Golbs et al. (1974) (10 per group) 40 mg/kg ip. Variety of gestation days. Table 47 (continued) Species Treatment End-point(s) Reference (number) Sprague-Dawley 0, 1, 10, 100 mg/kg body weight fetal examination Lechner & (6 or 7 per group) orally. 3 months before and during Abdel-Rahman gestation (1984) Sprague-Dawley 0, 20, 37.5 mg/kg body weight fetal examination Hart (1972) (22 or 23 per group) per day. Gestation day 6-15 [doses too low] Rats unknown dose (1/50 LD50). fetal examination Dinerman et al. Gestation days 9, 11, or 13 (1970) [insufficient data] Rats 0, 10.6, 106 mg/kg body weight fetal examination Shtenberg et al. per day. Gestation day 1-20 (1973) [insufficient data] Guinea-pigs HRA/HART 0, 50, 100, 200 mg/kg body fetal examination Weil et al. (1973) (9-4 per group) weight per day orally. 0, 100, 200, 300 mg/kg body weight per day in the diet Coulston strain 300 mg/kg body weight per day. fetal examination Robens (1969) (26 gestation day treatment Gestation day 11-20 and a variety [insufficient data] 11-20; other treatments of other treatment periods within unknown) this time Table 47 (continued) Species Treatment End-point(s) Reference (number) Rabbit New Zealand White 0, 150, 200 mg/kg body weight fetal examination Murray et al. (1979) (15-20 per group) per day orally. Gestation day 6-18 New Zealand White 0, 10, 30 mg/kg body weight per fetal examination Shaffer & Levy (9-12 per group) day. Gestation day 9-16 (1968) [doses too low] New Zealand White 0, 50, 100, 200 mg/kg body fetal examination Robens (1969) [small (4-9 per group) weight per day orally. Gestation number of animals] day 5-15 Dog Beagle 0, 3.125, 6.25, 12.5, 25, examination of pups Smalley et al. (1968) (6-13 per group) 50 mg/kg per day in the diet throughout gestation Beagle 0, 2.0, 5.0, 12.5 mg/kg per day in examination of pups Imming et al. (1969) (7-9 per group) the diet throughout gestation Pig Hormel-Hanford 0, 4, 8, 16 mg/kg in the diet 20 examination of fetuses (I) Earl et al. (1973) (5-16 per group) days before/7 days after breeding or piglets after birth (II) throughout gestation Table 47 (continued) Species Treatment End-point(s) Reference (number) Pig (30) up to 30 mg/kg body weight per ? Smalley (1968) day [insufficient data] Monkey Rhesus 0, 2, 20 mg/kg body weight per examination of gestational Dougherty et al. (4-6 per group) day orally throughout gestation course and new born (1971) Rhesus 0, 20, 32 mg/kg body weight per examination of gestational Coulston et al. (1974) (15-16 group) day orally. Gestation day 20-38 course and new born Sheep (25 and 26 per group) 159, 297.5 mg/kg diet examination of lambs Panciera (1967) after birth [significance of defects impossible to assess] Hamster Golden Syrian 125 mg/kg body weight per day fetal examination Robens (1969) (6 or 8 per group) on gestation day 6-8; 250 mg/kg [number tested too body weight per day on gestation small] day 7 or 8 Table 48. Non-mammalian studies Species Dosage End-points Reference Fish Medaka 0.5, 1.0, 2.5, 5.0, 10.0, and embryonic development Solomon & Weis (1979) (Oryzias latipes) 20.0 mg/litre, 4-cell through 10 eggs per group blastula Amphibian Xenopus laevis 0.1, 1.0, 10.0 mg/litre; embryos to embryonic development, Elliott-Feeley & Armstrong 10-12 per group hatching or tadpoles for 24 h posthatching activity (1982) Birds Chicken 0, 0.01, 0.1, 1.0, 10.0 mg/kg embryonic development Olefir & Vinogradova (1968) 4-6 per group body weight White Leghorn chicken 0, 250, 500 mg/kg diet to pullets egg production, embryonic Lillie (1972) 20 per group for 36 weeks and hatchlings for development, hatchability, 4 weeks posthatching development White Leghorn chicken 0, 1.0, 2.5, 5.0, and 10.0 mg/egg embryonic development Swartz (1981) 38-40 per group embryonic development injected into yolk sac after fertilization, prior to incubation for 5 or 12 days White Leghorn chicken 10 mg/egg after fertilization prior primordial germ cell Swartz (1985) 8 per group to incubation migration White Leghorn chicken 1.0, 0.3 mg/egg and lower examination of embryos Eto et al. (1980) [insufficient data] Table 48 (continued) Species Dosage End-points Reference 720 eggs 0, 1, 2, and 4 mg/egg examination of hatchlings Ghadiri & Greenwood (1966) [insufficient data] Duck and chicken 10-1000 µg/egg 0, 4, 7, 10, and examination of embryos or Khera (1966) 13 day eggs hatchlings [insufficient data] Chicken - examination of embryos Dinerman et al. (1970) [insufficient data] Quail Coturnix coturnix 0, 50, 150, 300, 600, 900, growth, reproduction, Bursian & Edens (1977) japonica 1200 mg/kg diet from hatching post-hatching viability 10 per group through 14 weeks Colinus virginianus 0, 300, 1000, 3000 mg/kg diet for reproduction, post-hatching Fletcher & Leonard (1986a) 36 per group 22 weeks viability, gross pathology Duck Anas platyrhyncos 0, 300, 1000, 3000 mg/kg diet for reproduction, post-hatching Fletcher & Leonard(1986b) 36 per group 22 weeks viability, gross pathology 10-1000 µg/egg 0, 4, 7, 10, and examination of embryos or Khera (1966) 13 days eggs hatchlings [insufficient data] 8.5.1 Mammalian reproductive toxicity studies 126.96.36.199 Mouse Studies on the effects of carbaryl (8.5-34 mg/kg per day), given orally for 5 days, to mature male Swiss Webster mice, indicated no effects on the weights of testes and accessory sex glands or uptake of C14-labelled testosterone by the prostate gland, as measured on the day after treatment (Thomas et al., 1974). These workers also examined the biotransformation of testosterone-1,2-3H in animals given 34 or 68 mg/kg per day, orally, for 5 days. They found a significant decrease in androstenedione synthesis in the high-dose group, indicating increased hepatic androgen hydroxylase activity (Dieringer & Thomas, 1974). 188.8.131.52 Rat Collins et al. (1971) studied the effects of carbaryl given in the diet (0, 2000, 5000, and 10 000 mg/kg) to Osborn-Mendel rats over 3 generations. The doses actually administered could only be estimated since there were no measurements of food intake. On the basis of a 15 g/day food intake and a 235 g rat, it can be estimated that animals received of the order of 125, 250, or 500 mg/kg per day. It should be noted, however, that the food intake of lactating rats greatly increases, so these figures may considerably under-estimate carbaryl exposure during this critical time period. The author used the number of animals mated rather than the number giving birth as the number for litter size and viability, therefore their calculations are overestimates. Nevertheless, the data do show impaired fertility in the high-dose group (which also exhibited growth rate reduction) as well as reduced postnatal survival. The number of live-born offspring and growth rate were reduced in the 5000 and 10 000 mg/kg diet groups. A three-generation study on Wistar rats was carried out by Weil et al. (1973) in which animals were given 0, 7, 25, 100, or 200 mg/kg per day in the diet or 0, 3, 7, 25, or 100 mg/kg per day orally. Maternal toxicity was seen in the dietary study at 200 mg/kg per day (decreased weight) and in the gavage study at 100 mg/kg per day (decreased weight and mortality). Postnatal toxicity was noted in the 100 mg gavage group (reduced litter size and viability), but not in lower dose groups. The dietary study indicated fewer effects in the maternal animals with only an initial loss in weight. No perinatal effects were noted. Vashakidze (1975) exposed male and female rats (number and strain unspecified) to 0, 1, 5, 10, 20, 40, and 50 mg carbaryl/kg, orally, for 1 month. Dose-related changes were noted in serum albumin (decrease), globulins (increase), ChE and AChE (decrease), aspartate transaminase (increase), and alanine transaminase (decrease). Reductions in stem cells as well as spermatozoa were noted at doses of 5 mg/kg or more. Adverse litter effects were seen in treated females. These effects included increased embryo/fetal death, decreased implantations, and prolonged estrus cycle. Luca & Balan (1987) administered carbaryl-beta-naphthol to Wistar rats in the diet for up to 18 months. The treated groups showed an increase in sperm shape abnormalities, though there were no clear dose- or time-relationships with effects. 184.108.40.206 Gerbil Collins et al. (1971) published the results of a 3-generation reproduction study in which animals were exposed to 0, 2000, 4000, 6000, or 10 000 mg/kg diet. Since all postnatal calculations used the number of animals mated rather than the number giving birth, the authors' data must be recalculated. When this is done, the magnitude of the effects reported is reduced. Adverse effects on various reproductive parameters are nevertheless seen, though the effects are not clearly related to dose levels in the 2000-6000 mg/kg diet groups. These data are difficult to interpret given the lack of information on maternal effects at doses other than the highest, where mortality was observed. 8.5.2 Mammalian developmental toxicity studies 220.127.116.11 Mouse Murray et al. (1979) administered 100 or 150 mg carbaryl/kg per day, by gavage, or 5660 mg/kg diet (calculated to be 1166 mg/kg per day) to CF-1 mice on gestation days (g.d.) 6-15. Maternal toxicity was noted in the 150-mg group (ataxia, lethality). Litter effects were noted in the 150 mg/kg per day group, where an increase in entirely resorbed litters was seen, and in the dietary group where there was decreased fetal weight. Pregnant mice were given 0, 100, 150, or 200 mg carbaryl/kg per day, orally, on gestation days 8, 12, or 6-15, and fetuses were examined at term (Mathur & Bhatnagar, 1991). Maternal death was noted at the high dose in the group receiving carbaryl on gestation days 6-15. Fetal weight reductions were seen at the high-dose levels in all groups, as was reduced ossification, open eyelids, and enlarged renal pelvis. These effects may be indicative of fetal growth retardation. 18.104.22.168 Rat Carbaryl was administered to Harlan Wistar rats at 0, 20, 100, or 500 mg/kg per day, orally, on gestation days 1-7, 5-15, or 1-21 (Weil & Carpenter, 1965; Weil et al., 1972). Maternal toxicity was evident as was reduced weight gain in the high-dose groups receiving the chemical on gestation days 5-15 or 1-2. No adverse fetal effects were seen. Golbs et al.(1975) treated Wistar rats with 0, 200, or 350 mg carbaryl/kg, orally, or 40 mg/kg ip on a variety of single or multiple gestation days. Reductions in fetal weight were seen in some groups. No other effects were noted. Sprague-Dawley rats were treated orally with 0, 1, 10, or 100 mg carbaryl/kg per day, three months before and during gestation, and litters were examined at term (Lechner & Abdel-Rahman, 1984). Maternal weight gain was significantly less in the 100 mg/kg group than in controls. No compound-related effects on fetuses were noted. 22.214.171.124 Guinea-pig Weil et al. (1973) administered carbaryl to guinea-pigs at dose levels of 0, 100, 200, or 300 mg/kg per day in the diet, or 0, 50, 100, or 200 mg/kg per day, orally. These dose levels were determined to be maximum non-maternally toxic doses in preliminary studies by the authors, though the 200 mg/kg dose did reduce maternal weight gain. No significant adverse embryo/fetal effects were seen in any treatment groups. 126.96.36.199 Rabbit Murray et al. (1979) tested the effects of carbaryl on New Zealand White rabbits during gestation. Dams had diarrhoea at the high dose of 200 mg/kg per day and animals at this dose level as well as at the lower dose level (150 mg/kg per day) gained less weight during gestation than controls. A significant increase in umbilical hernia was noted in fetuses at the 200 mg/kg per day group. 188.8.131.52 Dog Smalley et al. (1968) administered 3.125, 6.25, 12.5, 25, or 50 mg carbaryl/kg in the diet, throughout gestation, to beagle dogs. Maternal toxicity was noted at all dose levels. This toxicity was described by the authors as dystocia and symptoms included delayed delivery, anorexia, and restlessness. A variety of birth defects were found at doses of 6.25 mg/kg or more. The defects included ectopic intestines, brachygnathia, acaudia, polydactyly, and intestinal agenesis. Another study was carried out by Imming et al. (1969) on the beagle dog. These workers used 0, 2.0, 5.0 or 12.5 mg/kg per day, orally, throughout gestation. As in the Smalley (1968) study, treated females exhibited toxicity during labour and single deaths were recorded at this time in all treated groups. Birth defects including umbilical hernia and gastrointestinal defects were seen at the 5.0 and 12.5 mg dose levels only. 184.108.40.206 Pig Two studies on pigs were carried out by Earl et al. (1973). In the first study, fetal pigs were examined from dams given 0, 4, 8, or 16 mg carbaryl/kg per day in the diet; in the second, dams were allowed to farrow after receiving 0, 16, or 32 mg/kg per day in the diet. Animals were exposed throughout most or all of gestation. Effects noted were not consistent across the studies and increased prenatal lethality seen in the first was not noted in the second, even at a higher dose level. A small number of malformations were noted, but no characteristic dose-related pattern was evident. 220.127.116.11 Monkey Dougherty et al. (1971) exposed a small number of female Rhesus monkeys to 0, 2, or 20 mg carbaryl/kg, throughout gestation. Of the 8 pregnant, treated females, 5 were reported as having aborted as opposed only 1 out of 5 in the controls. The Coulston et al. (1974) study included a larger number of animals per dose, an additional lower dose level (0.2 mg/kg per day), and a shorter dosage period (days 20-38 of gestation). No adverse effects were noted in the second study. These studies are not comparable for the evaluation of the abortifacient potential of carbaryl, since the dosing began later in gestation in the second study. The authors noted that the determination of abortion in the Rhesus monkeys may not have been reliable. 8.5.3 Reproductive and developmental toxicity studies in non-mammalian species 18.104.22.168 Fish Medaka ( Oryzias latipes) embryos were exposed to nominal carbaryl concentrations of 0.5, 1.0, 2.5, 5.0, 10.0, 20.0, or 30.0 mg/litre in the water from the 4-cell through to the blastula stages (Solomon & Weis, 1979). Exposure of the eggs resulted in increased cardiovascular anomalies (heart defects, circulatory defects, and edema) at dose levels of 5 mg/litre or more. 22.214.171.124 Amphibian Elliott-Feeley & Armstrong (1982) treated both embryos and tadpoles of Xenopus laevis with nominal carbaryl concentrations in the water of 0.1, 1.0, or 10.0 mg/litre. Defects were seen in embryos exposed to 10 mg/litre, which also resulted in significant lethality. Embryo growth was reduced at all dose levels. Carbaryl-exposed tadpoles exhibited decreased activity at all dose levels. 126.96.36.199 Birds Chicken eggs have been used by a number of workers to test the potential of carbaryl to affect avian development. Olefir & Vinogradova (1968) injected eggs with carbaryl at 0.01, 0.1, 1.0, or 10.0 mg/kg and examined embryos at different times after injection. Death and anomalies were recorded at 5, 10, 15, and 20 days of incubation at all doses above 0.01 mg/kg. Lillie (1973) administered 0, 250, or 500 mg/kg diet to pullets for 36 weeks beginning at 32 weeks of age. The adult birds showed reduced weights at both levels of carbaryl in the adults and at 500 mg in the progeny. No embryotoxicity or other effects were noted. Swartz (1981) examined the effects of carbaryl on chicken embryo development. They examined chick embryos at different days after injection, for 5 or 12 days post-fertilization. They found vehicle-related increased mortality (carbaryl was more toxic when administered in sesame oil than in acetone) for both periods of exposure. Scattered anomalies were recorded in surviving embryos. In another study (Swartz, 1985) primordial germ cell migration was followed after injection of 10 mg carbaryl per egg prior to incubation. Results did not indicate any significant adverse effects on the primordial germ cells or reproductive organs. Bursian & Edens (1977) studied the effects of carbaryl on the fertility and post hatching viability of Japanese quail ( Coturnix coturnix japonica). Birds were exposed from hatching to 14 weeks of age (breeding maturity). Fertility and the hatchability of eggs were measured. The animals received 0, 50, 150, 300, 600, 900, or 1200 mg carbaryl/kg diet. Growth of the adult birds was reduced at the 900 and 1200 mg/kg dose levels. There were no significant effects on any reproductive parameter. Fletcher & Leonard (1986a) investigated the effects of carbaryl on reproduction in bobwhite quail ( Colinus virginianus) exposed to 0, 300, 1000, or 3000 mg/kg diet for 22 weeks. No adverse effects were described in any factors related to hatchability, posthatching viability, or gross pathology of newly hatched birds. These workers used a similar protocol to study the effects of carbaryl on mallard ducks ( Anas platyrhyncos) (Fletcher & Leonard, 1986b). The 3000 mg/kg diet level was toxic with some lethality, decreased numbers of eggs, and thinner egg shells. No effects were seen at the 300 or 1000 mg/kg diet levels. 8.5.4 Appraisal In summary, mammalian studies on the reproductive or developmental toxicity of carbaryl clearly show that this compound is capable of inducing adverse effects in utero and during the reproductive process. These effects are always seen only at dose levels at which there is concurrent maternal toxicity, with the possible exception of a few studies on the rat which have not been replicated by other workers. For a number of species, the dams appear to be more sensitive than their litters. In general, the adverse effects noted in developmental toxicology studies cannot be simply attributed to maternal toxicity (Chernoff et al., 1990). However, the pattern of maternal and fetal toxicity occurring at the same dose levels indicates that the developing mammalian embryo/fetus is not especially susceptible to carbaryl. Carbaryl has been shown to be embryotoxic for fish, amphibians, and birds, at some exposure concentrations. 8.6 Mutagenicity of carbaryl and N-nitrosocarbaryl In this section on mutagenicity, and in the section on carcinogenicity (8.7.1), the two compounds, carbaryl and nitrosocarbaryl, are discussed (separately), since the formation of N-nitrosocarbaryl was reported to occur in the stomach of rats and guinea-pigs, in the presence of sodium nitrite and carbaryl, under acid conditions (Elespuru & Lijinsky, 1973; Beraud et al., 1979; Rickard & Dorough, 1984). See also section 8.9.3. 8.6.1 Genotoxicity assays in vitro 188.8.131.52 Primary DNA damage (a) Carbaryl Carbaryl did not cause DNA damage in different wild type strains and DNA recombination lacking strains of Bacillus subtilis (Table 49). Carbaryl was reported to be non-genotoxic for B. subtilis Marburg 17A Rec+ and Marburg M45T Rec- strains, highly sensitive to frameshift mutagens, even at the highest tested concentration of 10 mg/plate (Uchiyama et al., 1975). Carbaryl (10-4 mmol/litre) did not affect the sedimentation profiles of DNA from human skin cells in culture (both normal and xeroderma pigmentosum), either immediately or 20 h after treatment (Regan et al., 1976). There are two controversial reports on the genotoxicity of carbaryl, evaluated by the induction of mitotic gene conversion in the diploid strain of Saccharomyces cerevisiae, heteroallelic at the two different loci ade2 and trp5. Siebert & Eisenbrand (1974) used an assay system with a 16-h incubation time and a concentration of carbaryl of 4.97 mmol/litre and did not observe any changes in the control frequency of mitotic gene conversion. However, Jaszczuk & Syrowatka (1979), reported a weak positive response with a lower concentration of carbaryl (2.5 mmol/litre) and shorter (5 h) incubation time. No converting activity of N-hydroxy carbaryl (2.5 mmol/litre) was found under the same assay conditions. Table 49. Bacterial assays of genetic toxicity of carbaryl Test Test organism Concentration Genetic Metabolic Result Reference (µg/plate) end-point activation DNA B. subtilis 700 mg/litre Rec assay - negative DeGiovanni-Donnelly et al. (1968) damage B. subtilis 20 Rec assay - negative Shirasu et al. (1976) B. subtilis 0.4, 4, 40, 400 Rec assay - negative Eto et al. (1982) Gene B. subtilis up to 10 000 Rec assay - negative Uchiyama et al. (1975) mutation E. Coli WP2 1000 Try- - negative Ashwood-Smith et al. (1972) E. Coli WP2 1000-3000 Try- - negative Nagy et al. (1975) E. Coli WP2 10 000 Try- - negative Uchiyama et al. (1975) H. influenzae 10 µmol/litre Novobiocim - negative Elespuru et al. (1974) S. typhimurium TA 98 up to 2000 His- - negative McCann et al. (1975) TA 98 up to 2000 His- Rat negative TA 98 50 nmol/litre His- - negative Blevins et al. (1977) TA 98 10-1500 His- - negative DeLorenzo et al. (1978) Table 49 (continued) Test Test organism Concentration Genetic Metabolic Result Reference (µg/plate) end-point activation TA 98 10-1500 His- Rat negative TA 98 0.25, 2, 5, 50, 1000 His- - negative Jaszczuk et al. (1979) TA 98 up to 5000 His- - negative Moriya et al. (1983) TA 98 up to 5000 His- Rat negative TA 98 0.2, 2, 20 His- - negative Eto et al. (1982) TA 98 0.2, 2, 20 His- Rat negative TA 98 5-2000 His- - negative Lawlor (1989) TA 98 5-2000 His- Rat negative TA 100 up to 2000 His- - negative McCann et al. (1975) TA 100 up to 2000 His- Rat negative TA 100 50 nmol/litre His- - negative Blevins et al. (1977) TA 100 10-1500 His- - negative DeLorenzo et al. (1978) TA 100 10-1500 His- Rat negative TA 100 0.25, 2, 5, 50, 1000 His- - negative Jaszczuk et al. (1979) TA 100 0.2, 2, 20 His- - negative Eto et al. (1982) Table 49 (continued) Test Test organism Concentration Genetic Metabolic Result Reference (µg/plate) end-point activation TA 100 0.2, 2, 20 His- Rat negative TA 100 5-2000 His- - negative Lawlor (1989) TA 100 5-2000 His- Rat negative TA 1535 up to 2000 His- - negative McCann et al. (1975) TA 1535 up to 2000 His- Rat negative TA 1535 2500 His- - positive Marshall et al. (1976) TA 1535 1000 His- Rat negative TA 1535 50 nmol/litre His- - negative Blevins et al. (1977) TA 1535 10-1500 His- - negative DeLorenzo et al. (1978) TA 1535 10-1500 His- Rat negative TA 1535 up to 5000 His- - negative Moriya et al. (1983) TA 1535 up to 5000 His- Rat negative TA 1535 5-2000 His- - negative Lawlor (1989) TA 1535 5-2000 His- Rat negative Table 49 (continued) Test Test organism Concentration Genetic Metabolic Result Reference (µg/plate) end-point activation TA 1536 2500 His- - negative Marshall et al. (1976) TA 1536 1000 His- Rat negative TA 1537 up to 2000 His- - negative McCann et al. (1975) TA 1537 up to 2000 His- Rat negative TA 1537 50 nmol/litre His- - negative Blevins et al. (1977) TA 1537 2500 His- - negative Marshall et al. (1976) TA 1537 1000 His- Rat negative TA 1537 10-1500 His- - negative DeLorenzo et al. (1978) TA 1537 10-1500 His- Rat negative TA 1537 0.25, 2, 5, 50, 1000 His- - negative Jaszczuk et al. (1979) TA 1537 up to 5000 His- - negative Moriya et al. (1983) TA 1537 up to 5000 His- Rat negative TA 1537 5-2000 His- - negative Lawlor (1989) TA 1537 5-2000 His- Rat negative TA 1538 100 His- - positive Egert & Greim (1976) Table 49 (continued) Test Test organism Concentration Genetic Metabolic Result Reference (µg/plate) end-point activation TA 1538 100 His- Mouse positive TA 1538 50 nmol/litre His- - negative Blevins et al. (1977) TA 1538 2500 His- - negative Marshall et al. (1976) TA 1538 1000 His- Rat negative TA 1538 10-1500 His- - negative DeLorenzo et al. (1978) TA 1538 10-1500 His- Rat negative TA 1538 up to 5000 His- - negative Moriya et al. (1983) TA 1538 up to 5000 His- Rat negative TA 1538 5-2000 His- - negative Lawlor (1989) TA 1538 5-2000 His- Rat negative Carbaryl was inactive in the rat primary hepatocytes unscheduled DNA synthesis assay (Cifone, 1989). It did not induce significant changes in the nuclear labelling of rat primary hepatocytes in two independent trials with applied concentrations ranging from 5 to 25 µg/ml. No DNA-damaging properties of carbaryl, assessed by its capacity for inducing unscheduled DNA synthesis (UDS) in cultured human lymphocytes, were reported by Rocchi et al. (1980). However, the authors did not use a standardized test protocol meeting established criteria for the UDS assay performance. They tested only one concentration of carbaryl (50 µg/ml), did not use any metabolic activation system, did not run negative and adequate positive controls concomitantly (other than the ultraviolet irradiation), and did not apply relevant criteria to assess data. The lack of DNA-damaging properties of carbaryl, as measured by the induction of UDS, was confirmed by Probst et al. (1981). They used a standardized protocol and autoradiographic techniques in metabolically competent, cultured rat hepatocytes with test concentrations ranging from 0.5 to 1000 nmol/ml. In contrast, Ahmed et al. (1977a) reported that carbaryl induced the UDS of the ultraviolet type (long patch repair) in a cultured human fibroblast VA-4 cell line with, and without, metabolic activation in concentration ranges of 1, 10, 100, and 1000 µmol/litre. However, this study had several experimental shortcomings. The protocol, the control value, and the results of compounds tested were not in accordance with comparable assays by other authors. (b) N-nitrosocarbaryl The DNA-damaging properties of N-nitrosocarbaryl in B. subtilis have been reported (Table 50). In one study (Uchiyama et al., 1975), more pronounced genotoxicity of N-nitrosocarbaryl to B. subtilis was registered compared with that of N-methyl- N'-nitro- N-nitrosoguanadine. However, the genotoxicity of N-nitrosocarbaryl was the lowest when compared with other concomitantly tested nitrocarbamates. In contrast to carbaryl, N-nitrosocarbaryl showed pronounced genotoxicity toward S. cerevisiae D4 (Siebert & Eisenbrand, 1974). N-nitrosocarbaryl, but not carbaryl, reacted with human DNA in cell culture to form alkaline-sensitive bonds (Regan et al., 1976). The DNA of nitrosocarbaryl-treated (10-4 mmol/litre) cells showed a substantial reduction in sedimentation rate immediately, and up to 20 h, after treatment. Presumably, the effect observed was related to the induction of numerous single-strand breaks in the DNA and the formation of DNA adducts. On the basis of selective labelling, the authors suggested that the methyl-containing moiety of nitroso-carbaryl was separated from the naphthalene ring in a human fibro-blast culture and bound irreversibly to DNA (Regan et al., 1976). 