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Diflubenzuron. Environmental Health Criteria Report 184. WHO International Programme on Chemical Safety. Published in 1996.
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UNITED NATIONS ENVIRONMENT PROGRAMME INTERNATIONAL LABOUR ORGANISATION WORLD HEALTH ORGANIZATION INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY ENVIRONMENTAL HEALTH CRITERIA 184 Diflubenzuron This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organisation, or the World Health Organization. First draft prepared by Dr M. Tasheva, Sofia, Bulgaria Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization, and produced within the framework of the Inter-Organization Programme for the Sound Management of Chemicals. World Health Organization Geneva, 1996 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. The Inter-Organization Programme for the Sound Management of Chemicals (IOMC) was established in 1995 by UNEP, ILO, the Food and Agriculture Organization of the United Nations, WHO, the United Nations Industrial Development Organization and the Organisation for Economic Co-operation and Development (Participating Organizations), following recommendations made by the 1992 UN Conference on Environment and Development to strengthen cooperation and increase coordination in the field of chemical safety. The purpose of the IOMC is to promote coordination of the policies and activities pursued by the Participating Organizations, jointly or separately, to achieve the sound management of chemicals in relation to human health and the environment. WHO Library Cataloguing in Publication Data Diflubenzuron. (Environmental health criteria ; 184) 1. Diflubenzuron - adverse effects 2. Diflubenzuron - toxicity 3. Insecticides - adverse effects 4. Insecticides - toxicity 5. Environmental exposure I. Series ISBN 92 4 157184 1 (NLM Classification: WA 240) 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 1996 Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. All rights reserved. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. CONTENTS ENVIRONMENTAL HEALTH CRITERIA FOR DIFLUBENZURON Preamble 1. SUMMARY AND EVALUATION; CONCLUSIONS AND RECOMMENDATIONS 1.1. Summary 1.1.1. Identity, physical and chemical properties, and analytical methods 1.1.2. Sources of human and environmental exposure 1.1.3. Environmental transport, distribution and transformation 1.1.4. Environmental levels and human exposure 1.1.5. Kinetics and metabolism in laboratory animals 1.1.6. Effects on laboratory mammals and in vitro test systems 1.1.7. Effects on humans 1.1.8. Effects on other organisms in the laboratory and field 1.2. Evaluation 1.2.1. Evaluation of human health risks 1.2.2. Evaluation of effects on the environment 1.2.3. Toxicological criteria for setting guidance values 1.3. Conclusions and recommendations 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS 2.1. Identity 2.2. Physical and chemical properties 2.3. Conversion factor 2.4. Analytical methods 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1. Natural occurrence 3.2. Anthropogenic sources 3.2.1. Production levels and processes 3.2.2. Formulations 3.2.3. Uses 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, TRANSFORMATION AND FATE 4.1. Appraisal 4.2. Transport and distribution between media 4.2.1. Soil mobility 4.2.2. Dissipation 4.2.3. Evaporation 4.2.4. Crop residue data 4.3. Transformation 4.3.1. Abiotic degradation 4.3.1.1 Photolysis 4.3.1.2 Hydrolysis 4.3.2. Biodegradation 4.3.2.1 Water 4.3.2.2 Soil 4.4. Bioaccumulation and biomagnification 4.5. Interaction with other physical, chemical or biological factors 4.6. Ultimate fate following use 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1. Environmental levels 5.1.1. Air 5.1.2. Water 5.1.3. Food and feed 5.1.4. Forest plants and litter 5.1.5. Aquatic organisms 5.2. General population exposure 5.3. Occupational exposure during manufacture, formulation or use 6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS 6.1. Absorption 6.2. Distribution 6.3. Metabolic transformation 6.3.1. Metabolites - distribution, excretion, retention and turnover 6.4. Elimination and excretion 6.5. Retention and turnover 6.5.1. Biological half-life 7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS 7.1. Single exposure 7.2. Short-term exposure 7.3. Long-term exposure 7.4. Skin and eye irritation; sensitization 7.5. Reproductive toxicity, embryotoxicity and teratogenicity 7.6. Mutagenicity and related end-points 7.7. Carcinogenicity 7.8. Other special studies 7.8.1. Special studies on met- and sulfhaemoglobin formation 7.9. Toxicity of metabolites 7.9.1. Carcinogenicity studies with 4-chloroaniline 8. EFFECTS ON HUMANS 9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD 9.1. Laboratory experiments 9.1.1. Microorganisms 9.1.1.1 Water 9.1.1.2 Soil 9.1.2. Aquatic organisms 9.1.2.1 Microorganisms 9.1.2.2 Plants 9.1.2.3 Invertebrates 9.1.2.4 Vertebrates 9.1.3. Terrestrial organisms 9.1.3.1 Plants 9.1.3.2 Invertebrates 9.1.3.3 Vertebrates 9.2. Field observations 9.2.1. Microorganisms 9.2.1.1 Water 9.2.1.2 Soil 9.2.2. Aquatic organisms 9.2.2.1 Plant 9.2.2.2 Invertebrates 9.2.2.3 Vertebrates 9.2.3. Terrestrial organisms 9.2.3.1 Invertebrates 9.2.3.2 Vertebrates 10. 