184.108.40.206 Gene mutation assay (a) Carbaryl As summarized in Table 49, carbaryl did not exert a mutagenic effect in studies with E. coli, H. influenzae, or Salmonella. Among the many reports on Salmonella, only two indicated a positive effect. Thus, Marshall et al. (1976) observed increased mutagenicity at 1000 µg/plate with S9 mix for TA1535. An evaluation of this data is difficult because no negative and positive control data were presented. Egert & Greim (1976) reported a positive response for TA1538 by 100 µg/plate. The mutagenicity of carbaryl in this study greatly increased when a non-standard metabolic activation system (mouse liver microsome) was used. Ahmed et al. (1977b) reported a positive mutagenic response to carbaryl in Chinese hamster V79 cells at a dose level of 0.01 mmol/litre, with no metabolic activation. There was a concentration-related effect of carbaryl on cell survival; less than 50% of the cells survived at concentrations >0.01 mmol/litre. Mutation studies carried out with a concentration of 0.01 mmol/litre showed an approximately 8-fold increase in ouabain resistance forward mutation in V79 cells as compared with the spontaneous mutation rate. However, Wojciechowski et al. (1982) found no ouabain-resistance with carbaryl in a cell-mediated mutagenesis assay when they used an exogenous activating system in which irradiated fetal cells of Syrian hamsters were co-cultivated with cells of Chinese hamsters V79. No mutagenic response was observed for carbaryl at concentrations of 0.01, 0.05, 0.1 mmol/litre with, or without metabolic activation. The toxicity of carbaryl at these concentrations ranged from 7% at the lowest concentration to 23% at the highest. Carbaryl produced a negative result for inducing forward mutations at the HPRT locus in Chinese hamster ovary cells both with, and without, metabolic activation (Young, 1990). Table 50. Bacterial assays of the genetic toxicity of nitrosocarbaryl Test Test organism Concentration Genetic Metabolic Result Reference (µg/plate) end-point activation DNA B. subtilis 0.4, 4, 40 Rec assay - positive Eto et al. (1982) damage B. subtilis up to 100 Rec assay - positive Uchiyama et al. (1975) Gene mutation E. Coli 30 R 0.1 mmol/litre Try- - positive Elespuru et al. (1974) E. Coli WP2 5, 10, 50, 100 Try- - positive Uchiyama et al. (1975) E. Coli K12 100 Try- - positive Egert & Greim (1976) E. Coli K12 100 Try- Mouse positive H. influenzae 10 µmol/litre Novobiocin - positive Elespuru et al. (1974) S. typhimurium TA 98 0.001-11 His- - positive/ Blevins et al. (1977) negative TA 9 10-1500 His- - negative DeLorenzo et al. (1978) TA 98 10-1500 His- Rat negative TA 98 0.25-1000 His- - positive Jaszczuk et al. (1979) Table 50 (continued) Test Test organism Concentration Genetic Metabolic Result Reference (µg/plate) end-point activation TA 98 0.02, 0.2, 2 His- - negative Eto et al. (1982) TA 98 0.02, 0.2, 2 His- Rat negative TA 98 0.01-100 His- - negative Rickard et al. (1982) TA 98 0.1-100 His- Rat negative TA 100 0.001-11 His- - positive Blevins et al. (1977) TA 100 0.02, 0.2, 2 His- - positive Eto et al. (1982) TA 100 0.02, 0.2, 2 His- Rat negative TA 100 0.1-100 His- - positive Rickard et al. (1982) TA 100 0.1-100 His- Rat positive TA 1535 0.5-100 His- - positive Marshall et al. (1976) TA 1535 50-1000 His- Rat positive TA 1535 0.001-11 His- - positive Blevins et al. (1977) TA 1535 0.25-1000 His- - positive Jaszczuk et al. (1979) TA 1535 0.1-100 His- - positive Rickard et al. (1982) TA 1535 0.1-100 His- Rat positive Table 50 (continued) Test Test organism Concentration Genetic Metabolic Result Reference (µg/plate) end-point activation TA 1536 0.5-100 His- - negative Marshall et al. (1976) TA 1536 50-1000 His- Rat negative TA 1537 0.001-11 His- - negative Blevins et al. (1977) TA 1537 0.5-100 His- - positive Marshall et al. (1976) TA 1537 50-1000 His- Rat positive TA 1537 0.25-1000 His- - positive Jaszczuk et al. (1979) TA 1538 100 His- - positive Egert & Greim (1976) TA 1538 100 His- Rat positive TA 1538 0.001-11 His- - negative Blevins et al. (1977) TA 1538 0.5-100 His- - positive Marshall et al. (1976) TA 1538 50-1000 His- Rat negative TA 1538 0.25-1000 His- - negative Jaszczuk et al. (1979) (b) N-nitrosocarbaryl N-nitrosocarbaryl has shown a positive response in a number of bacterial assays (Table 50). In Salmonella, N-nitrosocarbaryl was mutagenic to strains responding to both base substitution (TA1535, TA100) and frame shift (TA1536, TA1537, TA1538, TA98). The mutagenic potential of N-nitrosocarbaryl toward Salmonella TA1535 was greatly diminished by the added exogenous activation system (Marshall et al., 1976). A decrease in mutagenic activity by the addition of S9 mix on frame shift mutagenesis has also been reported (Marshall et al., 1976; Jaszczuk et al., 1979). 220.127.116.11 Chromosomal aberration assays and sister chromatid exchange (a) Carbaryl Animals Carbaryl induced a clastogenic response in the three in vitro bioassays (Ishidate & Odashima, 1977; Kazarnovskaya & Vasilos, 1977; Önfelt & Klasterska, 1983). All the positive responses were observed at toxic dose levels (30-80 µg/ml; 50, 100 µmol/litre); exogenous metabolic activation was not required for activity. Thus, Ishidate & Odashima (1977) observed a strong positive response for carbaryl (30 µg/ml) in a chromosomal aberration assay using a Chinese hamster fibroblast cell line. Carbaryl produced predominantly chromatid type gaps, breaks, translocations, rings, and fragmentation, 48 h after treatment. Carbaryl was negative for inducing chromosomal aberrations in CHO cells without metabolic activation but was positive under metabolic activation conditions (Murli, 1989). Kazarnovskaya & Vasilos (1977) reported a positive response for carbaryl in cultures of human embryonic fibroblast: levels of 40 and 80 µg/ml caused a 4-fold and 10-fold increase, respectively, in the frequency of chromosomal aberrations, at 24 h exposure time, compared with a control rate of 2% (without S9-mix). Again, the aberrations produced were mainly of a chromatid type; the type most frequently observed was single fragments. An increased number of paired fragments was found only at 80 µg carbaryl/ml. Carbaryl (80 µg/ml) increased the percentage of cells with chromosomal coiling (9.8% in the control group; 17.9% in the test group) and aneuploidy (3% in the control group; 29.2% in the test group). Previously, Kazarnovskaya & Vasilos (1977) had shown that carbaryl suppressed mitosis, changed the rate of the mitotic phase, and significantly increased the number of pathological forms of mitosis in a human embryonic fibroblast culture, with a dose-time response. Onfelt & Klasterska (1983) observed induction of viable aneuploid/polyploid cells and multiple chromatid exchanges after treatment of V79 Chinese hamster cells with carbaryl. The compound exerted a pronounced chromosome-breaking effect, at a concentration of 100 µmol/litre, 26 and 50 h after treatment. There was an increased frequency of multiple chromatid exchanges and fragments, as well as pulverisation and more diffuse signs of chromosome damage. The effects of carbaryl on chromosome structure and distribution were almost abolished by the simultaneous addition of Aroclor-induced 2 or 10% rat S9-mix and glutathion. In two in vitro assays for chromosome damage using Chinese hamster lung fibroblasts, carbaryl was found to be positive (Ishidate et al., 1981; Onfelt & Klasterska, 1984). In the latter study, the effect of carbaryl on the incidence of sister chromatid exchange was decreased by the addition of rat liver microsomes. Söderpalm & Önfelt (1988) related the mitotic aberrations in V79 Chinese hamster cells, in part, to a reduction in the intracellular levels of glutathione, and increased lipid peroxidation. They also hypothesized that the anticholinergic effects of carbaryl may play a role in the cleavage process. Plants A negative clastogenic response for carbaryl in a concentration range of 50-200 mg/litre was reported by Ma et al. (1984) who used the Tradescantia micronucleus test. At present, this test is considered the most established and standardized plant assay system for the purposes of in situ monitoring of environmental mutagen pollution (Ma et al., 1984). Carbaryl induced chromosomal effects in different plant assays (Wuu & Grant, 1966; Amer & Farah, 1968; Brankovan, 1972). Carbaryl increased by approximately 10-fold the number of aberrant cells in the root tips of barley seedings ( Hordeum vulgare) in C1 and C2 generations after the seeds were treated with concentrations of 500, 1000, or 1500 mg carbaryl/litre for 6 and 12 h. The cytogenetic effects induced included mostly metaphase and anaphase fragments and anaphase bridges and were time-dependent (Wuu & Grant, 1966). The mitotic, cytogenetic effects of carbaryl were seen in Vicia faba (Amer & Farah, 1968) and sugar corn (Brankovan, 1972). Carbaryl caused chromosomal abnormalities (chromosome lagging; stickiness, mainly in diakinesis, polyploidy, fragments, anaphase bridges) in different meiotic states in the pollen mother cells of Vicia faba after spraying flower buds of different ages (2 weeks, 1 month) with saturated aqueous solutions of a commercial carbaryl preparation. The percentage of the chromosomal aberrations increased with increase in the frequency of spraying (every week) or 2 weeks for l month; daily for 8 days) and then decreased when the recovery time was increased (Amer & Farah, 1968). 8.6.2 Genotoxicity in vivo 18.104.22.168 Host-mediated assay (a) Carbaryl Usha Rani et al. (1980) reported that carbaryl did not have a gene-mutation potential in vivo, when given orally in a toxic dose of 438 mg/kg, 3 times daily for 3 days, to 6 Swiss male mice, which were subsequently injected with Salmonella strain G46. These data are consistent with those that showed that carbaryl was not mutagenic for Salmonella in vitro. 22.214.171.124 Drosophila melanogaster and other insects (a) Carbaryl There are several reports of bioassays for carbaryl genotoxicity in which Drosophila melanogaster was used (Brzeskii, 1972; Brzeskii & Vaskov, 1972; Hoque, 1972; Woodruff et al., 1983; Omer et al., 1986). In studies by Hoque (1972), carbaryl at 1.5 and 10 mg/litre was given to female Drosophila. It was reported that the treatment caused a changed sex ratio, changes in eye colour, and various chromosomal aberrations in the offspring. The small amounts of material used and the lack of any detailed presentation of the findings preclude any evaluation or conclusion. At high doses (0.3 ml from a 1% suspension of 85% commercial product in glucose), carbaryl caused a slight increase in mutation frequency in the F1 generation males of a Drosophila line, which were studied at different stages of spermatogenesis. There were no deletions or disturbed fertility (Brzeskii, 1972; Brzeskii & Vaskov, 1972). Woodruff et al. (1983), who used a sensitive experimental protocol that incorporated the mating scheme with repair-deficient females, reported that carbaryl was not mutagenic for Drosophila. F1 male progeny of males that had ring X chromosomes and double marked Y chromosomes, were treated with carbaryl at 200 mg/litre and mated with mus-302, repair-deficient females of Drosophila. The male progeny did not show any induced complete (ring chromosome) or partial (Y chromosome markers) chromosome loss. The mutagenic activity of carbaryl in Drosophila melanogaster was studied by Omer et al. (1986). The results indicated that carbaryl does not increase the rate of dominant and sex-linked recessive lethal mutations. Carbaryl did not cause chromosome abnormalities in the meiotic cells of male grasshoppers, when they were given a single toxic dose (not precisely indicated) of 0.25 ml of the supernatant of an aqueous suspension/solution of carbaryl by ip injection; however, there were morphological disturbances in the spermatocytes (Venkat Reddy et al., 1974). (b) N-nitrosocarbaryl Results obtained by Omer et al. (1986) from the nitrosocarbaryl-treated populations of Drosophila melanogaster suggested that nitrosocarbaryl increased significantly the percentage of dominant lethal mutations above the spontaneous mutation frequency. The results obtained from the 0.05, 0.10, and 0.15% nitrosocarbaryl-treated populations also showed a significant increase in the percentages of sex-linked recessive mutations. 126.96.36.199 Chromosomal aberrations and sister chromatid exchange (a) Carbaryl There are several negative chromosomal studies on carbaryl and N-nitrosocarbaryl in somatic and germ cells in vivo in mammals (Venkat Reddy et al., 1974; Degraeve et al., 1976; Seiler, 1977, Usha Rani et al., 1980; Dzwonkowska & Hübner, 1986). In all these studies, sufficiently high dose levels of carbaryl were used (up to the LD50) in order to define the negative cytogenetic responses. Using the micronucleus test and the cytogenetic analysis of metaphase chromosomes, Degreave et al. (1976) found no clastogenic effects in the bone marrow of mice that had been given a single oral dose of carbaryl (0.2 ml), an intraperitoneal dose (0.5 ml) of carbaryl, or 7 oral doses of carbaryl solution (1x10-3 mg/litre) alone, or with sodium nitrite (2x10-3 mg/litre). A negative response for a high dose of carbaryl (146 mg/kg, orally, 2 times per 24 h; 30-h sampling time) was reported by Usha Rani et al. (1980) with an adequate protocol for the mouse bone marrow micronucleous test. No increase in the rate of the micronucleated, polychromatic erythrocytes in the bone marrow of mice was found during in vivo nitrosation of carbaryl after its oral administration at the maximum tolerated dose of 100 mg/kg, together with an excess of sodium nitrite (100 mg/kg) (Seiler, 1977). A dose of 64 mg carbaryl/kg produced a negative result in an in vivo assay of chromosomal aberrations in the bone marrow cells of the Syrian hamster (Dzwonkowska & Hubner, 1986). No chromosome aberrations were observed in the bone marrow of Syrian hamsters (6 per dose), given single intraperitoneal injections of doses up to the LD50 (64, 128, 320, and 640 mg/kg) of a commercial mixture of carbaryl/lindane (40:10) (Dzwonkowska & Hubner, 1986). Daily oral doses of technical carbaryl (10 mg/kg) suspended in peanut oil were given to male, albino rats for a period of 5 days. There were no significant chromosomal changes in the bone marrow cells of the exposed animals (Dikshith, 1991). 188.8.131.52 Dominant lethal assays in rodents (a) Carbaryl Epstein et al. (1972) studied the mutagenicity of carbaryl using the dominant lethal test. In this assay, male ICR/Ha Swiss mice were treated orally with 1000 or 50 mg carbaryl/kg daily for 5 successive days and then caged with 3 untreated virgin females, which were replaced weekly for 8 consecutive weeks. The frequency of early fetal deaths and preimplantation losses in the test groups were within the limits of the control values. Therefore, dominant lethal mutations were not induced in mice given sufficiently high oral doses of carbaryl. 8.6.3 Other end-points 184.108.40.206 Cell transformation (a) Carbaryl Transformation of the fibroblast clone A31 of the BALB/3T3 mouse was not induced by carbaryl when given in non-toxic (1.5 and 10 µg/ml) or moderately cytotoxic (20 and 40 µg/ml) doses, over 24 h (Quarles & Tennant, 1975). (b) N-nitrosocarbaryl N-nitrosocarbaryl showed transforming activity, at concentrations of 10-20 µg/ml, which was cytotoxic for the BALB/3T3, A31 cells. That 3-10 cell passages are necessary for detecting the transforming event suggests that the transformation frequency for N-nitrosocarbaryl in this test system is low. Transformed cells showed morphological alterations, loss of contact inhibition, and growth in soft agar, as well as carcinogenic activity in normal newborn, irradiated, suckling, or athymic BALB/C mice. The tumour incidence (anaplastic sarcomas) was relatively low; in several instances, tumours regressed in normal mice, but not in athymic mice, after 2-3 weeks of growth. N-nitrosocarbaryl did not activate detectable amounts of the endogenous murine leukaemia viruses carried by the BALB/3T3 cells or viral protein. However, viral antigen production in the transformed cells was induced by iododeoxyuridin, which indicated the presence of the viral genome. On the basis of this N-nitrosocarbaryl transforming activity in mammalian cells in culture, Quarles & Tennant (1975) suggested that the compound may be an active, though weak, carcinogen in vivo. 220.127.116.11 Aneuploidy induction (a) Carbaryl Tests of the chemical induction of spindle fibre inactivation and c-mitosis (colchicine-mitosis) in Allium revealed the existence of an unspecific physico/chemical mechanism, based on the partitioning of compounds into hydrophobic compartments of the cell. This means that chemical compounds, in general, cause c-mitosis according to their lipophilic characteristics, as indicated by the octanol/water partition coefficient. Thus, a close correlation exists between the lipophilicity of compounds and the dose at which spindle disturbance occurs. However, besides this unspecific effect on the spindle fibres mechanism, there are some compounds that exhibit specific effects causing inactivation of the spindle fibre mechanism at lower doses than indicated by their lipophilicity. This can occur via different mechanisms, such as specific binding to actin (colchicine) or binding to sulfhydryl groups (organic mercury). Onfelt (1987) analysed the c-mitotic effects of 22 compounds in V79 hamster cells. Five of these compounds fell outside the regression line for lipophilicity/c-mitosis, among them colchicine, methyl mercury, and carbaryl. Further analyses revealed that carbaryl owes its pronounced c-mitotic action to its reactivity with sulfhydryl groups. It is therefore not surprising that several authors have reported mitotic disturbances caused by carbaryl in various experimental systems. Onfelt & Klasterska (1983) reported that a significant increase in the aneuploidy/polyploidy cells was obtained with both 50 and 100 µmol carbaryl/litre, 26, 59, and 74 h after treatment. Carbaryl caused mitotic disturbances in Allium cepa, Vicia faba, Gossypium barbadense, Nigella damascena (Amer, 1965; Amer et al., 1971; Degraeve et al., 1976). Amer (1965) reported c-mitotic effects of carbaryl in the roots of Allium cepa that had been treated for 4 or 24 h with different concentrations of pure (0.5, 0.25%) and commercial products (85% sprayable powder). The increased rate of abnormal meta-telophases and ana-telophases depended on the concentration and temperature of the test solutions. The types of induced abnormal metaphases included star-metaphases and a few prophase-metaphases. Two types of anaphases, the c-type and the multipolar type, as well as multinuclear interphase cells, were observed. Continuous treatment with carbaryl for 24 h nearly arrested mitosis. There was a full recovery of mitosis in 48 h and induced signs of polyploidy appeared. Consistent with these findings are the c-mitotic effects of carbaryl in Vicia faba and Gossypium barbadense (Amer et al., 1971). The mitotic index of Vicia faba, after root treatment, decreased with increased concentrations of carbaryl (25, 50, or 100 mg/litre for 4 h. There was no effect from carbaryl after seed-soaking for different lengths of time. Mitotic anomalies (mostly disturbed meta- and anaphases) in the roots of Vicia faba and Gossypium barbadense showed a concentration-time response. Carbaryl caused mitotic disturbances (c-mitotic effect, multipolar anaphases) or cytotoxicity (pycnotic nuclei, tissue degeneration) after root treatment of Nigella damascena with concentrations of 2.5x10-4 and 1x10-3 mg/litre. When NaNO2 (2x10-3) was added to carbaryl solutions of 1x10-2 and 1x10-3 mg/litre, the effects increased, possibly because of the formation of nitrosocarbaryl (Degreave et al., 1976). No effect was observed after grain treatment. Soaking sugar corn seeds for 48 h with a 0.12 or 0.25% aqueous solution of 50% commercially prepared carbaryl, resulted in a dose-response induction of aberrations of anaphase chromosomes of types not seen in the controls (bridges with, or without, fragments, dicentrics). Repeated treatment of young plants with a 0.25% solution of carbaryl for 6 h during meiosis caused typical c-mitotic effects in metaphase and anaphase through the arrest of cell division and spindle inactivation. Some of the induced aberrations persisted until the generative stage. They were fixed and incorporated through the embryonal and generative development stages and thus increased pollen sterility (Brankovan, 1972). Carbaryl was reported to increase the number of aberrant forms of mitosis (mostly c-mitotic effect) in the small intestine, cornea, and spleen of rats given oral doses of 400 mg/kg. The authors also reported that at lower doses of carbaryl (40 or 80 mg/kg), there was an increased incidence of bridge and chromosome lagging in anaphase and telophase, in addition to a high percentage of c-mitosis. The authors reported that there were no effects at 20 mg/kg. Only the result following the 400 mg/kg dose was fully reported (Vasilos et al., 1975). 8.6.4 Appraisal Carbaryl has been evaluated for its potential mutagenicity in a number of tests in vitro as well as in vivo, in bacterial, yeast, plant, insect, and mammalian systems, testing a variety of end-points. The available evidence indicates that carbaryl has no DNA-damaging properties. No confirmed induction of mitotic recombination, gene conversion, and UDS in prokaryotes ( H. influenzae, B. subtilis) and eukaryotes ( S. cerevisiae, A. nidulans, cultured human lymphocytes, and rat hepatocytes) in vitro has been reported. Negative results were obtained in tests for gene mutations in a number of bacterial assays, with the exception of two cases. In several studies of gene mutations in mammalian cells in vitro, carbaryl only produced one equivocal positive result in a cell culture study. However, the study had several shortcomings and the result has not been confirmed in any other comparable studies. Chromosomal damage with high dosages of carbaryl, has been reported in vitro in human, rat, and hamster cells and in plants. No such effects have been observed in mammalian tests in vivo, even at doses as high as 1000 mg/kg. Carbaryl has been shown to induce disturbances in the spindle fibre mechanism in plant and mammalian cells in vitro. The relevance of plant assays for extrapolation to humans is unclear. It can be concluded that the available data-base does not support the presumption that carbaryl poses a risk of inducing genetic changes in either the somatic or the germinal tissue of humans. The nitrosated product of carbaryl, N-nitrosocarbaryl, is capable of inducing mitotic recombination and gene conversion in prokaryotes (H. influenzae, B. subtilis) and eukaryotes (S. cerevisiae) in vitro and gave positive results in E. coli spot tests. Furthermore, experimental results indicate that N-nitrosocarbaryl binds to DNA, causing alkali-sensitive bonds and single-strand breakage. Nitrosocarbaryl has not been established as a clastogen in vivo (bone marrow and germ cells), even at high toxic doses. 8.7 Carcinogenicity 8.7.1 Carcinogenicity studies of carbaryl in rodents 18.104.22.168 Mouse Seven early carcinogenicity studies with carbaryl have been reported (Table 51) involving different strains of mice, subcutaneous injection, skin painting, ip and oral routes of administration, different dose levels and dose schedules, and various lengths of exposure and observation. None of these negative studies (one marginal response) had sufficient data or data reporting and it was not possible to evaluate them. Table 51. Carcinogenicity studies on carbaryl in rodents Chemical Species, strain Sex Route and Dose Duration Significant Evaluation Reference (number) mode of (mg/kg) of study tumour/organ of individual administration study Carbaryl Mouse, A/Jax, C3H male SC 1/week 200 mg 20 weeks none inconclusive Carpenter et al. 30 in group (total) (1961) Carbaryl Mouse, NS not skin painting not 24 months none inconclusive Weil & Carpenter stated stated (1962) Carbaryl Mouse, CD-1 male oral, diet 0.01% 80 weeks none inconclusive Mellon Institute female 0.04% 2 years (1963) Carbaryl Mouse, A,C3,HA not intraperitoneal 60 2 years none inconclusive Makovskaya et al. stated 1/week (1965) Carbaryl Mouse, (C57,B16x male oral gavage daily 4.64 18 months none inconclusive Innes et al. C5H/Anf)F1 female 7 days-4 weeks 14 mg/kg (1969) (C57B1/6xAKR)F1 72 of age; in diet body 4 weeks-18 weight months of age Carbaryl Mouse, A/He: 16 male intraperitoneal 6 mg 20 weeks marginal (±) inconclusive Shimkin et al. 3/week for (total) lung tumours (1969) 4 weeks response Carbaryl Mouse, A/J: 124 female oral, diet 1000 10 weeks none inconclusive Triolo et al. (1982) Carbaryl Mouse CD-1 male diet 100,1000 53 weeks none no effect Hamada (1991b) technical female 8000 Table 51 (continued) Chemical Species, strain Sex Route and Dose Duration Significant Evaluation Reference (number) mode of (mg/kg) of study tumour/organ of individual administration study Carbaryl Rat, CF-N 120 male oral, diet 0.005, 2 years none inconclusive Carpenter et al. per level female 0.01, 0.02, (1961) 0.04% in diet Carbaryl Rat, Sprague-Dawley male diet 250, 1500 52 weeks none negative Hamada (1991b) technical 80 or 90 per group and 7500 female ß-Carbaryl Rat, mongrel 60 male oral gavage twice 30 22 months fibrosarcoma, positive but Andrianova & 48 control a week skin; polymorphic inconclusive Alexeev (1970) cell sarcoma, stomach; osteosarcoma with multiple mestastases 48 single 20 mg 22 months fibrosarcoma, positive but 48 control subcutaneous skin inconclusive implantation Most of these studies involved the lung adenoma test in strain A mice. This test is no longer considered to be a satisfactory carcinogenicity bioassay because of the high rate of background tumours that occur in untreated animals (Clayson, 1987). Carbaryl did not increase the incidence of lung tumours in two strains of male mice, A/Jax and C3H, which were given 20 consecutive, weekly, subcutaneous injections of 10 mg carbaryl (Carpenter et al., 1961). As only one dose of carbaryl was tested and the initial number of animals entered was insufficient, no conclusion concerning the carcinogenic potential of carbaryl can be drawn from this study. There was no tumour development in mice (unspecified strain and sex) after carbaryl application by skin painting for 24 months (dose not specified, 48% water suspension) (Weil & Carpenter, 1962). This study also lacked details about the experimental procedure and pathology used and, so, was placed in the inconclusive category. Carbaryl did not induce tumours in the lungs, liver, kidney, heart, spleen, pancreas, and the thyroid and adrenal glands in two strains of mice, A and C3HA, treated ip, once per week for about 2 years, with toxic doses of 60 mg/kg (Makovskaya et al., 1965). The study involved a sufficient number of animals in the carbaryl group (400), untreated control group (100), and urethan positive control group (150), killed at 1, 3, 6, 9, 12, 15, 18, and 24 months after the beginning of the study and studied histopathologically. However, no numerical data on the background lung tumours in the untreated control animals were presented. This lack of data, coupled with the absence of tumours in the lungs of the carbaryl-treated mice strain A, which are known for their high rate of naturally occurring lung adenomas, makes the negative response of little significance. Carbaryl yielded a marginal tumour response in the pulmonary tumour induction test on mice (strain A) given a total dose of 6 mg by ip injections, 3 times per week for 4 weeks (Shimkin et al., 1969). Again, the lung adenoma test on strain A mice was not reliable for evaluating possible carcinogenicity and, thus, the results obtained from this study are not definitive. No carcinogenic response was obtained with carbaryl administered orally, by gavage, and/or in the diet to four different strains of mice at doses ranging from 14 to 1000 mg/kg diet (Melon Institute, 1963; Innes et al., 1969; Triolo et al., 1982). There was no increase in the total tumour incidence and no changes in tumour patterns in either sex of CD-1 mice fed diets containing doses of 0.01 or 0.04% (100 or 400 mg/kg) carbaryl for 80 weeks and 2 years, respectively (Mellon Institute, 1969). The survival rate in both test groups was too low for a response to be observed; no information was given for one-third of the animals; histopathology was performed only on animals that were suspected of having tumours. Because of these deficiencies, no conclusion can be drawn. Carbaryl did not significantly increase the incidence of any type of spontaneously occurring tumours (hepatomas, lung adenomas, lymphoid sarcomas) in either sex of two hybrid strains of mice (C57B1/6xC3HAnf) F1 and (C57B1/6xAKR) F1, treated orally with 4.64 mg/kg by gavage (mice from 7 days to 4 weeks of age), and in the diet (mice from 4 weeks to 18 months of age) with 14 mg/kg diet (Innes et al., 1969). Another inadequate feeding study of carbaryl carcinogenicity was conducted by Triolo et al. (1982), using the model of lung tumour induction in strain A/J female mice. In two studies, the feeding level of 1000 mg carbaryl/kg, incorporated in the diet of mice for 20 weeks, did not cause a significant increase in the incidence of background lung adenomas, nor did it induce tumours in the glandular stomach or other tissues (spleen, intestinal tract). However, there were a number of confounding factors in defining the response in this study including: large variations in the incidence of lung adenomas in the four separate control groups; in particular, one such group had no tumours, despite high historical control levels in strain A mice. In addition, only one dose level was studied (1000 mg/kg), on the basis of which it is not possible to establish a dose-response relationship. In the second study, in which the same feeding level and exposure period were used, carbaryl increased the lung benzo( a)pyrene hydroxylase activity, which was associated with a modest increase in the rate of lung tumours induced by the oral administration of 3 mg BP twice, on days 7 and 21, respectively of the study. However, there was great variability in the incidence of BP-induced lung adenomas in the 3 control groups; the first (7.2±0.8) was well above the values presented for the test group carbaryl + BP (5.7±1.4); and the second fell within the limits of the incidence of spontaneous tumours in the corn-oil control group, (1.17±1.11), which make the results inconclusive. A new mouse oncogenicity study is in progress, under proprietary sponsorship. The study design was as follows: carbaryl was administered in the diet to male and female CDR-1 mice at rates of 0, 100, 1000, or 8000 mg/kg diet, for up to 104 weeks. Groups consisted of 80 mice/sex per group. Ten mice/sex per group were sacrificed for clinical pathology evaluation after 52 weeks of treatment. Results of this interim sacrifice are presented in section 8.3. The remaining animals were designated for continued exposure to the end of the 104-week treatment period. The study design and evaluation parameters are consistent with guidelines set out by the US EPA and OECD, with additional study parameters, for studies of this nature (Rhône-Poulenc, 1992).a 22.214.171.124 Rats Two reports by Carpenter et al. (1961) and Andrianova & Alexeev (1970) are controversial. They studied the carcinogenicity of carbaryl given to rats by oral gavage, in the diet, or by subcutaneous implantation. Both studies had insufficiencies in the protocols used. Carpenter et al. (1961) noted no significant increase in the total tumour incidence in either sex of CF-N rats fed a diet containing 0.005, 0.01, 0.02, or 0.04% carbaryl, for 2 years. Female mice had more pituitary tumours than male mice, but there was no significant difference in the incidence of tumours between the control and test groups. The small initial number of animals used, their relatively old age (60 days), their low survival rate, and the lack of detailed pathology are complicating factors in defining a negative response. However, Andrianova & Alexeev (1970) reported that carbaryl, administered by both oral gavage and subcutaneous implantation, produced positive carcinogenic responses in mongrel rats. Male rats given oral doses of 30 mg carbaryl/kg, twice weekly for 22 months, developed skin fibrosarcoma, polymorphic cell sarcoma in the stomach, and osteosarcoma with multiple metastases. Carbaryl caused high lethality (80%) during the exposure period. The authors did not state whether the gross pathology was examined. One fibrosarcoma was discovered among 46 control animals, at 11 months. No data were presented on the number of control animals that lived for 22 months, so a comparison of survival rates in control and test groups could not be made. In a parallel study, 20 mg of carbaryl in a purified paraffin capsule was implanted subcutaneously in 48 male rats. At the end of the 22-month exposure period, subcutaneous fibrosarcomas, in sites far from the implantation area, and on the back and neck, were diagnosed in 2 out of 10 surviving animals. There was no control group (Andrianova & Alexeev, 1970). The carbaryl used was obtained from a plant in the USSR and was of technical grade and 97.65% purity. No information about the chemical composition and impurities was given. Because of these deficiencies, this study is inconclusive. a Information to Task Group. These studies have not yet been reviewed by the IPCS. The company performing these studies has indicated that there is a significant increase in tumors at the highest dose in both species. A new combined long-term toxicity and oncogenicity study on rats is in progress, under proprietary sponsorship. The study design is as follows: carbaryl was administered in the diet to male and female Sprague-Dawley rats at rates of 0, 250, 1500, or 7500 mg/kg diet, for up to 104 weeks. Groups consisted of 80 rats/sex per group for low- and middle-dose groups and 90 rats/sex per group for the high-dose and control groups. Ten animals/sex per group were sacrificed after 26 and 52 weeks exposure and evaluated for clinical pathology and histopathology. In addition, 10 animals/sex from the control and high-dose groups were used as a recovery group, kept on a basal control group diet before sacrifice at 56 weeks. The results of the interim sacrifices are presented in section 8.3. The remaining animals were designated for continued exposure to the end of the 104-week treatment period. The study design and evaluation criteria are consistent with guidelines set out by the US EPA and OECD, with additional study parameters, for studies of this nature (Rhône-Poulenc, 1992).a 126.96.36.199 Overall appraisal of carbaryl carcinogenicity Carbaryl has been studied for its carcinogenic potential in numerous studies on rats and mice via various routes of administration. Most of these studies are old and do not meet contemporary standards. Only one paper from the existing body of publications clearly reports a tumorigenic action of carbaryl. Carbaryl induced malignant tumours in an unidentified strain of rat by the oral and subcutaneous routes of administration. This study does not meet contemporary standards, because of insufficient reporting of control data. New studies, designed to meet with contemporary standards, are in progress on rats and mice. Descriptions of the studies are given in sections 188.8.131.52 and 184.108.40.206. 8.7.2 Carcinogenicity studies of N-nitrosocarbaryl Carbaryl is a secondary amine and is, therefore, capable of nitrosation in the presence of nitro donor groups, such as sodium nitrate, to give a nitrosamide. This nitrosamide, nitrosocarbaryl, has been proved to be mutagenic and carcinogenic, at high doses in a Information to Task Group. These studies have not yet been reviewed by the IPCS. The company performing these studies has indicated that there is a significant increase in tumors at the highest dose in both species. animals (see Table 52). A condition of this nitrosation is an acidic pH (less than 2), which is comparable to the one found in the human stomach. However, nitrosocarbaryl is not stable at this pH. Its maximum stability is between pH 3 and 5, at which pH no significant amount of carbaryl can be nitrosated. Carbaryl was nitrosated in several studies, in vitro as well as in vivo, in the guinea-pig, which has a stomach acidity similar to that of humans. See also section 8.9.3. 220.127.116.11 Rats N-nitrosocarbaryl elicited a carcinogenic response in both sexes of two strains of rats (Wistar and Sprague-Dawley) by two routes of administration (oral gavage and subcutaneous injection) (Eisenbrand et al., 1975, 1976; Lijinsky & Taylor, 1976; Preussmann et al., 1976; Lijinsky & Schmähl, 1978). A local carcinogenic effect of N-nitrosocarbaryl was reported by Eisenbrand et al. (1975) in Wistar rats given a single subcutaneous injection of 1000 mg/kg. Fifteen out of 16 treated animals developed sarcomas at the injection site; there was no histopathological evidence of any systemic carcinogenic effects. The dose of N-nitrosocarbaryl was highly toxic and 14 out of 16 animals had died by day 450. No spontaneous tumours were observed in control animals. When given to rats orally in single doses of 200-1500 mg/kg, N-nitrosocarbaryl did not induce tumours during 21 months of observation. No signs of toxicity were noted in any of the test groups. Further studies involving repeated oral administration of N-nitrosocarbaryl to Sprague-Dawley rats gave unequivocal evidence of local carcinogenic effects, produced by both oral and subcutaneous routes of administration. (Eisenbrand et al., 1976; Lijinsky & Taylor, 1976, 1977; Preussmann et al., 1976; Lijinsky & Schmähl, 1978). The nonglandular part of the stomach (forestomach) was the target organ of the local carcinogenic action of N-nitrosocarbaryl, when it was given orally to rats. All stages of malignant transformation in the forestomach, from hyperplasia to squamous cell carcinoma, were observed in male Sprague-Dawley rats treated with oral doses of 130 mg N-nitrosocarbaryl/kg, twice weekly, until spontaneous death. There was a substantial lowering of the survival rate of treated animals because of the pronounced toxicity. By the time of maximum increase of the tumour incidence (after 200 days), most of the animals were dead. The average survival time of tumour-bearing animals was 167 days after the onset of the study. The low background tumour incidence (3 out of 29) in control animals included lymphosarcomas and leukaemia. Table 52. Carcinogenicity studies on N-nitrosocarbaryl in rodents Chemica Species, strain Sex Route and Dose Duration Significant Evaluation Reference (number) mode of (mg/kg) of study tumour/organ of individual administration study N-nitroso Rat, Wistar male subcutaneous, 1000 450 days polymorphic cell positive Eisenbrand et al. carbaryl 8: male; female single injection sarcoma at (1975) 8: female injection site; spindle cell sarcoma 37 male, oral gavage, 200-1500 21 months none inconclusive female single N-nitroso Rat, Sprague-Dawley male oral gavage, 130 (5000 till squamous cell positive Eisenbrand et al. carbaryl 31 twice per week total) spontaneous sarcoma, (1976) death papilloma, Preussmann et al. hyperkeratoses, (1976) forestomach N-nitroso Rat, Sprague-Dawley female oral gavage, 50 mg 110 weeks squamous cell positive Lijinsky & Taylor carbaryl 12 once a week (total) carcinoma (1976) for 10 weeks forestomach, papilloma, oesophagus 12 male oral gavage, 300 mg 90 weeks squamous cell twice a week (total) carcinoma for 20 weeks forestomach, papilloma trachea N-nitroso Rat, Sprague-Dawley male oral gavage, 600 80 weeks carcinoma positive Lijinsky & carbaryl 16: male; female once a week (total) forestomach Schmahl (1978) 16: female for 10 weeks In a comparative study of the carcinogenic potency of N-nitroso- N-alkylcarbamate esters, Lijinsky & Taylor (1976) showed that N-nitrosocarbaryl was a strong carcinogen, similar in action to its highly potent carcinogenic analogue N-nitrosomethylurethane. A high incidence (75-80%) of squamous-cell carcinomas in the forestomach was detected in female Sprague-Dawley rats given 10, weekly, equimolar oral doses (0.22 mmol) of both compounds. Nitrosomethylurethane was a more potent carcinogen, because the animals with tumours died earlier than those in the nitrosocarbaryl group. The same rate of carcinomas in the forestomach was induced in male rats treated orally with a considerably higher dose of N-nitrosocarbaryl (1.3 mmol total), over twice as long a period (20 weeks). The stronger carcinogenic effect of this dose in male animals was expressed by the shorter latent period of induced tumours and higher lethality compared with female animals. The local carcinogenic effect of N-nitrosocarbaryl in rats, the target organ being the forestomach, was seen in another oral carcinogenicity study of a series of nitroso- N-methylcarbamate insecticides carried out by Lijinsky & Schmähl, (1978). The oral gavage of 10 weekly doses of 60 mg nitrosocarbaryl/kg to both sexes of Sprague-Dawley rats led to a high incidence (over 70%) of carcinomas in the forestomach, with no evidence of other systemic carcinogenic effects. No carcinogenic response for carbaryl was obtained with the transplacental carcinogenicity test after in vivo nitrosation in pregnant Sprague-Dawley rats (Lijinsky & Taylor, 1976). Pregnant animals were given 30 mg carbaryl/rat, orally, for 10 days (4-18 days of gestation). Two other groups of animals received the same dose of carbaryl, together with 0.6-1ml of 4% sodium nitrite on days 4-6 and 14-18 of pregnancy. The distribution of tumours in the rats of various groups was that normally seen in Sprague-Dawley rats. It is likely that an insufficient amount of nitrosocarbaryl was formed under the conditions of in vivo nitrosation, or that no significant amount of nitrosocarbaryl crossed the placenta. A low rate of in vitro nitrosation of carbaryl under conditions similar to those that exist in the human stomach was reported by Eisenbrand et al. (1975). The reaction of 10-3 mol/litre carbaryl with a 5-fold molar excess of sodium nitrite in 0.1N HC1 (pH, 1) led, after 15-60 min, to yields of only 1.2 and 1.7% of the maximum possible conversion to nitrosocarbaryl. Reduction of the concentration of carbaryl and sodium nitrite by a factor of 10 decreased the yield of nitrosocarbaryl by about one-half. The yields of N-nitrosocarbaryl obtained by the in vitro nitrosation of carbaryl, under the described conditions, are low and the potential carcinogenic risk of in vitro nitrosation and similar nitrosation reactions in vivo is difficult to evaluate at present. 18.104.22.168 Mice Nitrosocarbaryl administered for 104 weeks to the skin of female CFLP mice (65 mice per group) in three doses (12.5, 50, and 200 µg/mouse) was found to be more potent regarding dermal carcinogenic efficiency than nitrosomethylurea, applied under the same conditions, though not as effective as benzo- a-pyrene (Deutsch-Wenzel et al., 1985). Conclusions were drawn on the basis of the number of animals bearing carcinomas, total cases with local tumours, observed/expected ratios and dose-response relationships of incidences of malignant tumours. These results were found to be in contrast to the ones obtained by Lijinsky & Winter (1981), who used a single total dose of 23 mg and found that nitrosocarbaryl was a much less effective inducer of mouse skin tumours than nitrosomethylurea. 22.214.171.124 Overall evaluation of the carcinogenicity of N-nitrosocarbaryl Sufficient evidence of carcinogenicity at the site of application was seen in multiple studies on both sexes of Sprague-Dawley rats, by different routes of administration (subcutaneous and oral gavage), using different dose levels. The non-glandular stomach was the target tumour site when nitrosocarbaryl was administered by oral gavage; subcutaneous injection of nitrosocarbaryl caused sarcomas at the injection site. The local carcinogenic effect of N-nitrosocarbaryl in rats, and the lack of any systemic carcinogenicity, characterizes it as a direct-acting alkylating agent. N-nitrosocarbaryl was active as a direct bacterial mutagen and interacted with human DNA in vitro. These data agree with data that show that N-nitrosocarbaryl is an effective in vivo genotoxin. 8.7.3 Carcinogenicity of ß-carbaryl Beta-carbaryl (N-methyl ß-naphthyl carbamate) was a component of technical carbaryl (alpha-carbaryl), suspected of having a carcinogenic structure and a tumorigenic potential in mice and rats (Zabezhinski, 1970). In life-time studies on mice, strain CC57W (24 months), and mongrel rats (33 months), using two routes of administration, oral gavage (10 mg/kg in mice; 25 mg/kg in rats) and subcutaneous injection (20 mg/kg in mice; 50 mg/kg in rats), ß-carbaryl showed a weak carcinogenic response. High rates of lethality (20-50%) were observed in both mice and rats, by the two routes of administration. No data for survival rates and the incidence of spontaneous tumours in control animals were presented. ß-carbaryl caused a low rate of tumours in rats by both routes of administration. The types of tumours (21%) observed after subcutaneous injection of ß-carbaryl included: fibrosarcoma at the injection site, subcutaneous rhabdomyosarcoma, intestinal sarcoma, leukaemia, reticuloses, and reticulosarcoma. The pattern of carcinogenic response after orally administered ß-carbaryl (25%) involved sarcoma in the liver, fibroadenoma and adenocarcinoma in the mammary glands, carcinoma in the thymus, and granulocellular carcinoma in the ovaries. No local tumours at the injection site were observed in control animals after subcutaneous administration of corn oil. The other tumours (carcinoma in the mammary glands and thymus, haematopoietic system malignancies) were unusual for control animals, as well. ß-carbaryl caused a higher tumour incidence in mice than in rats by both subcutaneous (60%) and oral (30%) routes. It should be mentioned, however, that most of the tumours observed (lung adenomas, leukaemia, and liver haemangiomas) occurred spontaneously in untreated mice. Since no control data were presented, the carcinogenic response to ß-carbaryl in mice is of uncertain significance. Because of these deficiencies, this study is inconclusive. 