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It is accepted that the following criteria should initiate the updating of an EHC monograph: new data are available that would substantially change the evaluation; there is public concern for health or environmental effects of the agent because of greater exposure; an appreciable time period has elapsed since the last evaluation. All Participating Institutions are informed, through the EHC progress report, of the authors and institutions proposed for the drafting of the documents. A comprehensive file of all comments received on drafts of each EHC monograph is maintained and is available on request. The Chairpersons of Task Groups are briefed before each meeting on their role and responsibility in ensuring that these rules are followed. WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR DIFLUBENZURON Members Dr T. Bailey, US Environmental Protection Agency, Washington DC, USA Dr A.L. Black, Department of Human Services and Health, Canberra, Australia Mr D.J. Clegg, Carp, Ontario, Canada Dr S. Dobson, Institute of Terrestrial Ecology, Monks Wood, Abbots Ripton, Huntingdon, Cambridgeshire, United Kingdom (Vice-Chairman) Dr P.E.T. Douben, Her Majesty's Inspectorate of Pollution, London, United Kingdom (EHC Joint Rapporteur) Dr P. Fenner-Crisp, US Environmental Protection Agency, Washington DC, USA Dr R. Hailey, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, USA Ms K. Hughes, Environmental Health Directorate, Health Canada, Ottawa, Ontario, Canada (EHC Joint Rapporteur) Dr D. Kanungo, Central Insecticides Laboratory, Government of India, Ministry of Agriculture & Cooperation, Directorate of Plant Protection, Quarantine & Storage, Faridabad, Haryana, India Dr L. Landner, MFG, European Environmental Research Group Ltd, Stockholm, Sweden Dr M.H. Litchfield, Melrose Consultancy, Denmans Lane, Fontwell, Arundel, West Sussex, United Kingdom (CAG Joint Rapporteur) Professor M. Lotti, Institute of Occupational Medicine, University of Padua, Padua, Italy (Chairman) Professor D.R. Mattison, University of Pittsburgh, Graduate School of Public Health, Pittsburgh, Pennsylvania, USA Dr J. Sekizawa, National Institute of Health Sciences, Tokyo, Japan Dr P. Sinhaseni, Chulalongkorn University, Bangkok, Thailand Dr S.A. Soliman, King Saud University, Bureidah, Saudi Arabia Dr M. Tasheva, National Centre of Hygiene, Medical Ecology and Nutrition, Sofia, Bulgaria (CAG Joint Rapporteur) Mr J.R. Taylor, Pesticides Safety Directorate, Ministry of Agriculture, Fisheries and Food, York, United Kingdom Dr H.M. Temmink, Wageningen Agricultural University, Wageningen, The Netherlands Dr M.I. Willems, TNO Nutrition and Food Research Institute, Zeist, The Netherlands Representatives of GIFAPa (Groupement International des Associations Nationales de Fabricants de Produits Agrochimiques) Dr M. Bliss, Jr., ISK Biosciences Corporation, Mentor, Ohio, USA Dr A.C. Dykstra, Registration Department BPID, Solvay-Duphar BV, CP Weesp, The Netherlands Dr H. Frazier, ISK Biosciences Corporation, Mentor, Ohio, USA Dr R. Gardiner, GIFAP, Brussels, Belgium Dr B. Julin, Regulatory Affairs, Du Pont de Nemours (Belgium), Agricultural Products Department, Mercure Centre, Brussels, Belgium Dr S.M. Kennedy (Environmental Science), Du Pont de Nemours (Belgium), Agricultural Products Department, Mercure Centre, Brussels, Belgium Dr J. Killeen, ISK Biosciences Corporation, Mentor, Ohio, USA Dr Th. S.M. Koopman, Toxicology Department, Solvay-Duphar BV, CP Weesp, The Netherlands Dr R.L. Mull, Du Pont Agricultural Products, Wilmington, Delaware, USA Dr J.L.G. Thus, Environmental Research Department, Solvay-Duphar BV, CP Weesp, The Netherlands Secretariat Ms A. Sundn Bylhn, International Register of Potentially Toxic Chemicals, United Nations Environment Programme, Chtelaine, Switzerland Dr P. Chamberlain, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland a Participated as required for exchange of information. Dr J. Herrman, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland Dr K. Jager, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland Dr P. Jenkins, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland Dr W. Kreisel, World Health Organization, Geneva, Switzerland Dr M. Mercier, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland Dr M.I. Mikheev, Occupational Health, World Health Organization, Geneva, Switzerland Dr G. Moy, Food Safety, World Health Organization, Geneva, Switzerland Mr I. Obadia, International Labour Organisation, Geneva, Switzerland Dr R. Pletina, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland Dr E. Smith, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland (EHC Secretary) Mr J. Wilbourn, International Agency for Research on Cancer, Lyon, France ENVIRONMENTAL HEALTH CRITERIA FOR DIFLUBENZURON The Core Assessment Group (CAG) of the Joint Meeting on Pesticide Residues met in Geneva from 25 October to 3 November 1994. Dr W. Kreisel of the WHO welcomed the participants on behalf of WHO, and Dr M. Mercier, Director, IPCS, on behalf of the IPCS and its cooperating organizations (UNEP/ILO/WHO). The Group reviewed and revised the draft monograph and made an evaluation of the risks for human health and the environment from exposure to diflubenzuron. The first draft of the monograph was prepared by Dr M. Tasheva, Sofia, Bulgaria. The second draft, incorporating comments received following circulation of the first draft to the IPCS contact points for Environmental Health Criteria monographs, was prepared by the IPCS Secretariat. Dr K.W. Jager and Dr P.G. Jenkins, both members of the IPCS Central Unit, were responsible for the overall scientific content and technical editing, respectively. The fact that Solvay-Duphar, BV, made available to the IPCS its proprietary toxicological information on diflubenzuron is gratefully acknowledged. This allowed the CAG to make its evaluation on a more complete database. The efforts of all who helped in the preparation and finalization of the monograph are gratefully acknowledged. ABBREVIATIONS ADI acceptable daily intake a.i. active ingredient AP alkaline phosphatase bw body weight 4-CPU 4-chlorophenylurea DFB diflubenzuron 2,6-DFBA 2,6-difluorobenzoic acid ECD electron capture detection G granular formulation GC gas chromatography GLC gas-liquid chromatography Hb haemoglobin HPLC high performance liquid chromatography MATC maximum acceptable toxicant concentration MCH mean cell haemoglobin MCHC mean cell haemoglobin concentration MCV mean cell volume NOAEC no-observed-adverse-effect concentration NOEL no-observed-effect level NPD nitrogen-phosphorus detector PCA para-chloroaniline (4-chloroaniline) PCV packed cell volume SAP serum alkaline phosphatase SGOT serum glutamic-oxaloacetic transaminase (aspartate aminotransferase) SGPT serum glutamic-pyruvic transaminase (alanine aminotransferase) TLC thin-layer chromatography WP wettable powder 1. SUMMARY AND EVALUATION; CONCLUSIONS AND RECOMMENDATIONS 1.1 Summary 1.1.1 Identity, physical and chemical properties, and analytical methods Diflubenzuron is a member of the benzoylphenylurea group of insecticides. Its insecticidal action is due to interaction with chitin synthesis and/or deposition. It forms odourless white crystals with a melting point of 230-232C. It is sparingly soluble in water (0.2 mg/litre at 20C) and is virtually non-volatile. It is relatively stable in acidic and neutral media but it hydrolyses in alkaline conditions. Diflubenzuron is produced by the reaction of 2,6-difluoro- benzamide with 4-chlorophenylisocyanate. Diflubenzuron residues may be measured in water, biological samples and soils by HPLC with UV detection or by GC with ECD for analysis of the intact molecule or following derivatization of the liberated 4-chloroaniline with trifluoroacetic anhydride. 1.1.2 Sources of human and environmental exposure Diflubenzuron is a synthetic compound used in agriculture, forestry and public health programmes to control insect pests and vectors. Different formulations of diflubenzuron are available for these uses. There is no relevant information on human exposure to diflubenzuron. 1.1.3 Environmental transport, distribution and transformation Diflubenzuron is usually applied directly to plants and water. Uptake of diflubenzuron through plant leaves does not occur. The adsorption of diflubenzuron to soil is rapid. It is immobilized in the top 10 cm layer of soil to which it is applied. It is unlikely to leach. Diflubenzuron is degraded in soils of various types and origin under aerobic or anaerobic conditions with a half- life of a few days. The rate of degradation depends greatly on the diflubenzuron particle size. The main metabolic pathway (over 90%) is hydrolysis leading to 2,6-difluorobenzoic acid and 4-chlorophenylurea; these are degraded with half-lives of about 4 and 6 weeks, respectively. Free 4-chloroaniline has not been detected in soils. Diflubenzuron degrades rapidly in neutral or alkaline waters. Studies of application of diflubenzuron to water show rapid partition to sediment; the parent compound and 4-chlorophenylurea may persist on sediment for more than 30 days. Diflubenzuron does not bioaccumulate in fish. 1.1.4 Environmental levels and human exposure Exposure of the general population to diflubenzuron via water or food as a result of its use in agriculture, against forest insects or in mosquito control is negligible. 