8.8 Special studies 8.8.1 Neurotoxicity The effect of carbaryl on the nervous system is primarily related to ChE inhibition. Carpenter et al. (1961) studied the delayed neurotoxic potential of carbaryl in chickens (Rhode Island hens) compared with that of triorthocresyl-phosphate. Single doses of 250, 500, 1000, or 3000 mg/kg body weight, 25-40% in lard were administered subcutaneously to chickens. At 2000 mg/kg, weakness was observed on day 1 or 2 after dosing. In one case, the chicken was unable to walk for 3 days. No evidence of demyelination was observed in any brain sciatic nerve or spinal cord section examined microscopically. According to the authors, there was a transient cholinergic effect caused by the slow absorption of carbaryl. The neurotoxic effect of carbaryl was studied in atropinized chickens (Gaines, 1969) to protect against acute effects of the subcutaneous injection of 800 or 1600 mg carbaryl/kg body weight. The higher dose caused leg weakness within 24 h, which recovered by day 24. Carbaryl solution in corn oil at a daily dose of 100 mg/kg was administered orally for 7 consecutive days to 35, 6-day-old, female broilers, hybrids between Peterson strain roosters and Hubbard hens (Farage-Elawar, 1989). Altered locomotion and abnormally shortened gait were observed on the 7th day, and cases of delayed paralysis 20-40 days, after the last treatment. Locomotion changes were found to have no association with the activities of brain acetylcholinesterase and neuropathy target esterase, 24 h after the first, second, third, and fifth doses as well as 1, 3, 6, 10, 20, 30, and 40 days after the last treatment, when no statistical deviations from the control values of both enzymes were registered. A similar, but lesser, effect on gait and stride length was observed when 45 mg carbaryl/kg was injected into chick eggs on day 15 of incubation (Farage-Elawar, 1990). This treatment resulted in significant inhibition of brain and plasma cholinesterase, and liver carboxylesterase. Administration of the same dose on day 5 of incubation resulted in 10% lethality. Brain NTE was not affected. The cause of this delayed alteration is not known. Effects of long-term carbaryl exposure on the neuromuscular system of pigs were reported by Smalley et al. (1969). Six Yorkshire pigs, 3 male and 3 female, received carbaryl in their diet (150 mg/kg body weight); the male pigs for 72 days and the female pigs for 83 days. Three pigs from the same litter were fed carbaryl at 150 mg/kg body weight daily, for 4 weeks, and then 300 mg/kg body weight, daily, for 46 days (2 male pigs), and for 85 days (the female pig). The signs of intoxication started after about 1´ months, and were mostly typical of neuromuscular system damage (see Table 44; section 8.3). Microscopic examination of the skeletal muscle revealed myodegeneration. In the myelinated tracts of the cerebellum, brain stem, and upper spinal cord, moderate to severe edema was associated with vascular degenerative changes. No demyelination of nerve tissue was observed. When carbaryl feeding was stopped and hydrochlorothiazide applied as a diuretic, signs of toxicity of carbaryl, such as ataxia, and partial paralysis, disappeared. Carbaryl disturbed the function of the myoneural synapses. It produced a decrease in the spontaneous activity and an increase in the permeability of the muscular fibre membrane for K+ and Na+ after multiple oral administrations of 8.5 mg/kg to rats(Kovtum & Sokur, 1970). Kovtun (1970) while analysing the effects of carbaryl on myoneural formations, pointed out that, at a single intake (425 mg/kg), multiple intakes during 2 months, or 1/100 LD50-8.5 mg/kg over 6 months, the frequency of tiny potentials of an edge plate was suppressed by 62-65%. On the basis of the data obtained it was concluded that carbaryl probably impairs the function of presynaptic nerve endings. At the same time, carbaryl does not affect the cholinereceptive membrane substance of muscular fibres. Carbaryl increases the rest potential of muscular fibres by 11-35%, depending of the carbaryl dose. This increase can be explained through high potassium ion accumulation inside the muscular fibres. Three different laboratories examined the effects of carbaryl on motor activity in rats. All three reported decreased activity after intraperitoneal administration, with ED50s that ranged from 13.3 to 17.6 mg/kg (Crofton et al., 1991). Takahashi et al. (1991) compared the effects of carbaryl on both young (3 months) and old (12 months) rats. A dose of 50 mg/kg decreased activity in an open field test, prolonged the duration of haloperidol-induced catalepsy, decreased body temperature, and increased the nociception threshold, as measured by a hot-plate test. A dose of 10 mg also prolonged haloperidol-induced catalepsy. The effects on body temperature and nociception were significantly greater in older rats. Studies were carried out on the mechanical response characteristics of the soleus muscle in situ. Female Holtzman rats (6 treated-control pairs) were given 56 mg carbaryl/kg orally. Carbaryl increased the tension developed during complete tetanus (by electric stimulus) and decreased the time constant of tension development (Santolucito & Whitcomb, 1971). The more forceful and rapid contraction of the skeletal muscle is probably related to an accelerated catecholamine release. EEGs on 4 Rhesus monkeys given 0.01 mg carbaryl/kg and on 3 given 1 mg/kg per day, orally, for 18 months, showed only a reduction in the amount of low-amplitude fast waves and an increased bilateral synchrony between the right and left hemispheres (Santolucito & Morrison, 1971). The authors did not relate these EEG changes to the dose. Belonozhko & Kuchak (1969) found some changes in the EEG in rats, such as desynchronisation of rhythms after a single application of carbaryl at 100 mg/kg. During repeated doses of 35 mg/kg for 90 days, no changes in the EEG were noted, due (according to the authors) to adaptation of the nervous system. Oral doses of 72 mg carbaryl/kg, administered to rats for 10 days, caused a decrease in serotonin and an increase in dopamine levels in the brain (Kuzminskaya et al., 1984). Morphological changes in the nervous system caused by carbaryl were dose-related. One to six months oral treatment of rabbits with 0.01 LD50 caused only haemodynamic disorders and cell infiltrations, a dose of 0.02 LD50 caused cell dystrophic changes, and a dose of 0.1 LD50 caused more progressive and serious disorders (Azizova, 1976). The behavioural effects of carbaryl were studied in rats and monkeys. Disturbance of discrete (shock) avoidance behaviour by carbaryl in rats was reported at ip doses > 2.5 mg/kg, the LD50 being 8 mg/kg (Goldberg et al., 1965). Carbaryl administered at doses of 8, 16, or 28 mg/kg, ip, decreased maze activity in CD male rats (10 in each group), whereas doses of 16 and 28 mg/kg reduced open field activity. After acute exposure, behavioural changes recovered within 60 min, whereas ChE of the blood and brain recovered after 240 min (Ruppert et al., 1983). Carbaryl at 10 mg/kg administered subcutaneously in 16, male, Long Evans strain rats decreased the number of times they approached the novel stimuli and explored the exploratory box, and increased habituation. In familiar situations, carbaryl increased activity (Albright & Simmel, 1977). Anger & Setzer (1979) studied the effects of oral and im administration of carbaryl on repeated chain acquisition in 5 male monkeys ( Macaccus mascicularis). Oral doses of 50 mg/kg did not alter the performance of repeated acquisition tasks. The im injection resulted in consistent changes in performance at 5 and 10 mg/kg, but did not cause any changes at 1 mg/kg. Carbaryl affected working memory (continuous delayed response and continuous non-match) in 28 male Sprague-Dawley rats (Heise & Hudson, 1985a,b). A dose of 10 mg carbaryl/kg, ip, affected performance of working memory procedures; with increasing dose, carbaryl nonselectively decreased response. Administration of 2.24 mg carbaryl/kg, ip for 14 days, did not affect the performance of rats in activity wheel cages, 24 h after the last treatment. Acute ip administration of 0.54 or 2.24 mg/kg significantly decreased the motor activity of rats in activity wheel cages. This decrease was reversed by atropine sulfate (Singh, 1973). Doses as low as 7.76 mg/kg ip caused mild tremors. Oral administration of carbaryl (200 mg/kg, 3 days/week) for a period of 90 days, though producing inhibition of ChE in blood (33%) and brain (11%), did not result in any kind of overt signs of toxicity in male albino rats (Dikshith et al., 1976). Desi et al. (1974), in long-term studies, investigated the effects of carbaryl on the learning process, on the performance of previously learned tasks in mazes, and on the EEG in 40 male Wistar strain rats. Carbaryl was given at doses of 10 or 20 mg/kg body weight per day in the diet (100-200 mg/kg food), for 50 days. Mild, but permanent and increased, functional deviations of the nervous system were found. Because of increased irritability in the CNS, the treated rats took less time than the untreated rats to find their food. Later, the task was performed with difficulty when the irritability of the CNS was reduced to below the normal level. The performance of the learned task was impaired. EEG deviations recorded at the end of the maze studies were slight, but permanent. They consisted of increased electric activity of the brain, increased number of moderately slow beta waves, and light flashes of 18 Hz accelerated electrical activity. Viter (1978) found behavioural changes in rats treated via inhalation for several months with 12 or 23 mg carbaryl/m3. The latency period of the conditioned reflex on nutrition was prolonged. Depression of investigative behaviour and spontaneous motor activity were noted. The behavioural effects of carbaryl on rats were examined by Moser et al. (1988) using a functional observation battery. Carbaryl was administered at doses of 3-30 mg/kg, ip, and tested 0.5, 3, 24, and 48 h afterwards. Carbaryl decreased spontaneous activity, CNS excitability, motor and sensory function, and body temperature and weight. Effects indicative of AChE inhibition were also observed. All responses were dose dependent. 8.8.2 Effects on the immune system 126.96.36.199 Appraisal on immunotoxicology The administration of carbaryl, or any other xenobiotic, at doses resulting in overt toxicity can be expected to result also in a variety of effects on the immune system. Carbaryl, when administered in vivo, at a dose not causing overt clinical signs has been reported to produce a variety of non-life-threatening effects on the immune system. The effects included cellular as well as humoral immunity, and several authors have suggested that they were was due to subtle, treatment-related stress. Many of the effects described were detected at doses close to the LD50. Shortcomings of several of these studies were a lack of consistency and, sometimes, overt contradiction between results, which prevents the description of a defined immunotoxic mechanism. Lifetime exposure to carbaryl did not result in increased occurrence of disease in rats or mice. No enhancement of viral infections was found with carbaryl, even at dosages close to the LD50. Most studies on rabbits and mice, at doses permitting survival, did not produce significant effects on the immune system. In vitro, a number of researchers have demonstrated viral enhancement by prior incubation with carbaryl. In vitro, carbaryl can enhance herpes varicella-zoster, but not herpes simplex. In goldfish cell culture, carbaryl has been shown to permit viral enhancement by compromising interferon synthesis. Inhibition of human serum complement activity as well as interleukine-2 driven proliferation of large granular lymphocytes have been demonstrated in vitro. These actions could be mediated through the inhibition of serine esterases involved in the processes. No increased viral infections have been seen in long-term in vivo feeding studies, previously conducted or in progress at present. Carbaryl does not enhance transformation of BALB/53T fibroblasts in culture or the expression of endogenous murine leukaemia virus. 188.8.131.52 In vivo studies Young mice weighing 10-12 g were infected with influenza by applying 3-4 drops (0.05 g) 1% influenza virus in physiological solution in the nose. The murine influenza virus strain A.P.R.8 was used in the primary dilution 1:32. After 2-3 days, this group of 30 mice was treated orally with carbaryl in sunflower oil, at a dose of 500 mg/kg (a dose that killed 3 out of 10 mice). Two control groups with the same number of mice, one treated with carbaryl alone, and one infected, were compared with the experimental animals for survival, blood biochemistry, and pathomorphological changes. The greatest number of mice dying were in the experimental group. AChE depression was more pronounced and recovery slower, and there were more histological changes in the livers of mice infected and intoxicated by carbaryl (Moreynis & Estrin, 1965). It is clear from this study that a summation of the effects of both factors occurred, but, because of the lack of statistical significance, it is impossible to judge the eventual effect of the enhancement. The effect of carbaryl on an experimental Erysipelothrix rhusiopathiae infection in rats was studied by Shabanov et al. (1983). Rats, treated with doses that increased gradually every 6 days from 2 to 5 mg/rat (mean weight of rats, 50-60 g) during 30 days, were infected iv with Erysipelothrix rhusiopathiae at 1.5-108 cells/rat. The mortality rate in carbaryl-treated rats was 36% versus 14% in control rats. Survival time was halved. Bacteraemia persisted for 10-12 days in treated groups, and only 5-6 days in the control group. Both the gross and the histopathological changes were more strongly manifested in the treated group. Carbaryl, at 0, 2, 20, or 200 mg/kg was administered orally to rabbits, daily, for 6 months. A decrease in the phagocytic activity of leukocytes and antibody formation (4-5 times) after immunization with a murine type of typhoid vaccine, was reported at the dose level of 200 mg/kg. At the lower dose of 20 mg/kg, there were different phases of reactivity; at the beginning of the first 2 months, there was an increase and later a decrease in immunological reactivity (Perelygin et al., 1971). The effects of oral ingestion of carbaryl on nonreaginic antibody production in BALB/c mice were studied after pretreatment with 150 mg carbaryl/kg diet for 10 weeks prior to commencement of the study. Carbaryl produced significant effects on systemic antibody production following oral immunization with sheep red blood cells (SRBC). It significantly increased both IgG1 and IgG2b titres. No reduction was seen in the synthesis of any antibody class. These results suggest that, at the doses used, carbaryl increased systemic antibody responses to orally ingested antigens (André et al., 1983). Changes in the immunological structures of lymphatic follicles in the spleen during carbaryl administration were studied by Dinoeva (1974, 1982), who used a micrometric method. Albino rats (30 treated-control pair) were given 1.5 mg carbaryl/kg, orally, daily for 6 months. Plethora in the spleen and retardation in lymphatic follicles were observed, which were similar to effects seen in the positive control group treated with cyclophosphamide (2 mg/kg). There were no changes in the structure and weight of the thymus in carbaryl-treated rats, while in the cyclophosphamide positive control group, the weight of the thymus was reduced 2.5 times. The authors suggested that there is a different mechanism of the immuno-suppressive effect of carbaryl that needs further research. A single dose of 500 mg carbaryl/kg inhibited the production of haemolysins and reduced the number of splenic germinal centres in White Leghorn chickens (Roszkowski et al., 1976). Dose-dependent immunosuppressive effects of continued dietary treatment of rabbits with carbaryl were studied (Street & Sharma, 1975). Rabbits (male White New Zealand, 7 in each group) were fed 0, 4, 20, 45, or 150 mg carbaryl/kg diet (corresponding to 0, 0.23, 1.0, 2.3, or 8.38 mg/kg body weight per day) for 57 days. The treatment reduced the germinal centres in the spleen and caused atrophy of the thymus cortex. The antigen-induced increase in serum gamma-globulin was decreased significantly (no dose relationship was noted), at 10 days. No changes were detected in the number of plasma cells in the lymph, haemolysin and haemoglutinin titres, skin sensitivity to tuberculin, leukocytes count, body weight, etc. Rats pretreated, orally, with 0.05 LD50 carbaryl, daily, for 2.5 months, were immunized with typhoid antigen. Administration of carbaryl continued for an additional 2 months. Signs of insufficiency of the immune system, expressed as a reduction in specific globulin production, were observed 3.5 months after carbaryl application began. Carbaryl did not affect immunogenesis at a dose of 0.001 LD50, daily, under the same exposure conditions. No overall evaluation was given (Olefir, 1977). A humoral immunity study in female BALB/C mice after oral administration of carbaryl was performed by Wiltrout et al. (1978). The effects of humoral immune competence were measured by the immunoplaque in gel technique. The number of antibody plaque-forming cells (PFC) per plate was counted and the PFC/spleen calculated. Different groups of mice received carbaryl, orally, at about 1 LD50 (153 mg/kg body weight), 5 days before immunization (type of antigen not specified), on the day of immunization, and 2 days after. Only the last treatment resulted in significant suppression of the humoral immune response. The ratio (experimental: control) of antibody plaque-forming cells was 0.34. The oral administration of 0.1 LD50 for 8 and 28 days did not cause any significant effects on humoral immune competence. Depression of the PFC/spleen level corresponds to a decline in the total splenic lymphocyte population. Akhundov et al. (1981) studied the effects of carbaryl on the immunological reactivity of 40 rabbits and 40 guinea-pigs given oral doses of 15 mg carbaryl/kg for 42 days. Heated vaccine Salmonella typhimurium, at doses of 250 or 500 million microbial cells, was applied 3 times at 7-day intervals, 21 days after the beginning of carbaryl administration. The auto-sensitizing effect of the vaccine was enhanced by carbaryl. Studies on guinea-pigs treated with 15 mg carbaryl/kg per day for 3 months showed decreased macrophagial migration activity. Pipy et al. (1983) evaluated the cellular and humoral mechanisms of carbaryl-induced reticuloendothelial phagocyte depression. The function of the reticuloendothelial system (RES) involved in specific and non-specific aspects of host resistance to infection, neoplasma, etc., was quantitatively evaluated by the rate of disappearance of colloids injected into the blood. Colloid particles are extracted from the blood exclusively by the RES, in particular liver and spleen macrophages. Plasma or serum factors called opsonins have a strong stimulatory influence on inert particle phagocytosis. Male Sprague-Dawley rats (200-240 g) were given single, iv injections of labelled carbaryl at 0.5-32 mg/kg body weight (7 different doses). The iv LD50 determined by the authors was 50 mg/kg body weight. Colloidal carbon was administered simultaneously. In another group, carbaryl was incubated with serum from a normal rat for 20 min before the injection. The authors call this carbaryl "opsonized". The results showed the ability of carbaryl to induce a state of reticuloendothelial phagocytic depression, mediated through depletion of opsonins. Carbaryl inhibits the cell-bound aromatic amino acid esterase of the serine esterase class. The authors concluded that, apart from a slight deficiency in plasma opsonins, the inhibitory effect of carbaryl on the phagocyte function was primarily due to a selective hepatic and splenic macrophage impairment, which could be related to inhibition of a cell-bound serine esterase. In another study by Pipy et al. (1982), more details are given of this possible mechanism of phagocytoxic blockade by carbaryl. Rats were treated with iv carbaryl at 8 or 16 mg/kg body weight. The time-dependence of the effects on phagocytic function and the activities of liver serine esterases ( N-benzoyl-DL-phenylalanine-ß-naphthyl esterase and acethylcholinesterase) was studied. Macrophages of the RES had decreased phagocytic capacities after administration of carbaryl. Depression of the phagocytosis of colloidal carbon persisted from 1 to 7 h and 1 to 24 h after administration of 8 and 16 mg/kg carbaryl, respectively. Liver ( N-benzoyl-DL-phenylalanine-ß- naphthyl esterase (cell-bound serine esterase) was susceptible to reversible dose-related inhibition by carbaryl. A correlation between the reactivation kinetics of liver serine esterases and phagocytic activity was demonstrated. 184.108.40.206 In vitro studies The replication of goldfish virus 2 (GFV-2) was enhanced in vitro by pre-treatment of a piscine culture with subtoxic concentrations (1 mg carbaryl/litre) (Shea, 1983). The mechanism of enhancement was studied in vitro in goldfish-derived cell lines and Air Bladder III infected with GFV-2 and pretreated with a carbaryl solution of 1 µg/ml. Interferon synthesis was studied as a possible mechanism of virus enhancement (Shea & Berry, 1984). The authors demonstrated that interferon synthesis was induced in CAR cells by GFV-2 cells. Antiviral protection provided by supernatants from infected CAR cultures with carbaryl, against infection of secondary CAR and Air Bladder III cultures was reduced. The authors interpreted this phenomenon as a result of general mild suppression of cellular metabolism, but other possible mechanisms, such as metabolic interaction, were not excluded. In another study, Shea (1985) demonstrated quantitative carbaryl inhibition of interferon synthesis. Supernatants from infected cultures, not treated with carbaryl, provided 10 times as much antiviral protection as compared with non-infected cultures. Carbaryl-treated infected culture provided only twice as much antiviral protection as uninfected cultures. A similar relationship was observed when comparing the amount of infectious viral progeny synthesized in the presence of supernatant from infected cultures not pretreated with carbaryl (10%), and from infected cultures pretreated with carbaryl (60% of the amount of virus synthesized in control cultures). The author speculated about the possibility of superimposition of a viral infection in a population exposed to pesticides. Characterization of varicella zoster virus enhancement by carbaryl was carried out in vitro by Abrahamsen & Jerkofsky (1981) and Jerkovsky & Abrahamsen (1983). In previous studies, the authors demonstrated that the replication of varicella zoster virus (a human herpes virus) is increased 12 to 15-fold by pretreatment of cultures of human embryonic lung cells (HEL) with carbaryl, and, that different strains of this virus show differences in sensitivity to enhancement. Wild-type strains recently isolated from clinical materials are more sensitive than laboratory adapted strains. In this study, the authors demonstrated that the maximum enhancement occurred 48-72 h post-inoculation and that the optimal time for the pretreatment of monolayers of HEL is 20-24 h. 1-naphthol also produced increased amounts of virus, but the treated cells cannot pass on to the daughter cells the ability to enhance virus replication. Rodgers et al. (1986) determined that the EC50 for the inhibition of a T-cell-mediated cytolytic (CTL) response by carbaryl in vitro was 65.9 µg/ml. The addition of a supernatant containing liver enzymes reduced the effects of several organophosphorous pesticides on the CTL response, but had no significant effect on the potency of carbaryl. Casale et al. (1989) compared the ability of carbaryl to interfere with the human serum complement mediated lysis of sheep blood cells. At a concentration of 3 mmol/litre and with 2-h preincubation, carbaryl inhibited lysis by 26-45%, depending on the antibody concentration. Carbaryl was a more potent lysis inhibitor than diisopropyl phosphorfluoridate or four other anticholinergic insecticides; the potency was not correlated with anticholinergic activity. The effects of carbaryl at concentrations of 0.5-500 µmol/litre on lymphocyte proliferation were studied in vitro (Bavari et al., 1991). Carbaryl inhibited (3H) thymidine incorporation, in a concentration-dependent manner, by as much as 50%. Alpha-naphthol also had a slight effect at 50 µmol/litre. The authors attributed the effect to inhibition of an esterase responsible for interleukin activation. 8.8.3 Effects in blood Carbaryl affects the coagulation process. Hyper- and hypocoagulation were reported in different studies (Hassan & Cueto, 1970; Gapparov, 1974; Lox, 1984; Krug & Berndt, 1985; Krug et al., 1988). Gapparov (1974) studied the indices of blood coagulation in dogs after oral treatment with a daily dose of 2 mg carbaryl/kg body weight, over 5 months. A clearly manifested hypercoagulation was established, which was connected with a rise in the general coagulation activity of the blood, higher thromboplastic activity, and prothrombin content, increased number of thrombocytes, accelerated coagulation time, and increased activity of the fibrinostabilizing factor. Also noted was a decrease in the recalcification time of blood plasma, fibrinogen concentration, and free heparin quantity, together with an inhibition of the fibrolytic activity of the blood. The author interpreted all these changes as being connected with the arousal of the parasympathetic system. The blood coagulation time was considerably shortened in rabbits that were given, orally, a mixture of carbaryl (5 mg/kg), DDT (5 mg/kg), and parathion (0.5 mg/kg), for 222 days. This effect corresponded to increased levels of 5-hydroxy-3-indolacetic acid (5-HIAA) and 4-hydroxy-3-methoxymandelic acid (VMA) in the urine, indicating an increased rate of metabolism of serotonin and catecholamine (Hassan & Cueto, 1970). The authors suggested that this effect was a manifestation of non-specific stress, since adrenocortical hormones shorten the coagulation time. Fifteen male Sprague-Dawley rats (250-300 g) were given drinking-water containing 10 mg carbaryl/litre for 30 days. Platelet count, prothrombin time, partial thromboplastin time, fibrinogen, and clotting factor activity for coagulation factors II, V, VII, VIII, IX, X, and XII were determined. A significant decrease in platelet count and in the factor VII clotting activity was observed compared with those in the same number of control rats. Microscopic evaluation of the liver revealed hepatocyte degeneration, central vein congestion, some leukocytic infiltration, and vacuolization of the cytoplasm. The author suggested that carbaryl might have harmful effects on haemostasis (Lox, 1984). Carbaryl has an in vitro inhibitory effect on arachidonic acid-induced platelet aggregation, which corresponds to its inhibition of thromboxane B2-formation. The results suggest that carbaryl affected platelet aggregation by inhibition of cyclooxygenase (the key enzyme of prostaglandin synthesis) by carbamoylation (Krug & Berndt, 1985). At a concentration of 10 µmol/litre, carbaryl completely blocked platelet aggregation and cyclo-oxygenase activity (Krug et al., 1988). The carbamoylation of several platelet proteins, including cyclo-oxygenase, may be responsible for this effect. Carbaryl produced, in vitro, a dose-dependent increase in methaemoglobin (Met Hb) formation at 10 and 100 mg/litre, as well as decreases in reduced glutathion levels in the erythrocytes of Dorset sheep with low erythrocyte glucoso-6-phosphate dehydrogenase (G-6-PD), which is similar to humans who have G-6-PD deficiency. Carbaryl posed oxidative stress to G-6-PD-deficient red cells, probably due to its major metabolite alpha-naphthol (Calabrese & Geiger, 1986). Decreases in the K+ ion concentration in erythrocytes (with more than 24%) and in haematocrits (from 43.5 to 38%) were found by Sokur (1971) in rats fed carbaryl 0.05 LD50/day for 2 months. In an in vitro study, Szczepaniak & Jeleniewicz (1981) found that carbaryl binds free blood amino acids (plasma and erythrocytes). These authors also performed a series of in vitro studies to investigate the effect of carbaryl on amino acids. A single application of 475 mg carbaryl/kg on 46 treated and 12 control rats produced significantly decreased amino acid values in the brain, except for valine and phenylalanine. All amino acids reached the control level after 72 and 120 h. Two hours after administration, erythrocyte amino acids also decreased >50% (Szczepaniak & Jeleniewicz, 1980; Jeleniewicz et al., 1984). A slight decrease in erythrocyte amino acid concentrations was observed after 30 days with 95 mg carbaryl/kg administered orally (Jeleniewicz & Szczepaniak, 1980). Blood serum amino acids in 24 rats decreased 4 h after a single application of 189.6 mg/kg. There was a larger decrease in valine, then in phenylalanine, alanine, aspergic acid, serine, and glycine (Szczepaniak et al., 1980). After 15-day dosing of 0.1 LD50(94.8 mg/kg) per day to 12 rats, there were no changes in levels in serum and erythrocytes, but after 30-day dosing in 8 rats, there was a slight decrease in the alanine concentration of the erythrocytes (Jeleniewicz & Szczepaniak, 1980). The effects of carbaryl on the thermoresistance and fractional content of blood serum proteins was studied by Subbotina & Belonozhko (1968). A single dose of 150 mg carbaryl/kg administered to rabbits and multiple applications of 100 mg carbaryl/kg for 2 months showed that, with a single application of carbaryl, there was an increase in protein thermocoagulation from 28% on day 1 to 67% on day 7, and with multiple applications of carbaryl there was a lowering of albumin levels and a rise in globulins (mostly alpha-globulins) in serum, on day 10. These changes were reversible. There was also a decrease in the coefficient of thermal dehydration. The changes in proteins in both studies show lowering of their lability. Carbaryl inhibited the incorporation of 3H-uridine and 14C-labelled amino acids into RNA and proteins in cultures of HeLa cells. The effect was dose-dependent. Incorporation was inhibited by 50% at a 150 µg/ml concentration after 30 min incubation. At a concentration of 350 µg/ml, only 10% of amino acids and 32% of uridine incorporation activity were retained (Myhr, 1973). Later, Blevins & Dunn (1975) showed that carbaryl caused general metabolic changes in Hela cells. A concentration of 1-2 mg carbaryl/litre stimulated all divisions and, at 4-8 mg/litre, inhibited growth. A decrease in cellular protein at 4 mg/litre was noted. Changes in the phospholipid fraction at 8 mg/litre were probably related to the alteration of the structure of cellular membranes. Disturbances in the development of human cells in culture were also reported by Shpirt (1973). An inhibitory effect of carbaryl on cell development was demonstrated in Ehrlich ascites tumours in mice. A reduced rate of incorporation of labelled uridine-5-3H-thymidine methyl-3H and L-leucine 14C in the RNA, DNA, and proteins in Ehrlich cells in vitro was also reported (Walker et al., 1975b). The authors suggested that this effect could be a basis for investigating the mechanism of the adverse effects that carbaryl has on reproduction. Carbaryl and N-nitrosocarbaryl appeared to have different action characteristics with regard to rat liver microsomal membrane alterations (Beraud et al., 1989b); carbaryl was ineffective on the lipoperoxidation indices while the nitroso compound had an inhibitory action on the formation of malonaldehyde and conjugated dienes as well as on the NADPH-dependent reductase activities. 8.8.4 Effects on the liver and other organs Several authors reported data on disturbances in the carbohydrate, protein-forming, and detoxicating functions of the liver. A single application of 300 mg carbaryl/kg in rats produced an increase in albumin and alpha-globulins, and a decrease in ß-and gamma-globulins (Zapko, 1970). Kagan et al. (1970) studied the effects of carbaryl on the liver of 180 rats and 18 rabbits. During an 11-month study, they gave a daily, oral dose of 38 mg carbaryl/kg to rats. After 1 month, they observed a rise in the alanine-aminotransferase and alkaline-phosphatase activities in serum, and a decrease in succinic dehydrogenase and glycogen in the liver. Doses of 0.76 mg/kg and 0.38 mg/kg, given in the diet to rabbits, caused retention of bromsulfophthaleine in the blood. The authors also reported a changed ratio in the protein fractions in serum and an increase in liver weight. The pathomorphological changes in the liver were destructive, necrobiotic, and proliferative. An effect on the liver was also demonstrated in the study of Lox (1987) (see section 8.8.3). Pavlova et al. (1968) found that, with acute and long-term exposures, carbaryl affected the oxidative processes in tissues, because of its direct action on the enzymes of cell respiration and possible disturbance in the membrane processes. They performed studies on rats treated with 0.2 LD50 for 3 days and on rats treated with 0.01 LD50 for 20 weeks. At the higher dose, there were decreases in the cytochromoxidase and succinedehydrogenase activities in the liver and brain mitochondria. A histochemical examination revealed a rather high, irregular activity of the cytochromoxidase in the heart, as well as increased succinic dehydrogenase activity. With the long-term dosing, the changes were not significant, but there was a lowering of the cytochromoxidase and succinedehydrogenase activities in the heart mitochondria. Development of experimental cholesterol arteriosclerosis in rabbits was facilitated by the application of 20 mg carbaryl/kg for 2.5 months (Lukaneva & Rodionov, 1973). Changes were found in the following indices: general cholesterol, ß-lipoproteins, ECG changes, and pathomorphological changes in the aorta and coronary vessels. Carbaryl (100 mg/kg body weight) given to dogs in their diet for 45 days caused disturbances in the secretion of the intestinal enzymes. There was an increase in enterokinase secretion, as well as in the excretion of alkaline phosphates and lipase into the intestinal juice. The no-observed-effect level (NOEL) for these effects was 700 µg/kg body weight, which corresponds to 7 mg carbaryl/kg diet. Three dogs were used in each group (Georgiev, 1967). 8.8.5 Effects on serum glucose Orzel & Weiss (1966) found that a rise in blood glucose correlated with the onset and duration of tremors and the degree of brain ChE inhibition in rats that were treated ip with 5 and 25 mg carbaryl/kg. The hyperglycaemic response is blocked by adrenal-ectomy and is unaltered by hypophysectomy. The authors suggested that the hyperglycaemia was related to increased secretion of epinephrine. Hyperglycaemia is thought to result from cholinergic stimulations as it is also found in acute intoxications with organophosphorous compounds (Kaloyanova, 1963). In their studies, Orzel & Weiss did not find changes in liver glycogen. Muscle glycogen was decreased only in non-fasted rats. Elevated levels of blood glucose and slightly reduced immunoreactive insulin were found with repeated oral exposure of rats at 3 mg/rat, weekly, for one year (Wakakura et al., 1978). Glycogen disappeared in most of the hepatocytes. The cells mainly contained related granular endoplasmic reticulum with swollen mitochondria. A single application of 30 mg/rat produced transient hypoglycaemia at 20 h followed by hyperglycaemia at 44 h. The effects of carbaryl on respiration, glycolysis, and glycogenesis in isolated hepatocytes of male Wistar rats were studied by Parafita et al. (1984) and Parafita & Fernandez Otero (1984). The cells were treated with concentrations of carbaryl of 0.01, 0.1, and 1.0 mmol/litre dissolved in 1% dimethylsulfoxide (DMSO). DMSO slightly stimulates the respiratory coefficients. Carbaryl decreased the metabolic output of CO2 at all concentrations; oxygen consumption was reduced by 40% in relation to the DMSO-treated groups only at the highest concentration (1.0 mmol/litre). Glucose remained unchanged in the presence of carbaryl. Endogenous production of lactic acid was not affected, and net metabolic production was strongly inhibited by both DMSO and carbaryl. At a maximum carbaryl concentration (1 mmol/litre), the net lactic acid production was completely blocked. Carbaryl inhibited lactate gluconeogenesis, and, to some extent, gluconeogenesis from fructose pyruvate and alanin. Glycerol glucogenesis was unaffected. Lactic dehydrogenase activity was reduced by 38% and glucose 6-phosphate synthetic activity was increased by 1.0 mmol carbaryl/litre. Aspartate aminotransferase activities (cytoplasmic and mitochondrial fractions) were inhibited by 0.1 and 1.0 mmol carbaryl/litre. The results indicated that carbaryl causes a decrease in glucose production by hepatic cells and suggested that carbaryl-induced hyperglycaemia in fasted animals is caused by deficiencies in the peripheral utilization of the glucose. 8.8.6 Interactions with the drug metabolizing enzyme system Carbaryl is a weak inducer of hepatic microsomal drug-metabolizing activity. Cress & Strother (1974) reported depressed hexobarbital sleeping time in mice given 0.125 or 0.5 LD50 carbaryl, orally, for 10 days. These authors studied the effects of a 2-week dietary administration of high levels of carbaryl (10 times higher than the dose given in the earlier study). Weanling male Swiss-Webster mice, weighing 17-20 g, were divided into 5 groups of 40 animals each. The daily consumption of carbaryl by each animal was approximately 119% of the acute LD50. This dose was well tolerated with no deaths or overt symptoms of cholinesterase inhibition, probably because carbaryl was administered over a 24-h period, instead of in a single injection. Feeding carbaryl resulted in a 44% increase in the rate of in vitro p-hydroxylation of aniline, and increased in vitro demethylation of benzphetamine. The hepatic levels of cytochrome P-450 and b5 were increased, but the microsomal protein concentration per gram of liver was not affected. No changes were reported in 1-naphthol-treated mice. Increased metabolic activity was demonstrated with phenobarbital and carbaryl. Phenobarbital sleeping time was shortened to 45 min (control 74 min) in carbaryl-fed mice. The rate of elimination for the carbaryl-treated animals was twice as high as that for control animals. The oral LD50 of carbaryl in carbaryl-pretreated animals (14 days feeding) was three times as high as in the control animals. 1-Naphthol did not affect the levels of cytochromes. Sleeping time and the LD50 for carbaryl were not different in control and 1-naphthol pretreated animals. Liver weight was low in carbaryl-fed mice, but higher as a fraction of body weight. The authors concluded that the degree of the enzyme induction was not high, because most indicators exceeded the control values by 50% only, except for the lethal dose level. The pretreatment of rats with 5 daily doses of 10 mg carbaryl/kg, administered ip, resulted in a 4.2-fold increase in the rate of benzo( a)pyrene metabolism (Lesca et al., 1984). Total microsomal P-450 content and benzphetamine demethylase activity were not significantly affected. The increased ability to hydroxylate aniline in mice under carbaryl treatment was reported by Guthrie et al. (1971). Exposure of murine 3T3 fibroblasts to carbaryl at a concentration of 10-6 mol/litre was found to increase aryl hydrocarbon hydroxylase activity (Lahmy et al., 1988). The magnitude of the effect increased with the duration of exposure. Carbaryl induction of microsomal enzyme systems in White Leghorn cockerels was demonstrated by Puyear & Paulson (1972). A decrease in phenobarbital sleeping time, an increase in liver weight and elevation of liver aniline hydroxylase activity were found after oral treatment with carbaryl at 100 mg/kg per day (in gelatine capsules) for 3 or 6 days. No effect was observed at a dose of 50 mg/kg. Thus, the sleeping time data indicated that the NOEL of carbaryl for Leghorn cockerels was between 50 and 100 mg/kg per day. Higher doses caused an increase in liver aminopyrine demethylase activity (200 or more mg/kg per day) and in liver cytochrome P-450 content (300 or 400 mg/kg per day). Microsomal protein was not changed. All study parameters returned to control values by day 11 after the treatment was terminated (Puyear & Paulson, 1972). El-Toukhy et al. (1989) reported that the administration by gavage of carbaryl to mice at a dose of 166 mg/kg resulted in small, but statistically significant (15-20%), decreases in liver pyridoxal phosphokinase and L-tryptophan 2,3-dioxygenase activity. Administration over a 5-day period of 83 mg carbaryl/kg per day had no significant effect. Hassan et al. (1990) administered 85 mg/kg, orally, either once or five times daily. Both the single and repeated regimens resulted in approximately 40% decreases in L-tryptophan 2,3-dioxygenase. Carbaryl was also found to be a competitive inhibitor of this enzyme in vitro. Inhibition of monoamino-oxidase (MAO) was reported in the presence of carbaryl in vitro in liver homogenate (Hassan et al., 1966). The authors suggested that MAO and the catalase system are involved in oxidative demethylation during carbaryl detoxification. Exposure of rat liver microsomes to carbaryl in vitro at a concentration of 0.5 mmol/litre resulted in decreases in the activities of several monooxygenases (Knight et al., 1986). The ability of carbaryl to inhibit the N-demethylation of ethylmorphine appeared to be competitive with a Ki of 2.4x10-4 mol/litre. However, when administered to rats at doses of up to 25 mg/kg, carbaryl did not have any significant inductive or inhibitory effect. Lechner & Abdel-Rahman (1986a) reported that the in vitro incubation of 2 and 4 mmol carbaryl/litre for 60 min reduced glutathione levels and increased glutathione S-alkyltransferase in a rat liver homogenate in a dose- and time-dependent manner. Carbaryl had no apparent effect on glutathione levels in whole blood with 120 min of incubation. Carbaryl inhibited N-demethylation and O-demethylation by rat liver microsomes with apparent Ki of 0.1 and 0.7 mmol/litre, respectively (Beraud et al., 1989a). Carbaryl had an antagonistic action on the release of microsomal ß-glucuronidase by malathion in a microsomal suspension in vitro (Lechner & Abdel-Rahman, 1985). Jacob et al. (1985) studied the effects of carbaryl on the metabolic activation of the environmental, carcinogenic, polycyclic aromatic hydrocarbons. Carbaryl was a weak inducer of metabolism of benz( a)anthracence in the rat liver, and altered its metabolite profile by shifting from 10, 11-oxidation to 5, 6- and 8, 9-oxidation. Since carbaryl did not induce bay-region activation, the authors concluded that tumour-promoting activity could not be expected. 8.8.7 Effects on the endocrine system The effect of carbaryl on the neuroendocrine system was studied in rats. Carbaryl was given, orally, at 7, 14, or 70 mg/kg body weight to male and female rats (each group contained 24 animals weighing 90-130 g) for a period of 1 year. Growth retardation, changes in the blood enzymes, and endocrine gland disturbances varied with the dosage, being especially marked at the end of the study in the group receiving 70 mg/kg body weight per day. Acetylcholinesterase and butylcholinesterase activities were inhibited significantly in these groups. Spermatozoon motility was reduced progressively with the duration of the exposure. In the prolonged estrus cycle, the diestrus period was particularly affected. An increase in the number of corpora lutea and atrophic follicles in the ovaries was correlated with this disturbance. There was a dose-dependent increase in the gonadotropic function of the hypophysis, which was determined by tests on immature mice. Hypophyseal homogenate from a rat given 70 mg carbaryl/kg increased the weight of the ovaries by 51.5%, and that of the uterus by 123%, compared with the weights in control mice. At all doses, histochemical studies of the hypophysis showed changes indicative of an increase in the activity of the cells producing a luteinizing gonadotrophy, i.e., an increase in the size of the cells, loss of granules, and hyalinization of the cytoplasm. Histological examination of the adrenal glands revealed an increase in the size and mitotic activity of cells in the zone glomerulosa. Enlarged cells with a large nucleus or two nuclei were present in the fascicular zone. Impairment of thyroid gland functional activity in the test group was indicated by the reduction in the rate of absorption and excretion of 131I and its rather low recovery, as well as by the corresponding histological findings, i.e., enlargement of follicles and more dense and basophilic colloid at all doses. Histological findings were more pronounced in the 70 mg/kg per day groups. It is likely that the effects of carbaryl on the reproductive organs is mediated by the endocrine glands. However, the possibility of a direct effect was not excluded by the authors (Rybakova, 1966; Shtenberg & Rybakova, 1968; Shtenberg et al., 1970). The effect of carbaryl on the thyroid gland in rats was studied by Shtenberg & Hovaeva (1970). Rats were given carbaryl, orally, at 0.7, 2, 5, or 17 mg/kg per day for 6 months. They were fed a normal iodine diet or an iodine-deficient diet. There was an initial increase in function in the first 3.5 months of exposure and a decrease after 6 months of exposure. Recovery was complete 2 months after the end of the exposure. Decrease in iodine uptake and reduction of function was 26.5% in rats given 5 mg/kg and 29.5% in rats given 15 mg/kg. Rats fed an iodine-deficient diet were more sensitive to carbaryl-induced changes in the thyroid. The functional states of the thyroid gland and adrenal cortex were affected by a daily administration of 36 mg carbaryl/kg to rats for 4 months. The maximum accumulation of 131I occurred after 6 h and, in the control animals, after 12 h. The 131I absorption rate normalized after 45 days, but its excretion was delayed. The concentration of sodium in the blood increased by 36.4% after day 15 and by 74% after 45 days, while the potassium concentrations were reduced. The daily excretion of 17-corticosteroids in the urine increased by 35.9%. The changes in the thyroid and adrenal glands were transient (Dyadicheva, 1971). Hassan (1971) studied the effects of carbaryl on the synthesis and degradation of catecholamines in the rat. Rats were given carbaryl in the diet at concentrations of 100 or 700 mg/kg for 7 months, or in peanut oil at single oral doses of 50, 80, or 250 mg/kg, or 3 single doses of 80 mg/kg body weight. On day 30, the rats given 700 mg/kg diet eliminated amounts of urinary 4-hydroxy-3-methoxy mandelic acid (VMA) increased by more than 300%; levels returned to normal after 195 days. A similar effect was demonstrated after single oral doses with a dose-effect relationship. The amount of 5-hydroxyindole acetic acid (5-HIAA) in the urine increased in rats that were treated with a single dose and 3 successive daily doses of 80 mg/kg per day. Adrenalectomy and hypophysectomy did not change the pattern of VMA excretion produced by carbaryl. There was an increased turnover rate (68%) of heart norepinephrine (NE-3H) in rats treated with 80 mg carbaryl/kg, 2 h before application of NE-3H. The corticosterone level was increased by about 125% at the same dose level. A carbaryl concentration of 16.2 µg/ml in the blood was enough to trigger maximal corticosterone secretion. The authors suggested a probable activation of the enzyme system involved in the catecholamine metabolism, because carbaryl may also directly affect the adrenergic nerve endings. Activation of the pituitary adrenal axis and an increase in sympaticoadrenergic activity with concomitant increased VMA elimination was a pharmacological effect due to carbaryl treatment. The effect of a single oral dose of 60 mg carbaryl/kg body weight on serotonin (5-HT) metabolism in the male Holtzman Albino rat brain was studied by Hassan & Santolucito (1971); 2, 4, 6, and 24 h after carbaryl application, whole brain samples were analysed for serotonin, 5-HIAA, and corticosterone. The concentrations of 5-HT and 5-HIAA in the brain increased by 20-30% (5-HT from 0.61 to 0.8; 5-HIAA from 0.25 to 0.31 µg/g) from 2 to 6 h and returned to normal after 24 h. The plasma corticosterone level was also increased by about 125%, 1 h after oral administration of carbaryl, and returned to normal after 20 h. The inhibitory effect of p-chlorophenylalanine on brain 5-HT formation was diminished by carbaryl treatment. The serotonin level in the brain of carbaryl-treated animals pretreated with p-chlorophenylalanine was 72% higher than in the control animals. Increased 5-HIAA levels were probably a result of an increased rate of synthesis of serotonin. The authors suggested the possible role of the increased synthesis of serotonin and tryptophan-5-hydroxylase activity, which could be related to stress conditions. Carbaryl activation of MAO is probably related to the increased formation of cerebral 5-HIAA. The uptake of noradrenaline in vitro by hypothalamic slices, isolated from rats exposed in vivo to near lethal levels of carbaryl, was increased in a dose-dependent manner (Jablonska & Brzezinski, 1990). Oral administration of 200 mg carbaryl/kg to rats was reported to raise striatal levels of noradrenaline and homovanillic acid, a metabolic product of dopamine, when measured 0.5-2 h later (Ray & Poddar, 1985b). The mechanism of the stimulation of corticosterone secretion is not clear. In vivo studies on male mice receiving 38 and 68 mg carbaryl/kg showed that carbaryl can increase certain hepatic androgen hydroxylase activities. In vitro incubation of carbaryl with prostate tissue and testosterone 1,2-3H stimulated the formation of dehydrotestosterone-3H, thus, suggesting a direct action on steroidogenesis. Comparing these results with the effects of other toxic substances, the authors concluded that carbaryl cannot exert any major changes in steroid metabolism, nor can it reduce hormonal disturbances (Dieringer & Thomas, 1974). Attia et al. (1991) studied the effect of carbaryl on rat melatonin production as alterations of the latter were found to have marked consequences in reproduction, immunology, and tumour growth. Male albino rats ( Rattus rattus), 8 animals in a group, were exposed to light/dark cycles of 14/10 h. They were treated by gastric gavage with carbaryl dissolved in corn oil for 6 successive days. The total doses were 50, 125, and 250 mg/kg. The animals were killed 2 and 4 h after the onset of darkness, which was 8 or 10 h after the application of the insecticide. Carbaryl was found to bring about an augmentation of the pineal melatonin content 4 h after the onset of darkness, which coincided with the stimulated N-acetyltransferase levels and hydroxyindole- O-methyltransferase activity. However, at the same time, carbaryl treatment at all doses significantly lowered the circulating melatonin titres; this was supposed to be related to increased hepatic melatonin metabolism due to the increased activity of the liver drug metabolizing enzyme system (Gaillard et al., 1977). These results and the established carbaryl-induced elevated pineal 5-hydroxytryptophan, serotonin, and 5-hydroxyindole acetic acid contents in the course of the night cycle, support the concept that carbaryl has a significant effect on pineal melatonin synthesis and secretion. Kadir & Knowles (1981) reported that carbaryl inhibited rat brain monamine oxidase activity in vitro. 8.8.8 Other studies Human serum albumin reacted in vitro with the ester group of carbaryl and catalysed the hydrolysis and liberation of 1-naphthol. This reaction is similar to an "esterase type" action (Casida & Augustinsson, 1959) called carbamoylation (Oonnithan & Casida, 1968). 8.9 Factors modifying toxicity, toxicity of metabolites 8.9.1 Factors modifying toxicity The toxicity of carbaryl can be modified by altering liver function by tranylcypromine treatment or 70% hepatectomy. A decrease in LD50 and increase in ChE activity were more pronounced after tranylcypromine treatment (Falzon et al., 1983). The carbaryl LD50 in animals was increased three-fold by pretreating animals with small doses of carbaryl (Cress & Strother, 1974). Phenobarbital, administered 24 h before carbaryl treatment, decreased the acute ip toxicity of carbaryl in mice, while 2-diethyl-amino-ethyl-2,2'-diphenyl-valerate-HC1 (SKF 525-A), given 1 h before carbaryl treatment, increased its acute intraperitoneal toxicity (Neskovic et al., 1978). Enzyme-mediated binding of carbaryl to rat hepatic microsomal protein occurred in vitro in the presence of NADPH and oxygen. Incorporation of radioactivity of 14C-ring labelled carbaryl was studied. A 2- to 3-fold increase in binding was produced by pretreatment of animals with MFO inducers, e.g., phenobarbital or 3-methylcholanthrene. SKF-525-A inhibited binding by approximately 77% of the radioactivity. It is likely that binding of active carbaryl metabolites formed by MFO occurs. The radiolabelled metabolic products were covalently bound to amino acid residues of microsomal protein: 99.3-99.7% of the bound radioactivity (Miller et al., 1979). A 50% decrease in microsomal ß-glucuronidase activity was observed 1 h after a single oral administration of 50 mg carbaryl/kg to female, Sprague-Dawley rats. The whole liver homogenate ß-glucuronidase content was reduced by 40% after daily treatment with the same dose of carbaryl for 7 days. This effect was attributed to the decreases in mitochondrial lysosomal and microsomal ß-glucuronidase. The action of carbaryl in depleting the endoplasmic reticulum of ß-glucuronidase led to an increase in serum ß-glucuronidase activity. This was demonstrated in an in vitro microsomal suspension study which showed that incubation of 4 mmol carbaryl/litre with microsomal suspensions effected a release of the ß-glucuronidase enzyme. The effect on the endoplasmic reticulum was further exemplified by the induction in microsomal UDP-glucuronyl transferase activity after 7 days of daily treatment with carbaryl at a dose of 25 mg/kg. Thus, carbaryl modifies the enzyme activities associated with the endoplasmic reticulum by decreasing specific activity (90%) towards ß-glucuronidase enzyme and activating the synthesis of endoplasmic reticulum protein connected with drug metabolism, such as the UDP-glucuronyl-transferase enzyme (Abdel-Rahman et al., 1985; Lechner & Abdel-Rahman, 1985). Osman & Brindley (1981) conducted bioassays to determine the carbaryl susceptibility and synergism with piperonyl butoxide in natural populations of three species of Lobops grassbugs, in order to estimate monoxygenase detoxification. Pretreatment of the insects with piperonyl butoxide inhibited the mixed-function oxidase system and decreased the values of the LC50 for the insects (Osman & Brindley, 1981). Mixed-function oxydase involvement in the biochemistry of synergistic insecticides (including carbaryl) has been reviewed by Casida (1970). Diet may modify carbaryl toxicity. Boyd & Boulanger (1968) reported an increased susceptibility to carbaryl toxicity in Albino rats (272 Wistar strain rats were used) fed a protein-deficient diet. Boyd & Krijnen (1969) also reported that the LD50 decreased from 589 mg/kg in rats fed an 81% casein diet to 67 mg/kg in rats fed a 0% casein diet; 285 male rats were used in this study. The combined effects of different ambient temperature levels and carbaryl were studied by Ahdaya et al. (1976). The LD50 values in mice injected ip were 263 mg/kg at 1 °C and 122 mg/kg at 38 °C (588 mg/kg is the LD50 at 27 °C). The thermoregulation ability of mice treated with carbaryl was affected more than that of mice treated with parathion which is a stronger inhibitor of ChE. The authors suggested that this effect was due to an overall reduction in the basal metabolic rate. Atropine sulfate decreased signs of parasympathetic stimulation in a group of pigs acutely poisoned with 1-2 g carbaryl/kg (Smally, 1970). During multiple administrations of carbaryl, drug-induced (hydrochlorothiazide) diuresis helped in the detoxification processes. ChE reactivations are contraindicated because they may aggravate the signs of carbaryl intoxication (Sanderson, 1961; Podolak & Warchocki, 1980). Application of atropine or trepazin, 20 min before oral intoxication with carbaryl in mice, decreased toxicity about 2 times. The results were similar when cholinolytics were applied 2 min after administration of carbaryl (Vyatchanikov & Alexachina, 1968). Atropine administered iv to rats at a dose of 8 mg/kg increased the carbaryl ip LD50 by a factor of about 7 (70 to 460 mg/kg) (Harris et al., 1989). Co-administration of either of two different oximes (2-PAM and HI-6) with atropine was found to provide significantly less protection than that afforded by atropine alone, indicating that oxime therapy is not a useful treatment for carbaryl poisoning. Co-administration of an oral dose of 10 mg malathion/kg in rats significantly reduced the rates of both absorption and elimination of an oral dose of 10 mg carbaryl/kg (Lechner & Abdel-Rahman, 1986a). Peak plasma levels of labelled carbaryl were observed about 1 h after the administration of carbaryl alone, and after 2 h when malathion was administered simultaneously. The terminal rate of elimination of carbaryl was decreased by a factor of about 4, with the half-life increasing from 16.96 h to 64.41 h. Concentrations of cimetidine in the range of 60-240 µg/ml were observed to decrease the clearance rate of carbaryl by perfused rat liver in a dose-dependent manner (Ward et al., 1988). Ray & Poddar (1985a,b, 1990) reported that the intraperitoneal administration of 1 mg haloperidol/kg, 12.5 to 100 mg 5-hydroxy-tryptamine (5-HTP)/kg, or 100 mg L-tryptophan/kg increased the incidence of tremors in rats following an oral dose of 50-200 mg carbaryl/kg. The potentiation of the tremorogenic effect of carbaryl by 5-HTP was demonstrated to be dose dependent. The potentiating effects of 5-HTP and haloperidol could be blocked by the coadministration of the serotonergic antagonist methysergide (20 mg/kg, ip) or the dopaminergic antagonist bromocriptine (10 mg/kg, ip), respectively. These results suggest the possible involvement of a central cholinergic, dopaminergic interaction in the carbaryl-induced tremor. The effects of humic acids in the detoxification of carbaryl during oral application are very slight: only 9.9-18.6% decrease in toxicity was observed (Golbs et al., 1984). The combined effects of carbaryl and sodium nitrite have been studied (Podolak-Majczak & Tyburczyk, 1984, 1986; Tyburczyk & Podolak-Majczak, 1984a,b; Tyburczyk & Podolak-Majczak, 1986). Dosing Wistar rats for 3 months with sodium nitrite at 20 mg/kg per day, and carbaryl at 0.1 LD50/day (60 mg/kg), the brain gamma-aminobutyryl acid level, methaemoglobin, blood serotonin, free blood tryptophan, serum and liver alanine amino-transferase activity levels increased and blood cholinesterase activity decreased. Decreases were observed in the vitamin E and A levels, glucose-6-phosphate dehydrogenase activity, and liver aminohexoses and hydroxyproline levels with a simultaneous increase in the serum aminohexoses level. This finding may indicate the increasing process of connective tissue catabolism and lisosomal membranes labilization. 8.9.2 Toxicity of metabolites Hydrolysis of the carbamate ester bond of carbaryl results in detoxification. The carbamate moiety is decomposed to carbon dioxide and methylamine, and the phenolic part is conjugated and excreted. Kuhr (1971) summarized the data on the toxicity of carbaryl metabolites (Table 53). Table 53. Toxicity of carbaryl and some of its metabolitesa LD50 (mg/kg) 7-day Molar I50 bovine NOELb anticholin-esterase Mouse (mg/kg) Rat oral Intraperitoneal Rat Carbaryl 270 29-42 125-250 5 x 10-8 4-Hydroxycarbaryl 1190 74 > 1000 4 x 10-7 5-Hydroxycarbaryl 297 56 > 1000 4.6 x 10-8 7-Hydroxycarbaryl 4760 > 1000 Hydroxymethylcarbaryl > 5000 630-780 250-500 1.4 x 10-5 1-Naphthol 2570 500-1000 1 x 10-3 aFrom: Kuhr (1971). bNOEL = No-observed-effect level. Many of the known metabolites are much less toxic than carbaryl. The pharmacological study of carbaryl in rats showed that the metabolites with a methylcarbamate moiety are inhibitors of plasma cholinesterase (Fernandez et al., 1982). None of the carbaryl metabolites was appreciably more active as a cholinesterase inhibitor than carbaryl itself (Oonnithan & Casida, 1968). 8.9.3 N-nitrosocarbaryl Carbaryl is a secondary amine and is, therefore, capable of nitrosation in the presence of nitro donor groups, such as sodium nitrate, to give a nitrosamide. This nitrosamide, nitrosocarbaryl, has been proved to be mutagenic and carcinogenic, at high doses in animals. Conditions of this nitrosation include a strongly acidic pH (less than 2), which is comparable with the pH in the human stomach. However, nitrosocarbaryl is not stable at this pH. Its maximum stability is between pH 3 and 5, pHs at which no significant amounts of carbaryl can be nitrosated. Carbaryl has been nitrosated in several studies, in vitro as well as in vivo, in the guinea-pig, which has a stomach acidity close to that in man. If significant yields of nitrosocarbaryl have been shown in these circumstances, the significance of these findings is still not clear for human risk evaluation. The pH in the human stomach is variable during food intake and probably, more importantly, the stomach contains a lot of food, which will minimize the contact between naturally occurring nitrite and carbaryl residues, but which will also afford a lot of nucleophilic sites for nitrosocarbaryl to react with. It is also noteworthy that all studies that were conducted with jointly administered carbaryl and nitrite yielded negative results for oncogenicity. Cranmer (1986) estimated the potential intake of nitrosocarbaryl at 6x10-9 mg/kg per day. Using different mathematical models, Cranmer estimated a cancer risk for nitrosocarbaryl between 10-6 (one-hit model) and 10-9 (probit model). However, if such oncogenic nitroso-carbamates were to be found in such quantities in the human stomach, an increased incidence of stomach cancer would have been expected during the period when drug and pesticide carbamates were widely used. However, during this period, the incidence of gastric cancer in the USA declined considerably. The bacterial metabolite N-nitrosocarbaryl may act as a noncompetitive inhibitor of the in vitro metabolism of aminopyrine, p-nitroanisole, and aniline by rat liver microsomes (Beraud et al., 1980, 1989a,b). N-nitrosocarbaryl was more effective as an inhibitor of microsomal activity than the parent compound. 8.10 Mechanism of toxicity - mode of action The mechanism of toxicity and the mode of action of carbaryl and carbamates, in general, have been described in EHC 64 (WHO, 1986). 8.10.1 Inhibition of cholinesterase activity As a carbamate compound, carbaryl is an inhibitor of cholinesterase (ChE) activity (for details see Reiner & Aldridge (1967) and Aldridge (1971)). A number of studies have been performed by Carpenter et al. (1961) to assess the extent of ChE inhibition by carbaryl in mammals. A single oral dose of carbaryl of 560 mg/kg produced a 43% inhibition of the erythrocyte ChE in rats in 0.5 h, and a 30% inhibition in brain AChE. However, they returned to normal in rats that survived 24 h after administration of the dose. Carbaryl did not depress plasma ChE significantly. Two groups of Beagle dogs were injected once, iv, with 10 or 15 mg/kg as an 8% solution in 95% alcohol. No significant effects were found on either erythrocyte or plasma ChE. On the 5th day, several administrations of the same doses depressed plasma ChE by 24% and erythrocyte AChE by 40%. It is doubtful that a long incubation time for the samples (2 h for plasma) played a role in the slight depression of ChE. Comparative data on ChE inhibition in the brain, plasma, and erythrocytes of rats that received single doses of carbaryl were reported by Mount et al. (1981). As shown in Table 54, there was a dose-dependent decrease in ChE in the brain, plasma, and erythrocytes. Brain AChE was significantly different in dead rats and in surviving rats given 800 mg/kg. AChE depression in the brain was >70% in the lethally poisoned rats. The inhibition of red blood cell AChE activity was the same as in plasma. Table 54. Inhibition of ChE in % in comparison with the controlsa Sprague-Dawley rats Post-dosing Brain Red blood Plasma (h) cells Controls 10 rats 0 0 0 450 mg/kg 0.5 56 44 51 (24 rats sacrificed) 1 74 29 53 2 74 42 68 4 66 48 61 8 55 25 31 24 27 60 38 48 22 23 46 96 6 19 -19 800 mg/kg 0.5 66 32 47 (34 rats sacrificed) 1 70 44 50 2 79 73 69 4 50 72 70 12 50 72 50 28 58 63 68 48 17 26 40 96 16 -6 16 800 mg/kg 3 80 - - (24 rats died) 4 85 - - 18 88 - - 25 71 - - 29 88 - - 30 84 - - 1200 mg/kg 1-36 88-91 - - (15 died) aAdapted from: Mount et al. (1981). The affinity of AChE of human brain caudate nucleus for carbaryl was studied in vitro and some inhibition constants were determined (Patocka & Bajgar, 1971). The value of the I50 affinity constant was calculated from the dependence of AChE inhibition on the molar concentration of the inhibitor in probit logarithm transformation. The Hill coefficient was obtained from Hill plots. The affinity constant for carbaryl pI50 was 5.59 and the Hill coefficient was 1.50. According to the authors, some results of this study suggest that AChE may be an allosteric enzyme. Intravenous administration of colloidal carbon in the rat, inhibiting the phagocytic activity of the RES, prolonged the duration of the anticholinesterase effect of carbaryl (Pipy et al., 1979). The authors speculated that the metabolism of carbaryl was decreased by the RES inhibition and, as a result, the reactivation speed of ChE was slower. Pregnant rats were treated orally with 1 or 5 mg carbaryl/kg from day 11 to the last day of gestation (Declume & Benard, 1977b; Declume et al., 1979). No cholinesterase depression in blood, brain, and liver were observed in either the dams or the newborn offspring. However, after administration of 50 mg/kg from day 19 to the last day of gestation, ChE inhibition was seen in both the mother and newborn offspring. Cambon et al. (1978) reported decreased cholinesterase activity in the blood, brain, and liver in the mother and the fetus after treatment of the dams at 6.25, 12.5, 25, and 50 mg/kg. Variability of cholinesterase levels in the controls and short-comings in the cholinesterase analysis protocol make these results difficult to interpret. 9. EFFECTS ON HUMAN BEINGS 9.1 General population exposure 9.1.1 Acute toxicity, poisoning incidents The clinical picture of carbaryl intoxication results from the accumulation of ACh at nerve endings (WHO, 1986). The signs and symptoms can be categorized into the following 3 groups: (a) Muscarinic manifestations - increased bronchial secretion, excessive sweating, salivation, and lacrimation; - pinpoint pupils, bronchoconstriction, abdominal cramps (vomiting and diarrhoea); and - bradycardia. (b) Nicotinic manifestations - fasciculation of fine muscles (in severe cases, diaphragm and respiratory muscles also involved); and - tachycardia. (c) Central nervous system manifestations - headache, dizziness, anxiety, mental confusion, convulsions, and coma; and - depression of respiratory centre. All these signs and symptoms can occur in different combinations and can vary in onset and sequence, depending on the chemical, dose, and route of exposure. The duration of symptoms is usually shorter than that observed in organophosphorus poisoning. Mild poisoning might include muscarinic and nicotinic signs only. Severe cases always show central nervous system involvement; the clinical picture is dominated by respiratory failure sometimes leading to pulmonary oedema, due to the combination of the above-mentioned syndromes. A review of carbaryl-related poisoning from 1966 to 1980 was made by US EPA. During this period, 193 cases of over-exposure to carbaryl as the sole active ingredient in the poisoning and 144 cases of over-exposure to a combination of carbaryl with other active ingredients were recorded (Weston, 1982). Not all case histories have been confirmed. There were 5 deaths. There is no evidence that carbaryl was involved in these deaths, except for one, which was acknowledged by the manufacturer. Hayes (1963) reported two incidents of poisoning: one, a 19-month-old child who swallowed an unknown amount of carbaryl, and the other, a man who swallowed 250 mg of carbaryl. Both developed moderately severe ChE inhibition symptoms within 20 min: constricted pupils, salivation, muscular incoordination in the child, and epigastric pain, profuse sweating, lassitude, and vomiting in the man. Both recovered after atropine treatment. Blood cholinesterase was inhibited. One death from carbaryl ingestion while drunk was reported by Farago (1969) who concluded that 2-PAM application hastened the fatal outcome (due to pulmonary oedema), 6 h after ingestion. Carbaryl was found in various tissues. Acute intoxication during the loading of an airplane was reported by Long (1971). In a case report of a suicide attempt involving about 25 g (500 mg/kg) of carbaryl, dicumarin and boric acid, Dickoff et al. (1987) described the occurrence of a peripheral neuropathy that resembled the syndrome observed following exposure to some organophosphorus compounds. Recovery continued for 9 months. Electrophysiological findings were consistent with axonal peripheral neuropathy. The cause-effect relationship was confounded because of combined exposure. 9.1.2 Controlled human studies Wills et al. (1968) carried out controlled studies in human volunteers, aged 25-57 years. In a preliminary study, oral doses of 0.5, 1, and 2.0 mg carbaryl/kg in capsules (single application to 2 subjects) were tolerated without any subjective or objective symptoms. In the main study, one group (5 subjects) took 0.06 mg carbaryl/kg daily, and one group (6 subjects) took 0.13 mg carbaryl/kg for 6 weeks. Physical examination, BSP removal from blood, EEG examination, routine blood and urinalysis were performed, and ChE of plasma and red blood cells was examined. No changes were found in the low-dose group. An increase in the ratio of amino acid nitrogen to creatinine in the urine, at the high dose, may represent a decrease in the ability of the proximal convoluted tubule to reabsorb amino acids. This change was reversible. The urine of some subjects was analysed (Knaak et al., 1968). The overall recovery (by the fluorometric method) of the carbaryl equivalents in the urine was 26-28%, and, with the colorimetric method, 37.8%, in subjects treated with 2 mg carbaryl/kg. The following metabolites were found chromatographically in a 4-h urine sample: alpha-naphthol glucuronide (10-15%), and sulfate (6-11%) and 4-(methylcarbamoyloxy)-alpha-naphthyl glucuronide (4%). Another metabolite, alpha-naphthyl methylimido-carbonate O-glucuronide, was identified by fluorometry. A human ingestion study was conducted to determine the relationship between a single oral dose of carbaryl and the rate of its urinary excretion as metabolites. Elimination was apparently first order over the dosage range of the studies (0.25-1 mg/kg). The model predicts that, 24 h after ingestion, approximately 41% of the dose can be accounted for as urinary metabolites (Hansen, 1978). In a study involving one subject, Ward et al. (1988) observed that pretreatment with a clinical regimen of cimetidine (3x200 mg over a three-day period) reduced presystemic (first-pass) clearance of a dose of 1 mg carbaryl/kg by about 46%. A scientist studying the anthelminthic activity of carbaryl, tested its human safety by ingesting 250 mg (about 2.8 mg/kg). After 20 min, he experienced epigastric pain and began to sweat profusely. He was treated with a total dose of 3 mg atropine and recovered completely 2 h after taking the carbaryl. Another scientist ingested 420 mg carbaryl (4.45 mg/kg), and after 85 min he had vision troubles, weakness, profuse sweating, and felt lightheaded. He was treated with a total dose of 4.8 mg atropine and recovered 4 h after the onset of symptoms (Hayes & Laws, 1991). 9.1.3 Long-term exposure Branch & Jacqz (1986a,b) reported the case of a 75-year-old man who was exposed to carbaryl for 8 months, inside his home, after repeated excessive applications of 10% dust formulation. He experienced a series of signs and symptoms compatible with cholinesterase depression in addition to a 40-lb (18-kg) weight loss. After exposure ceased, the patient's condition improved markedly. However, a few months later he started to experience modification of his sleep pattern and peripheral neuropathy and cerebral atrophy were demonstrated. Other pathologies, such as recurrent gastric ulcer, cardiac fibrillations, a recent head injury due to an automobile accident, and other less defined pathologies were present in this patient which provide other, more likely, causes for these later symptoms. The uncertainties associated with long-term exposure to levels sufficient to result in sustained suppression of plasma pseudocholinesterase activity and possible brain damage are discussed by Avashia (1987) and Branch (1987). 9.2 Occupational exposure 9.2.1 Epidemiological studies The first report on workers exposed to carbaryl was published by Best & Murray (1962). For 19 months, from the start of carbaryl production, they studied men working on the production, handling, and shipping of carbaryl. The most exposed group were bagging workers occasionally exposed to carbaryl dust under abnormal conditions (40 mg/m3). These showed a slight depression in blood ChE activity, but this was below the rate at which clinical symptoms might be expected. They found that 41% of 689 urine specimens contained >1000 µg/100 ml total 1-naphthol (>400 µg/100 ml indicates absorption), with no clinical or subjective symptoms. In cases of acute intoxication, 3140 µg 1-naphthol/100 ml urine were found. Knaak et al. (1965) using fluorometric analysis in conjunction with chromatographic separation, found 5 times more sulfate (25 mg/litre) than glucuronides (5 mg/litre) of 1-naphthol in the urine of exposed workers. Vandekar (1965) in a village-scale trial in Nigeria, assessed the risk for the population of exposure to carbaryl. A slight depression of plasma ChE was found in all spraymen, the day after the spraying. Levels of 1-naphthol derivatives in their urine did not increase on days 1 and 2 after spraying but increased slightly on day 6. In a study on 19 agricultural workers (Yakim, 1967), whole blood ChE activity was measured before, and after, 3-4 days exposure to airborne carbaryl. Men exposed to a mean airborne carbaryl concentration of 2 mg/m3 showed a decrease of 20-24% in ChE activity. Signalmen exposed to 4 mg/m3 (mean) showed a 13-30% decrease in ChE activity. No objective signs of ill health were observed. In the same study, a mean carbaryl concentration of 0.7 mg/m3 was reported in the cabin of an aeroplane used to apply carbaryl, but no changes were reported in the biological parameters of the pilots. During the agricultural application of carbaryl dust at a maximum exposure level of 19 mg/m3 dust on cotton fields, Adylov (1966) reported a decrease in catalase activity, and a 20% decrease in ChE activity on day 14. In cases of occupational overexposure to carbamates, mild symptoms appear long before a dangerous dose is absorbed, which is why severe occupational intoxications with carbaryl are rare. Tobin (1970) gives reasons for the lack of severe intoxications: (1) there is a very short time (´ h or less) between exposure to carbamates and the onset of symptoms; (2) lack of symptoms progression, because of a large margin between the median effective dose and the lethal dose of carbamates and the early detection of intoxication. Workers exposed to carbaryl used on pets for flea control, experienced diarrhoea, increased salivation, cough, difficulty in breathing, and phlegm (Ames et al., 1989). Vandekar (1965) reported a skin rash in a sprayman who was accidentally splashed with a carbaryl formulation. Although carbamate compounds generally have not caused dermatitis or allergic skin reactions, Vandekar suggested that they can appear in certain individuals after unusually heavy exposure. One out of a group of 30 farmers with contact dermatitis was identified by a patch test as having a positive allergic reaction to a 1% solution of carbaryl (Sharma & Kaur, 1990). No allergic reaction were observed in a control population (No.=20). To identify possible effects on reproduction, Whorton et al. (1979) studied a cohort of 47 male workers who had worked for at least 1 year in the production and packaging of carbaryl. A semen sample was used for sperm count. Testosterone follicle-stimulating hormone, and luteinizing hormone were determined by radioimmunoassay. The range of airborne carbaryl concentrations in the workplace was 0.03-14.21 mg/m3 with a mean of 4.9 mg/m3. This cohort showed no seminal or blood abnormalities related to carbaryl exposure. Although a small excess (not significant at alpha=0.05) was observed in a small number of oligospermic men in the exposed group, there was no evidence that testicular function or fertility in the male workers was affected under these conditions of exposure. Wyrobek et al. (1981) used the same cohort of exposed workers to study sperm abnormalities. Semen was collected from 50 men, occupationally exposed to carbaryl for 1-18 years. Semen samples were analysed for changes in sperm motility, sperm count, morphology, and frequency of sperm-carrying double fluorescent bodies (YFF). The YFF test represents sperm with two Y chromosomes due to meiotic nondysjunction. The exposed workers showed changes in sperm morphology with a higher proportion of sperm with abnormal head shapes in comparison with the control group of newly hired unexposed workers. There was no dose dependence as judged by job classification. A negative correlation between number of years working in the carbaryl area and the percentage of abnormal sperm was observed. Workers who had once been exposed to carbaryl, but who had not been exposed for an average of 6.3 years, showed a marginally significant elevation in sperm abnormalities, possibly not reversible. MacLeod (1982) reviewed the Wyrobek et al. (1981) study and did not find any essential differences in the distribution of the sperm types in the control and the carbaryl-exposed groups. No significant changes in sperm count and fertility were reported in 100 workers (Thomas, 1981). 10. PREVIOUS EVALUATION BY INTERNATIONAL BODIES The latest IARC evaluation of carbaryl was made during 1987 (IARC, 1987). It was concluded that there were no data on cancer in humans and inadequate evidence of carcinogenicity in experimental animals. Carbaryl could not be classified with regard to its carcinogenicity to humans (Group 3). The FAO/WHO Joint Meeting on Pesticide Residues (JMPR) evaluated carbaryl at its meetings in 1963, 1965, 1966, 1967, 1968, 1969, 1970, 1971, 1973, 1975, 1976, 1977, 1979 and 1984 (FAO/WHO, 1964, 1965, 1967, 1968, 1969, 1970, 1971, 1972, 1974, 1976, 1977, 1978, 1980, 1985). Since 1973, an acceptable daily intake (ADI) of 0-0.01 mg/kg body weight has been established. This estimate is based on the following experimental data: no-effect level for rats: 200 mg/kg in the diet = 10 mg/kg per day; for dogs: 100 mg/kg in the diet = 1.8 mg/kg per day; and for human beings: 0.06 mg/kg per day (FAO/WHO, 1965, 1967, 1974). Maximum residue levels (MRLs) for carbaryl were recommended by FAO/WHO (1986b) (see Table 55). The values recommended for tolerance levels represent the sum of free carbaryl, combined carbaryl, conjugated naphthol, and conjugated methylcarbaryl, expressed as total toxic residues of carbaryl. A WHO study group on occupational health recommended 5 mg carbaryl/m3 as a tentative, health-based, maximum permissible level in the working environment. A biological limit of 30% inhibition of ChE activity in whole blood, plasma, or red cells with respect to pre-exposure levels should not be exceeded (WHO, 1982). In the WHO recommended classification of pesticides by hazard, technical carbaryl is classified in Class II as moderately hazardous in normal use (WHO, 1992). WHO/FAO (1975) issued a data sheet on carbaryl (No. 3). IRPTC in its series "Scientific reviews of Soviet literature on toxicity and hazards of chemicals" has published a review on carbaryl (IRPTC, 1982, 1989). Table 55. Maximum Residue Limits (MRLs) established by the Codex Alimentariusa Commodity MRL (mg/kg) Animal feedstuffs (green alfalfa, clover, corn, forage, cow pea 100 foliage, grasses, peanut hay, sorghum forage, soybean vines, sugarbeet tops, bean and pea vines) Bran (wheat) 20 Apricots, blackberries, boysenberries, nectarines, peaches, 10 raspberries, asparagus, okra, leafy vegetables (except brassica), nuts (whole), olives (fresh), sorghum grain, cherries, plums, kiwi fruit Blueberries, citrus fruit, cranberries, strawberries 7 Apples, bananas (pulp), grapes, beans, peas (including pod), 5 brassica, tomatoes, peppers, aubergines, pears, poultry skin, barley, oats, rice (in husk and hulled), rye, wheat Cucumbers, melons (Cantaloupe), pumpkins, squash 3 Root crop vegetables (beets, carrots, radishes, rutabagas, 2 parsnips), peanuts (ground-nuts, whole), wholemeal flour Cottonseed (whole), sweet corn (kernels), nuts (shelled), olives 1 (processed), soybeans (dry mature seed), cow-peas Poultry meat, eggs (without shells) 0.5 Potatoes, meat of cattle, sheep, and goats, sugarbeets, wheat 0.2 flour (white) Milk and milk products 0.1 aFrom:FAO/WHO (1986b). 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TREATMENT OF CARBAMATE PESTICIDE POISONING IN MAN (From EHC 64: Carbamate Pesticides - A General Introduction) All cases of carbamate poisoning should be dealt with as an emergency and the patient should be hospitalized as quickly as possible. Extensive descriptions of the treatment of poisoning by anticholinesterase agents are given in several major references (Kagan, 1977;Taylor, 1980; Plestina, 1984). The treatment is based on: (a) minimizing the absorption; (b) general supportive treatment; and (c) specific pharmacological treatment. I.1 Minimizing the absorption When dermal exposure occurs, decontamination procedures include removal of contaminated clothes and washing of the skin with alkaline soap or with a sodium bicarbonate solution. Particular care should be taken in the cleaning of the skin area where venupuncture is performed. Blood might be contaminated with carbamates and therefore inaccurate measures of ChE inhibition might result. Extensive eye irrigation with water or saline should also be performed. In the case of ingestion, vomiting can be induced, if the patient is conscious, by the administration of ipecacuanha syrup (10-30 ml) followed by 200 ml of water. However, this treatment is contraindicated in the case of pesticides dissolved in hydrocarbon solvents. Gastric lavage (with the addition of bicarbonate solution or activated charcoal) can also be performed, particularly in unconscious patients, taking care to prevent aspiration of fluids into the lungs (i.e., only after a tracheal tube has been put in place). The volumes of the fluids introduced in the stomach should be recorded and samples of gastric lavage frozen and stored for subsequent chemical analysis. If the formulation of the pesticide involved is available, it should also be stored for further analysis (i.e., detection of toxicologically relevant impurities). A purge to remove the ingested compound can be administered. I.2 General supportive treatment Artificial respiration (via a tracheal tube) should be started at the first sign of respiratory failure and maintained for as long as necessary. Cautious administration of fluids is advised as well as general supportive and symptomatic pharmacological treatment and absolute rest. I.3 Specific pharmacological treatment I.3.1 Atropine Atropine should be given, beginning with 2 mg iv repeated at 15 to 30-min intervals. The dose and the frequency of atropine treatment varies from case to case, but should maintain the patient fully atropinized (dilated pupils, dry mouth, skin flushing, etc.). I.3.2 Oxime reactivations Although it might be suspected that oxime cholinesterase reactivators would be as helpful in carbamate poisoning as they are in organophosphorous poisoning, this is not the case. There is experimental evidence that the pyridinium oxime 2-PAM is not effective in carbamate poisoning and there is some evidence that it makes poisoning by certain carbamates, including carbaryl, worse. I.3.3 Diazepam Diazepam should be included in the therapy of all but the mildest cases. Besides relieving anxiety it appears to counteract some aspects of CNS-derived symptoms that are not affected by atropine. Doses of 10 mg sc or iv are appropriate and may be repeated as required. Other centrally acting drugs and drugs that may depress respiration are not usually recommended in the absence of artificial respiration procedures. References to Annex I KAGAN, J.S. (1977) [The toxicity of organophosphorus pesticides.] Moscow (in Russian). PLESTINA, R. (1984) Prevention, diagnosis, and treatment of insecticide poisoning. Geneva, World Health Organization. (Unpublished WHO document VBC/84.889). TAYLOR, P. (1980) Anticholinesterase agents. In: Goodman, L.S. & Gilman, A., ed. The pharmacological basis of therapeutics. 6th ed., New York, Macmillan Publishing Co., pp. 100-119. RESUME ET EVALUATION, CONCLUSIONS ET RECOMMANDATIONS 1. Résumé et évaluation 1.1 Identité, propriétés et méthodes d'analyse Carbaryl est la dénomination commune d'un dérivé de l'acide carbamique, le N-méthylcarbamate de 1-naphtyle. Le produit technique consiste en un solide cristallin blanc, peut volatil et peu soluble dans l'eau, qui est stable à la lumière et à la chaleur mais qui s'hydrolyse facilement en milieu alcalin. Il existe une norme FAO pour le carbaryl qui stipule un degré de pureté de 98%, avec une limite pour les impuretés ( N-méthylcarmate de ß-naphtyle) de 0,05%. Pour les analyses portant sur le carbaryl et ses métabolites, il existe de nombreuses méthodes: chromatographie sur couche mince, spectrophotométrie, chromatographie en phase gazeuse, chromatographie en phase liquide à haute pression et spectrométrie de masse à ionisation chimique. La limite de détection peut descendre en-dessous du nanogramme et le taux de récupération dépasse généralement 80%. 1.2 Production et usages On utilise le carbaryl depuis une trentaine d'années comme insecticide agissant par contact et par ingestion avec certaines propriétés endothérapiques; il permet de lutter contre de nombreux vecteurs et ravageurs. La principale unité de production se trouve aux Etats-Unis d'Amérique. Plus de 290 fabricants proposent une gamme de formulations de carbaryl qui dépasse les 1500 produits. 1.3 Transport, distribution et transformation dans l'environnement Dans la plupart des conditions, le carbaryl ne persiste pas dans l'environnement. Dans l'eau, son temps de demi-hydrolyse dépend de la température, du pH et de la concentration initiale; il varie de quelques minutes à plusieurs semaines. Le principal produit de décomposition est le 1-naphtol. L'accumulation du carbaryl, exprimée sous la forme d'un facteur de bioconcentration dans l'environnement aquatique, et plus précisément chez les poissons d'eau douce, varie de 14 à 75. Le carbaryl s'adsorbe plus facilement sur les sols à forte teneur organique que sur les sols sableux. Aux doses d'emploi usuelles, et lorsqu'il est appliqué conformément "aux bonnes pratiques agricoles", il se dissipe rapidement, avec une demi-vie de 8 jours à un mois dans les conditions normales. Il arrive que le carbaryl soit entraîné par les pluies ou par les travaux agricoles de la surface vers les couches sous-jacentes (à un mètre de profondeur). Le carbaryl peut contaminer la végétation, soit au cours de l'épandage soit par migration à partir d'un sol contaminé. La décomposition du carbaryl dans l'environnement dépend de son degré de volatilisation, de photodécomposition et de dégradation chimique ou microbienne dans le sol, l'eau et les végétaux. la vitesse de décomposition est plus rapide en climat chaud. 1.4 Concentrations dans l'environnement et exposition humaine Dans la population générale, le carbaryl est principalement absorbé par la voie alimentaire. Les résidus présents dans des échantillons de rations totales sont relativement faibles, et ils vont de traces à 0,05 mg/kg. Aux Etats-Unis d'Amérique, on estime que pendant les premières années d'utilisation du carbaryl, l'ingestion journalière de carbaryl était de 0,15 mg/personne/jour (dans 7,4% des composites); cette dose est tombée à 0,003 mg/personne/jour en 1969 (dans seulement 0,8% des composites). Pendant la période d'épandage, on peut retrouver parfois du carbaryl dans les eaux de surface et les retenues d'eau. La population peut être exposée au carbaryl lors d'opérations de lutte de contre les ravageurs ou les vecteurs tant au domicile que sur les aires de loisir. Les travailleurs peuvent être exposés au carbaryl lors de la fabrication, de la formulation, de l'emballage, du transport et du stockage du produit ainsi que pendant son épandage. Les concentrations dans l'atmosphère des lieux de travail au cours de la production varient de < 1 mg/m3 à 30 mg/m3. Il peut y avoir une importante exposition cutanée chez les travailleurs de l'industrie et les ouvriers agricoles en cas de mesures de protection insuffisantes. 1.5 Cinétique et métabolisme Le carbaryl est rapidement absorbé au niveau des poumons et des voies digestives. Une dose de carbaryl dans l'acétone appliquée sur la peau de volontaires humains a été absorbée par voie percutanée à raison de 45% en 8 heures. Toutefois, les données obtenues in vitro concernant la pénétration cutanée ainsi que les données toxicologiques indiquent que l'absorption percutanée s'effectue généralement à un rythme beaucoup plus lent. Les principales voies métaboliques du carbaryl consistent en une hydroxylation du cycle et une hydrolyse. Il en résulte la formation de nombreux métabolites qui subissent ensuite une conjugaison avec formation de sulfates, de glucuronides et de mercapturates hydrosolubles qui sont excrétés dans l'urine. L'hydrolyse conduit à la formation de 1-naphtol, de dioxyde de carbone et de méthylamine. L'hydroxylation produit du 4-hydroxycarbaryl, du 5-hydroxycarbaryl, du N-hydroxyméthylcarbaryl, du 5,6-dihydro-5,6-dihydroxycarbaryl et du 1,4-naphtalènediol. Le principal métabolite chez l'homme est le 1-naphtol. Dans les conditions normales d'exposition, il est improbable que le carbaryl s'accumule chez les animaux. Il est excrété principalement par la voie urinaire étant donné que la détoxification de son produit d'hydrolyse, le 1-naphtol, s'effectue essentiellement par la formation de conjugués hydrosolubles. Dans le cas des métabolites du carbaryl, le cycle entérohépatique joue également un rôle considérable, en particulier après administration par voie orale. Le produit d'hydrolyse, l'acide N-naphtolcarbamique, se décompose spontanément en méthylamine et dioxyde de carbone. La méthylamine subit ensuite une déméthylation en dioxyde de carbone et formiate, ce dernier étant ultérieurement excrété, en majeure partie dans l'urine. Des métabolites du carbaryl sont également présents en faible proportion de la dose absorbée dans la salive et le lait. 1.6 Effets sur les êtres vivants dans leur milieu naturel Les valeurs de la CL50 pour les crustacés varient de 5 à 9 µg/litre (puces d'eau et mysidés), 8 à 25 µg/litre (orchesties) et 500 à 2500 µg/litre (écrevisses). Chez les insectes aquatiques, les limites de sensibilité sont du même ordre. Les plécoptères et éphémèroptères (perles et éphémères) en constituent les groupes les plus sensibles. Les mollusques sont moins sensibles avec des valeurs de la CE50 de l'ordre de quelques mg/litre. Dans le cas des poissons, la plupart des valeurs de la CL50 se situent entre 1 et 30 mg/litre. Les salmonidés constituent le groupe le plus sensible. La toxicité aiguë est faible pour les oiseaux. La DL50 pour la sauvagine et le gibier à plumes en général est > 1000 mg/kg. D'après les tests, l'oiseau le plus sensible est un francolin (Francolinus levaillanti) (DL50 = 56 mg/kg). Rien n'indique que les oiseaux aient eu à souffrir de l'effet des épandages effectués sur les zones forestières à la dose de 1,1 kg de carbaryl/ha. Le carbaryl est très toxique pour les abeilles et les lombrics. Dans le cas des abeilles, la DL50 par voie orale est de 0,16 µg/insecte (soit 1-2 mg/kg). On est fondé à penser que le carbaryl puisse avoir une influence temporaire sur la composition en espèces des écosystèmes terrestres et aquatiques. Par exemple, une étude a montré que les effets exercés sur certaines communautés d'invertébrés terrestres pourraient persister au moins 10 mois après un seul épandage. 1.7 Effets sur les animaux d'expérience et les systèmes d'épreuve in vitro La toxicité aiguë, exprimée sous la forme de la DL50, varie considérablement selon l'espèce, la formulation et le véhicule du produit. La DL50 estimative par voie orale pour le rat varie de 200 à 850 mg(kg. Les chats sont plus sensibles, avec une DL50 de 150 mg/kg. Les porcs et les singes le sont moins, avec une DL50 > 1000 mg/kg. Une concentration de 792 mg de matière active par m3, qui constitue la valeur maximale qu'on puisse obtenir pour un aérosol, a produit, au cours d'une exposition de 4 heures, une mortalité de 20% (1/5) chez des rattes. A des concentrations de 20 mg/m3, des aérosols de carbaryl ont provoqué une réduction de l'activité cholinestérasique chez des chats lors d'une exposition de 4 heures, mais cette concentration n'a eu aucun effet observable chez des rats. Le carbaryl est légèrement irritant pour l'oeil et n'a que peu ou pas de pouvoir sensibilisateur. Lors d'études à long terme, la concentration sans effet nocif observable a été évaluée à 10 mg/kg de poids corporel (200 mg/kg de nourriture) pour le rat et à 1,8 mg/kg de poids corporel (100 mg/kg de nourriture) pour le chien. Chez le chat, soumis à une exposition de longue durée par inhalation, la concentration sans effet nocif observable est de 0,16 mg/m3. Le carbaryl a un faible potentiel d'accumulation. 1.7.1 Reproduction On a montré que le carbaryl avait des effets indésirables sur la reproduction des mammifères et le développement périnatal chez un certain nombre d'espèces. Ces effets sur la reproduction consistent en une réduction de la fécondité, une diminution de l'effectif des portées et une réduction de la viabilité postnatale. Les effets toxiques du carbaryl sur le développement se traduisent pas un certain nombre de morts foetales, une réduction du poids foetal et la présence de malformations. A l'exception d'un petit nombre d'études, les effets nocifs sur la reproduction et le développement n'ont été constatés en totalité qu'à des doses manifestement toxiques pour la mère, et dans un certain nombre de cas, la mère était plus sensible au carbaryl que l'embryon ou le foetus. Ces effets toxiques sur la femelle gestante consistaient en une mortalité accrue, une réduction de la croissance et des dystocies. Les données indiquent que, par rapport à l'organisme adulte, la fonction de reproduction et le processus de développement des mammifères n'est pas particulièrement sensible au carbaryl. 1.7.2 Mutagénicité Un certain nombre de tests in vitro et in vivo ont été effectués sur la carbaryl afin d'en évaluer le pouvoir mutagène; divers points d'aboutissement de ces effets ont été étudiés sur divers systèmes (bactéries, levures, végétaux, insectes et mammifères). Les données disponibles montrent que le carbaryl n'a aucune tendance à endommager l'ADN. On ne dispose d'aucun rapport confirmant les caractères mutagènes suivants: induction de recombinaisons mitotiques, conversion génique, et synthèse non programmée de l'ADN chez les procaryotes (H. influenzae, B. subtilis) ni chez les eucaryotes ( S. cerevisiae, A. nidulans, lymphocytes humains en culture et hépatocytes de rat) in vitro. La recherche de mutations géniques, qui a donné lieu à un grand nombre d'épreuves sur systèmes bactériens, a toujours donné des résultats négatifs, sauf dans deux cas. Lors de plusieurs études portant sur les mutations géniques et effectuées sur des cellules mammaliennes in vitro, le carbaryl n'a donné qu'un seul résultat positif équivoque dans une de ces études. Toutefois, cette étude présentait un certain nombre de défauts et ce résultat n'a pas été confirmé par ceux d'autres études comparables. Des lésions chromosomiques ont été observées in vitro sur des cellules humaines, des cellules de rat et de hamster ainsi que des cellules végétales exposées à des fortes doses de carbaryl. Aucun effet de ce genre n'a été observé lors d'épreuves in vivo sur des systèmes mammaliens, même à des doses atteignant 1000 mg/kg. On a montré que le carbaryl perturbe le mécanisme d'élongation des fibres du fuseau chez les cellules végétales et mammaliennes in vitro. On ne sait pas encore très bien si les résultats des épreuves effectuées sur des végétaux sont extrapolables à l'homme. Compte tenu de la base de données sont on dispose actuellement, on peut conclure que rien n'autorise à soupçonner le carbaryl de présenter un risque d'effets mutagènes pour les cellules somatiques ou germinales de l'homme. Un dérivé nitrosé du carbaryl, le N-nitrosocarbaryl, peut provoquer des recombinaisons mitotiques et des conversions géniques chez les procaryotes (H. influenzae, B. subtilis) et les eucaryotes (S. cerevisiae) in vitro, et il donne des résultats positifs lors de "spot tests" effectués sur E. coli. En outre, les résultats expérimentaux indiquent que le N-nitrosocarbaryl se lie à l'ADN provoquant la formation de liaisons alcalino-sensibles et des cassures monocaténaires. Il n'est pas établi que le nitrosocarbaryl soit un agent clastogène in vivo (cellules de la moelle osseuse et cellules germinales) même à doses toxiques élevées. 1.7.3 Cancérogénicité De nombreuses études ont été consacrées au pouvoir cancérogène du carbaryl chez le rat et la souris. La plupart de ces études ont donné des résultats négatifs mais il s'agit de travaux anciens qui ne satisfont pas aux normes actuelles. Quoiqu'il en soit, de nouvelles études portant sur ces mêmes animaux et qui, elles, satisfont aux exigences modernes, sont actuellement en coursa. La dernière évaluation du CIRC (CIRC, 1987) a conclu que l'existence de cancers imputables au carbaryl n'était pas documentée chez l'homme et qu'on ne possédait pas de preuves suffisantes d'un pouvoir cancérogène de cette substance chez les animaux de laboratoire. Il n'a donc pas été possible de classer le carbaryl en fonction de son pouvoir cancérogène pour l'homme (Groupe 3). On a montré que le N-nitrosocarbaryl induisait des tumeurs locales chez les rats (sarcomes au point d'injection ou carcinomes spinocellulaires au niveau de la portion cardiaque de l'estomac, lorsque la substance est administrée par voie orale). Etant donné la biochimie du carbaryl chez l'homme, on peut considérer comme négligeable le risque de cancérisation par le N-nitrosocarbaryl résultant d'une exposition humaine au carbaryl. 1.7.4 Effets sur les différents organes et systèmes a) Système nerveux Les effets du carbaryl sur le système nerveux tiennent essentiellement à l'inhibition de la cholinestérase qui est généralement passagère. Ces effets ont été étudiés sur des rats et des singes. Des doses de 10 à 20 mg de carbaryl par kg, administrées pendant 50 jours par voie orale, ont provoqué des perturbations dans l'apprentissage et l'exécution de certaines épreuves par les rats traités. ______________ a Ces études n'ont pas encore fait l'objet d'un examen par le PISC. La société qui effectue ces travaux indique qu'aux doses les plus fortes étudiées, on note une augmentation significative de la fréquence des tumeurs chez les deux espèces. Lors d'une petite étude portant sur des porcs, du carbaryl administré pendant 72 à 82 jours en mélange avec la nourriture à raison de 150 mg/kg de poids corporel, a produit un certain nombre d'effets neuromusculaires. On a observé en outre chez des poulets ayant reçu de fortes doses de carbaryl, une faiblesse réversible des pattes. Aucun signe de démyélinisation n'a été observé dans le cerveau, le nerf sciatique ou les coupes de moelle épinière examinées au microscope. On n'a pas observé d'effets de ce genre à l'issue d'études à long terme sur des rongeurs. b) Système immunitaire Administré in vivo à des doses provoquant des signes cliniques manifestes, le carbaryl exerce divers effets sur le système immunitaire. Nombre de ces effets ont été observés à des doses voisines de la DL50. Dans la plupart des études où les doses utilisées permettaient la survie des lapins et des souris examinés, on n'a pas observé d'effets significatifs sur le système immunitaire. Plusieurs de ces travaux présentaient des défauts, par exemple un manque de cohérence et parfois des contradictions manifestes dans les résultats, défauts qui ne permettent pas de dégager des résultats obtenus un mécanisme immunotoxique bien défini. c) Sang Le carbaryl affecterait l'hémostase mais les résultats concernant le sens de cet effet sont contradictoires. On a observé que le carbaryl produisait une augmentation de la formation de méthémoglobine liée à la dose dans des érythrocytes de moutons présentant un déficit en glucose-6-phosphate-déshydrogénase. La sérum-albumine humaine réagit in vitro sur le groupement ester du carbaryl. Le carbaryl se lie aux acides aminés libres. d) Foie On a fait état de troubles affectant le métabolisme des glucides et la synthèse des protéines au niveau du foie ainsi que d'une perturbation de la fonction détoxifiante de cet organe chez les mammifères. Le carbaryl induit faiblement l'activité pharmacométabolisante des microsomes hépatiques. Il y a raccourcissement de la durée du sommeil induit par le phénobarbital. Les taux hépatiques de cytochrome P-450 et b5 sont augmentés. Ces modifi-cations du métabolisme hépatique pourraient expliquer en partie le triplement de la DL50 pour le carbaryl observé chez des rats préalablement traités par cette substance. e) Fonction gonadotrope Il a été signalé que le carbaryl augmentait la fonction gonadotrope de l'hypophyse chez le rat. 1.7.5 Mécanisme fondamental de la toxicité Le carbaryl est un inhibiteur de l'activité cholinestérasique. Cet effet est lié à la dose et rapidement réversible. On n'a pas observé de vieillissement de la cholinestérase carbamylée. Tous les métabolites reconnus du carbaryl ont une activité anticholinestérasique sensiblement plus faible que le carbaryl lui-même. 1.8 Effets sur l'homme Le carbaryl est facilement absorbé par inhalation et après administration par voie orale mais moins facilement pas la voie percutanée. Etant donné que l'inhibition de la cholinestérase est le principal mécanisme de l'action du carbaryl, le tableau clinique d'une intoxication par cette substance est dominé par les symptômes correspondants, à savoir: augmentation de la sécrétion bronchique, sueurs profuses, salivation et larmoiement; myosis, bronchoconstriction, crampes abdominales (vomissements et diarrhée); bradycardie; fasciculation des petits muscles (dans les cas graves le diaphragme et les muscles respiratoires sont également atteints); tachycardie, céphalées, vertiges, angoisses, confusion mentale, coma et dépression des centres respiratoires. Ces signes d'intoxication se manifestent rapidement après l'absorption et disparaissent aussi vite une fois que l'exposition a cessé. Des études contrôlées sur des volontaires humains ont montré que des doses uniques inférieures à 2 mg/kg étaient bien tolérées. Une dose unique de 250 mg (2,8 mg/kg) a suscité des symptômes modérés d'inhibition cholinestérasique (douleurs épigastriques et sueurs) en l'espace de 20 minutes. Les sujets ont complètement récupéré dans les 2 heures suivant un traitement par le sulfate d'atropine. En cas d'exposition excessive d'origine professionnelle au carbaryl, on observe des symptômes légers bien avant qu'une dose dangereuse ne soit absorbée, ce qui explique que les cas graves d'intoxication professionnelle par le carbaryl soient rares. Lors des épandages agricoles, l'exposition percutanée peut être importante. Cependant on n'observe en général aucun effet irritant local, encore que l'on ait décrit des éruptions cutanées à la suite l'éclaboussures accidentelles de carbaryl en formulation liquide. Les données concernant les effets du carbaryl sur le nombre de spermatozoïdes et la modification de leur morphologie chez les travailleurs de l'industrie sont contradictoires. Aucun effet indésirable sur la reproduction n'a été signalé. L'indicateur biologique le plus sensible de l'exposition au carbaryl est l'apparition de 1-naphtol dans les urines et la diminution de l'activité cholinestérasique du sang. On peut donc utiliser la concentration urinaire du 1-naphtol comme indicateur biologique à condition qu'il n'y ait pas de 1-naphtol sur le lieu de travail. Lors de certains cas d'exposition professionnelle, on a constaté que 40% des échantillons d'urine contenaient plus de 10 mg de 1-naphtol total par litre. Dans un cas d'intoxication aiguë, on en a trouvé 31 mg/litre d'urine. On considère qu'il y a danger à partir de 10 mg/litre et que les symptômes apparaissent à partir de 30 mg de 1-naphtol par litre d'urine (Fiche d'information sur le carbaryl, OMS, 1973, VBC/DS/75.3). La mesure de l'activité cholinestérasique peut constituer un test très sensible pour la surveillance médical des travailleurs, à condition que le dosage soit effectué peu après l'exposition. 2. Conclusions On estime que le carbaryl est peu dangereux pour l'homme en raison de sa faible tension de vapeur, de sa décomposition rapide, de la désinhibition spontanée également rapide de la cholinestérase et en raison du fait que les symptômes d'intoxication apparaissent bien avant qu'une dose dangereuse ne se soit accumulée dans l'organisme. On ne dispose pas encore de bonnes études de cancérogénicité qui satisfassent aux normes actuelles en la matière. 2.1 Exposition de la population générale Les quantités résiduelles de carbaryl qui demeurent dans les denrées alimentaires et l'eau de boisson après l'utilisation normale de cet insecticide en agriculture, sont très inférieures à la dose journalière acceptable (DJA) (0,01 mg/kg de poids corporel/jour) et il est improbable qu'elles puissent constituer un risque pour la santé de la population dans son ensemble. 2.2 Sous-groupes de population exposés à un risque élevé Lorsqu'on utilise du carbaryl à des fins de santé publique, soit au domicile soit sur des aires de loisir, il y a un risque d'exposition excessive si l'on ne suit pas les règles concernant son emploi. 2.3 Exposition professionnelle En faisant respecter des méthodes de travail raisonnables et notamment un certain nombre de précautions de sécurité et des mesures de protection individuelle, avec une surveillance convenable, il n'existe aucun risque qui puisse résulter d'une exposition professionnelle au cours de la fabrication, de la formulation et de l'épandage du carbaryl. Les produits non dilués doivent être manipulés avec de grandes précautions car toute faute de manipulation peut entraîner une contamination cutanée. Sur le lieu de travail, la concentration atmosphérique ne doit pas dépasser 5 mg/m3. 2.4 Effets sur l'environnement Le carbaryl est toxique pour les abeilles et les lombrics. On ne doit pas procéder à son épandage pendant la floraison. En utilisation normale, le carbaryl ne devrait pas poser des problème écologique. Il est adsorbé en grande partie sur les particules de sol et il ne passe pas facilement par lessivage dans les eaux souterraines. Il subit une décomposition rapide dans l'environnement et n'est donc pas persistant. L'emploi de carbaryl ne devrait pas entraîner d'effets nocifs à court terme sur l'écosystème. 3. Recommandations * La manipulation et l'épandage du carbaryl doivent s'effectuer en observant les précautions qui s'imposent pour tous les pesticides. On suivra rigoureusement les instructions d'utilisation qui figurent sur l'emballage. * La fabrication, la formulation, l'emploi et l'élimination du carbaryl doivent se faire avec toutes les précautions voulues pour réduire au minimum la contamination de l'environnement. * Les travailleurs qui sont soumis à une exposition régulière doivent faire l'objet d'un contrôle médical périodique. * La chronologie des épandages de carbaryl doit être réglée de manière à éviter tout effet sur les espèces non visées. * Il importe d'effectuer des études de cancérogénicité satisfaisant aux normes modernes. RESUMEN Y EVALUACION, CONCLUSIONES Y RECOMENDACIONES 1. Resumen y evaluación 1.1 Identidad, propiedades y métodos analíticos El 1-naftil N-metil carbamato, derivado del ácido carbámico, se conoce por el nombre común de carbarilo. El producto de calidad técnica es un sólido cristalino blanco, de baja volatilidad y escasa hidrosolubilidad, y estable a la luz y al calor, pero fácilmente hidrolizable en medio alcalino. La FAO ha establecido una especificación mínima de pureza del 98%, con un límite de impureza del 0,05% para el ß-naftil N-metil carbamato. Para analizar el carbarilo y sus metabolitos, se pueden utilizar numerosas técnicas, como la cromatografía de capa fina, la espectrofotometría, la cromatografía de gases, la cromatografía líquida de alta presión y la espectrometría de masas con ionización química. Pueden alcanzarse límites de detección inferiores a un nanogramo, y la recuperación supera por lo general el 80%. 1.2 Producción y usos El carbarilo se viene utilizando desde hace unos 30 años como insecticida de contacto y de ingestión, posee algunas propiedades sistémicas y permite combatir una amplia serie de plagas. La planta de fabricación más importante está en los Estados Unidos. Más de 290 fabricantes transforman el carbarilo para integrarlo en más de 1500 productos diferentes. 1.3 Transporte, distribución y transformación en el medio ambiente En la mayoría de las situaciones, el carbarilo no persiste en el entorno. En el agua, su semivida por hidrólisis depende de la temperatura, del pH y de la concentración inicial, y varía entre varios minutos y varias semanas. El principal producto de degradación es el 1-naftol. Se ha estudiado la acumulación de carbarilo en peces de agua dulce, y expresada como factor de bioconcentración en el medio acuático se ha cifrado en valores comprendidos entre 14 y 75. El carbarilo es adsorbido más fácilmente en los suelos que poseen un alto contenido orgánico que en los suelos arenosos. A un ritmo habitual de aplicación conforme con unas "prácticas agrícolas adecuadas", la disipación es rápida, con una semivida de entre 8 días y un mes en condiciones normales. Ocasionalmente, por efecto de la lluvia y del cultivo agrícola, el carbarilo es transportado de la superficie al subsuelo (a un metro de la superficie). El carbarilo contamina la vegetación durante el rociamiento o por desplazamiento hasta las plantas a través del suelo contaminado. La degradación del carbarilo en el entorno depende del grado de volatilización, fotodescomposición y degradación química y microbiana que se produzca en el suelo, el agua y las plantas. La descomposición es más rápida cuando el clima es cálido. 1.4 Niveles ambientales y exposición humana La principal fuente de ingestión de carbarilo entre la población general son los alimentos. Los residuos hallados en muestras de la ingesta alimentaria total son relativamente escasos, pues oscilan entre cantidades ínfimas y 0,05 mg/kg. En los Estados Unidos, la ingesta diaria durante los primeros años de aplicación de carbarilo fue de 0,15 mg/día por persona (lo contenían el 7,4% de los compuestos); la cifra se redujo a 0,003 mg/día por persona en 1969 (sólo lo contenían un 0,8% de los compuestos). Durante el periodo de aplicación se encuentra carbarilo ocasionalmente en las aguas superficiales y los embalses. La población general puede estar expuesta al carbarilo durante las operaciones de lucha contra las plagas, en su vivienda o en zonas recreativas. Los trabajadores pueden estar expuestos al carbarilo durante su fabricación, formulación, envasado, transporte y almacenamiento, así como durante y después de su aplicación. Las concentraciones halladas en la atmósfera del lugar de trabajo durante su producción oscilaron entre < 1 mg/m3 y 30 mg/m3. Si las medidas de protección son inadecuadas, los trabajadores industriales y agrícolas pueden sufrir exposiciones cutáneas importantes. 1.5 Cinética y metabolismo El carbarilo es absorbido rápidamente por los pulmones y el tracto digestivo. En voluntarios se observó una absorción cutánea del 45% a las 8 horas de aplicar una dosis del producto diluida en acetona. Sin embargo, los datos referentes a la penetración cutánea in vitro y a la toxicidad indican que la absorción cutánea se produce por lo general a una velocidad mucho menor. Las principales vías metabólicas del carbarilo son la hidroxilación del anillo y la hidrólisis. El resultado son numerosos metabolitos que experimentan conjugación, con formación de sulfatos, glucurónidos y mercapturatos hidrosolubles, que se excretan por la orina. Como resultado de la hidrólisis se forman 1-naftol, dióxido de carbono y metilamina. La hidroxilación da lugar a 4-hidroxicarbarilo, 5-hidroxicarbarilo, N-hidroximetilcarbarilo, 5-6-dihidro-5-6-dihidroxicarbarilo y 1,4-naftalendiol. El metabolito principal en el hombre es el 1-naftol. En condiciones normales de exposición, el carbarilo rara vez se acumula en los animales. El producto se excreta principalmente por la orina, debido a que la detoxificación del producto de su hidrólisis, el 1-naftol, se produce principalmente por transformación en conjugados hidrosolubles. La circulación enterohepática de los metabolitos del carbarilo es también considerable, sobre todo tras su administración oral. El producto de la hidrólisis, el ácido carbámico N-naftol, se descompone espontáneamente en metilamina y dióxido de carbono. La posterior desmetilación de la metilamina da lugar a dióxido de carbono y formato, siendo este último excretado principalmente por la orina. Un pequeño porcentaje de las dosis de carbarilo absorbidas aparecen como metabolitos en la saliva y la leche. 1.6 Efectos en otros organismos en el medio ambiente En los crustáceos, las CL50 oscilan entre 5 y 9 µg/litro (pulgas de agua, mísidos), 8 y 25 µg/litro (anfípodos), y 500 y 2500 µg/litro (cangrejos de río). El margen de sensibilidad es parecido en los insectos acuáticos; Plecoptera y Ephemeroptera (gusarapa y cachipollas) son los grupos más sensibles. Los moluscos son menos sensibles, situándose su CE50 en niveles de unos pocos mg/litro. En cuanto a los peces, la mayoría de las CL50 están comprendidas entre 1 y 30 mg/litro; el grupo más sensible son los salmónidos. En el caso de las aves la toxicidad aguda es baja. La DL50 para las aves de caza, sean acuáticas o terrestres, es > 1000 mg/kg. El ave más sensible analizada es el mirlo de alas rojas (DL50 = 56 mg/kg). En zonas forestales rociadas con 1,1 kg de carbarilo por hectárea no se observó ningún efecto en las aves locales. El carbarilo es muy tóxico para las abejas y las lombrices de tierra. La DL50 oral para las primeras es de 0,18 µg/abeja (aproximadamente 1-2 mg/kg). Hay indicios de que el carbarilo puede alterar temporalmente la composición de especies en los ecosistemas tanto terrestres como acuáticos. Por ejemplo, un estudio reveló que en determinadas colonias de invertebrados terrestres sus efectos pueden persistir durante por lo menos 10 meses tras una sola aplicación. 1.7 Efectos en animales de experimentación y en sistemas de prueba in vitro La toxicidad aguda, expresada como DL50, varía considerablemente según las especies, fórmulas y vehículos. Las estimaciones de la DL50 oral en la rata oscilan entre 200 y 850 mg/kg. Los gatos son más sensibles, pues presentan una DL50 de 150 mg/kg. Los cerdos y los monos son menos sensibles, pues su DL50 es > 1000 mg/kg. La exposición a 792 mg de ingrediente activo de carbarilo nebulizado, que es la máxima concentración a la que se llegó durante 4 horas, provocó la muerte de una de cinco ratas hembra. Aerosoles de carbarilo a concentraciones de 20 mg/m3 dieron lugar a una disminución de la actividad colinesterasa (ChEA) en gatos durante exposiciones únicas de 4 horas, pero esa misma concentración no tuvo efectos observables en ratas. El carbarilo produce leves irritaciones oculares y tiene un potencial de sensibilización escaso o nulo. Estudios prolongados revelaron un NOEL de 10 mg/kg de peso corporal (200 mg/kg ingesta alimentaria) en las ratas, y de 1,8 mg/kg de peso corporal (100 mg/kg dieta) en los perros. El NOEL por inhalación prolongada es de 0,16 mg/m3 en los gatos. El potencial de acumulación de carbarilo es bajo. 1.7.1 Reproducción Se ha demostrado que el carbarilo tiene efectos adversos sobre la reproducción y el desarrollo perinatal en diversas especies de mamíferos. Los efectos sobre la reproducción comprenden problemas de infertilidad, una disminución del tamaño de las camadas y una reducción de la viabilidad postnatal. Los efectos tóxicos sobre el desarrollo observados son un aumento de la mortalidad in utero, una disminución del peso del feto y la aparición de malformaciones. Salvo en un reducido número de estudios, todos los efectos adversos sobre la reproducción y el desarrollo se observaron sólo a dosis manifiestamente tóxicas para la madre, y en varios casos ésta resultó ser más sensible al carbarilo que su prole. Entre los efectos tóxicos para la madre cabe citar la letalidad, una disminución del crecimiento y la distocia. Los datos disponibles indican que los procesos de reproducción y desarrollo de los mamíferos no son especialmente sensibles al carbarilo en comparación con la susceptibilidad del organismo adulto. 1.7.2 Mutagenicidad Se ha evaluado la posible mutagenicidad del carbarilo mediante diversas pruebas in vitro e in vivo, empleando para ello bacterias, levadura, plantas, insectos y mamíferos, y analizando diversos puntos finales. Según los datos disponibles, el carbarilo no es lesivo para el ADN. No se ha notificado ningún dato que confirme que ha habido inducción de la recombinación mitótica, conversión génica o síntesis imprevista de ADN en procariotas (H. influenzae, B. subtilis) y eucariotas ( S. cerevisiae, A. nidulans, linfocitos humanos en cultivo, y hepatocitos de rata) in vitro. Se obtuvieron resultados negativos en las pruebas de detección de mutaciones génicas en un gran número de ensayos realizados con bacterias, salvo en dos casos. En varios estudios de mutagenicidad del carbarilo llevados a cabo con células de mamífero in vitro, se obtuvo sólo un resultado positivo equívoco en un estudio de células en cultivo. Ese estudio, sin embargo, presentaba varias deficiencias y sus resultados no han sido confirmados en estudios comparables. Se han notificado lesiones cromosómicas a altas dosis de carbarilo en células humanas y de rata y hámster in vitro, así como en plantas. No se han observado efectos de ese tipo en pruebas in vivo con mamíferos, ni siquiera a dosis de hasta 1000 mg/kg. Se ha mostrado que el carbarilo altera el mecanismo de las fibras del huso en células de plantas y mamíferos in vitro. Es dudoso el interés de realizar ensayos con plantas para extrapolar sus resultados al hombre. Cabe concluir que los datos disponibles no corroboran la suposición de que el carbarilo plantea un riesgo de inducción de cambios génicos en las células somáticas o germinales del hombre. El producto nitrosado del carbarilo, el N-nitrosocarbarilo, puede inducir fenómenos de recombinación mitótica y conversión génica en los procariotas (H. influenzae, B. subtilis) y eucariotas (S. cerevisiae) in vitro, y arroja resultados positivos en las pruebas in situ con E. coli. Además, resultados experimentales indican que el N-nitrosocarbarilo se une al ADN, causando la ruptura de los enlaces alcalisensibles y de las cadenas simples. No se ha demostrado que el nitrosocarbarilo sea clastógeno in vivo (médula ósea y células germinales), ni siquiera a dosis tóxicas elevadas. 1.7.3 Carcinogenicidad Se han realizado numerosos estudios en la rata y el ratón para determinar los posibles efectos carcinógenos del carbarilo. Los resultados de la mayoría de esos estudios fueron negativos, pero se trata de trabajos realizados hace muchos años y que no satisfacían los criterios actuales. Se están realizando nuevos estudios conformes con los actuales criterios en ratones y ratas.a En la más reciente evaluación del CIIC (CIIC, 1987) se llegaba a la conclusión de que no había información sobre el cáncer en el hombre y de que los indicios de carcinogenicidad en animales de experimentación eran insuficientes. No se podía clasificar el carbarilo en lo que se refiere a su carcinogenicidad para la especie humana (Grupo 3). Se ha mostrado que el N-nitrosocarbarilo induce la aparición de tumores localmente en la rata (sarcoma en el lugar de la inyección o carcinoma de células escamosas del antro cardiaco al administrarlo por vía oral). Teniendo en cuenta las transformaciones químicas que sufre el carbarilo en el hombre, el riesgo de carcinogenicidad por N-nitrosocarbarilo como consecuencia de la exposición a esta sustancia puede considerarse insignificante. 1.7.4 Efectos en distintos órganos y sistemas a) Sistema nervioso Los efectos del carbarilo sobre el sistema nervioso se deben principalmente a la inhibición de la colinesterasa y son por lo general temporales. En unos estudios sobre los efectos en el sistema nervioso central de ratas y monos se observó que la administración oral de 10-20 mg/kg durante 50 días provocaba trastornos del aprendizaje y del comportamiento en las ratas. En un pequeño estudio realizado con cerdos, el carbarilo (150 mg/kg de peso corporal en la alimentación durante 72-82 días) tuvo diversos efectos neuromusculares. Se observó una debilidad reversible de las patas en pollos sometidos a altas dosis de carbarilo. No se observaron signos de desmielinización en los cortes de cerebro, nervio ciático o médula espinal examinados al microscopio. Tampoco se notaron efectos de esa índole en estudios prolongados realizados con roedores. b) Sistema inmunitario Se ha notificado que el carbarilo administrado in vivo a dosis que producen claros signos clínicos tiene diversos efectos en el sistema inmunitario. Muchos de los efectos descritos se detectaron a dosis cercanas a la DL50. La mayoría de los estudios llevados a cabo con conejos y ratones a dosis compatibles con la supervivencia no han revelado efectos de importancia en el sistema ______________ a El IPCS aún no ha examinado esos estudios. La sociedad encargada de realizarlos ha indicado que se observa un aumento significativo de tumores a la dosis màs elevada en las dos especies. inmunitario. Sin embargo, varios de esos estudios adolecían de incoherencia y a veces de contradicción patente entre los resultados, lo cual no permite describir un mecanismo inmunotóxico bien definido. c) Sangre Se ha señalado que el carbarilo afecta a la coagulación, pero existe cierta controversia en cuanto al tipo de efecto. En eritrocitos de oveja con déficit de glucosa-6-fosfato deshidrogenasa, el carbarilo provocó un aumento dosis-dependiente de la formación de metahemoglobina (Met-Hb). La albúmina sérica humana reaccionó in vitro con el grupo éster del carbarilo. El carbarilo se une a los aminoácidos libres de la sangre. d) Hígado Se han señalado trastornos del metabolismo de los carbohidratos y de la síntesis de proteínas, así como de la función de detoxificación en el hígado de ciertos mamíferos. El carbarilo es un inductor ligero de la actividad de metabolización de medicamentos que reside en los microsomas hepáticos. Se observa un acortamiento del periodo de sueño inducido por el fenobarbital. Los niveles hepáticos de citocromo P-450 y b5 aumentan. Los cambios del metabolismo hepático son tal vez parcialmente responsables de la triplicación de la DL50 por carbarilo en ratas tratadas previamente con dicho compuesto. e) Función gonadotrópica Se ha señalado que el carbarilo estimula la función gonadotrópica de la hipófisis de la rata. 1.7.5 Mecanismo principal de toxicidad El carbarilo inhibe la acción de la colinesterasa, efecto que depende de la dosis y es fácilmente reversible. No se observó ningún fenómeno de "maduración" de la colinesterasa carbamilada. Todos los metabolitos identificados del carbarilo son considerablemente menos activos que éste como inhibidores de la colinesterasa. 1.8 Efectos en el hombre El carbarilo es fácilmente absorbido por inhalación y por vía oral, y menos fácilmente por vía cutánea. Como su principal mecanismo de acción es la inhibición de la colinesterasa (ChE), en el cuadro clínico de la intoxicación predominan los síntomas de esa inhibición, tales como: hipersecreción bronquial, aumento de la sudación, de la salivación y del lagrimeo; pupilas puntiformes, broncoconstricción, espasmos abdominales (vómitos y diarrea); bradicardia; fasciculación de los músculos finos (que también afecta en los casos graves al diafragma y a los músculos respiratorios); taquicardia; cefalea, vértigo, ansiedad, confusión mental, convulsiones y coma; y depresión del centro respiratorio. Los signos de intoxicación aparecen rápidamente tras la absorción y desaparecen pronto al cesar la exposición. En estudios controlados realizados con voluntarios se observó una buena tolerancia a dosis únicas inferiores a 2 mg/kg. Una dosis única de 250 mg (2,8 mg/kg) provocó síntomas moderados de inhibición de la ChE (dolor epigástrico y sudación) al cabo de 20 minutos. La recuperación completa se produjo tras dos horas de tratamiento con sulfato de atropina. En los casos de sobreexposición ocupacional al carbarilo, se observan síntomas leves mucho antes de que llegue a absorberse una dosis peligrosa; de ahí que raras veces se produzcan casos graves de intoxicación ocupacional por este compuesto. Durante las aplicaciones agrícolas, la exposición cutánea puede desempeñar un papel importante. No suelen observarse efectos irritantes locales, pero se ha descrito la aparición de exantema cutáneo tras la salpicadura accidental con formulaciones de carbarilo. Los datos acerca de los efectos del carbarilo sobre el número y la morfología de los espermatozoides en trabajadores industriales son discordantes. No se han notificado efectos adversos sobre la reproducción. El indicador biológico más sensible de la exposición al carbarilo es la aparición de 1-naftol en la orina y una disminución de la actividad ChE de la sangre. Si no hay 1-naftol en el entorno de trabajo, los niveles urinarios de este producto se pueden emplear como indicador biológico. Durante la exposición ocupacional, el 40% de las muestras de orina contenían más de 10 mg de 1-naftol/litro. En un caso de intoxicación aguda se hallaron 31 mg/litro en la orina. El nivel de riesgo es > 10 mg/litro, y el nivel de aparición de síntomas, 30 mg 1-naftol/litro de orina (hoja de datos sobre el carbarilo, OMS, 1973, VBC/DS/75.3). El análisis de la actividad ChE puede ser una prueba de gran sensibilidad a efectos de vigilancia, siempre que se realice poco después de la exposición. 2. Conclusiones Se considera que los riesgos que plantea el carbarilo para el ser humano son escasos, debido a su baja presión de vapor, a su rápida degradación y a la recuperación espontánea y rápida de la colinesterasa inhibida, así como al hecho de que los síntomas aparecen normalmente mucho antes de que pueda haberse acumulado en el organismo una dosis peligrosa. Aún no se dispone de estudios satisfactorios sobre la carcinogenicidad conformes con los criterios actuales. 2.1 Exposición de la población general Los niveles de residuos de carbarilo presentes en los alimentos y el agua de bebida, resultantes de su uso normal en la agricultura, son muy inferiores a la ingesta diaria admisible (IDA) (0,01 mg/kg de peso corporal al día), y difícilmente pueden representar un riesgo para la salud de la población general. 2.2 Subpoblaciones de alto riesgo El uso del carbarilo con fines de salud pública en la vivienda o en zonas recreativas puede ser causa de sobreexposición si se descuidan las normas aconsejadas para su aplicación. 2.3 Exposición ocupacional Si se vela por el cumplimiento de unas prácticas laborales razonables, en particular las medidas de seguridad, la protección del personal y una supervisión adecuada, la exposición ocupacional durante la fabricación, formulación y aplicación de carbarilo no planteará riesgos. Las concentraciones no diluidas se deben manejar con sumo cuidado, dado que unas prácticas laborales incorrectas pueden ser causa de contaminación cutánea. Las concentraciones en el aire del lugar de trabajo no deberían superar los 5 mg/m3. 2.4 Efectos en el medio ambiente El carbarilo es tóxico para las abejas y las lombrices. No debería aplicarse a los cultivos durante la floración. Si se emplea normalmente, el carbarilo no debería suscitar preocupación desde el punto de vista del medio ambiente. El carbarilo se adsorbe en buena parte en el suelo y no se lixivia fácilmente hacia las aguas subterráneas. Se degrada rápidamente en el entorno, por lo que no tiende a persistir. El uso de carbarilo no debería tener efectos nocivos a corto plazo en el ecosistema. 3. Recomendaciones * La manipulación y la aplicación del carbarilo se deberían realizar adoptando las precauciones previstas para todo plaguicida y siguiendo minuciosamente las instrucciones suministradas en el envase para emplear correctamente el producto. * Deberían controlarse cuidadosamente la fabricación, la formulación, el uso y la eliminación del carbarilo, con objeto de reducir al mínimo la contaminación del medio ambiente. * Los trabajadores expuestos regularmente al producto deberían someterse a chequeos periódicos. * Debería determinarse la época de aplicación del carbarilo de manera que no afecte a las especies que no se desee combatir. * Deberían realizarse estudios de carcinogenicidad que satisfagan los criterios actuales.
See Also: Carbaryl (IARC Summary & Evaluation, Volume 12, 1976) Carbaryl (ICSC) Carbaryl (PDS) Carbaryl (PIM 147)