1.1.5 Kinetics and metabolism in laboratory animals In experimental animals, diflubenzuron is absorbed from the digestive tract and to a lesser extent through the skin. There is a saturable absorption mechanism in the rat gastrointestinal tract. Consequently a large proportion of orally administered diflubenzuron is found in the faeces. Diflubenzuron has widespread distribution in the tissues, but it does not accumulate. The metabolic fate of diflubenzuron has been studied in various species. The major route of metabolism in mammals is via hydroxylation. Hydrolysis of diflubenzuron may occur at any of the three carbon-nitrogen bonds. In pigs and chickens the major route of hydrolysis is at the ureido bridge. In rats and cows the major metabolic pathway is hydroxylation. The major metabolites in sheep, swine and chickens are 2,6-difluorobenzoic acid and 4-chloro- phenylurea; minor metabolites are 2,6-difluorobenzamide and 4-chloroaniline. In rats and cattle 80% of the metabolites are 2,6-difluoro-3-hydroxydiflubenzuron, 4-chloro-2-hydroxy-diflubenzuron and 4-chloro-3-hydroxydiflubenzuron. The metabolic studies indicate that little or no 4-chloroaniline is formed in rats or cattle. The major route of elimination is via the faeces, ranging from 70 to 85% in cats, pigs and cattle. In sheep elimination is roughly equally distributed between the urine and faeces. Urinary excretion in rats and mice decreases proportionally with increasing dosage level. Less than 1% of an oral dose is recovered in exhaled air. Only trace residues are found in milk. No human studies on the kinetics and metabolism of diflubenzuron, including the extent of biotransformation to 4-chloroaniline, are available. 1.1.6 Effects on laboratory mammals and in vitro test systems Diflubenzuron has low acute toxicity by any route of exposure. It has been classified by WHO as a "product unlikely to present an acute hazard in normal use", based on an acute oral LD50 of more than 4640 mg/kg body weight in rats. The acute dermal LD50 in rats is greater than 10 000 mg/kg body weight while the acute inhalational LC50 for rats is greater than 2.49 mg/litre. No signs of intoxication have been observed during the 14-day period following single administration of diflubenzuron by various routes to a variety of animal species. Diflubenzuron is not a skin irritant (in rabbits) and not a skin sensitizer (in guinea-pigs). It is marginally irritating to the eyes of rabbits. Diflubenzuron causes methaemoglobinaemia and sulfhaemo- globinaemia. Dose-related methaemoglobinaemia has been demonstrated after oral, dermal or inhalatory exposure to diflubenzuron in various species. This effect is the most sensitive toxicological end-point in experimental animals. The NOEL based on methaemoglobin formation is 2 mg/kg body weight per day in rats and dogs and 2.4 mg/kg body weight per day in mice. In long-term toxicity studies with mice and rats, treatment-related changes were principally associated with oxidation of haemoglobin or with hepatocyte changes. In carcinogenicity studies in mice and rats at dietary levels up to 10 000 mg/kg in the diet, there were no treatment-related effects on tumour incidence. Specifically, there were no mesenchymal neoplasms of the spleen or liver as observed in carcinogenicity studies with 4-chloroaniline. In several reproductive toxicity studies on rats, mice, rabbits and three avian species, no effects were seen on reproduction and there was no embryotoxicity. Teratogenicity studies in rats and rabbits demonstrated no teratogenic effects. Diflubenzuron and its main metabolites have been examined in a variety of in vitro and in vivo mutagenicity tests. Neither diflubenzuron nor its major metabolites have a mutagenic effect. The minor metabolite, 4-chloroaniline, was shown to be positive in several in vitro mutagenicity assays using various end-points. It is carcinogenic in rats and mice. The neoplastic lesions related to administration of 4-chloroaniline were benign and malignant mesenchymal tumours in the spleens of male rats and haemangiomas and haemangiosarcomas, primarily in the spleen and liver of male mice. 1.1.7 Effects on humans The diflubenzuron metabolite, 4-chloroaniline, has been reported to cause methaemoglobinaemia in exposed workers and in neonates inadvertently exposed. Some individuals who are deficient in NADH-methaemoglobin reductase may be particularly sensitive to 4-chloroaniline and hence to diflubenzuron exposure. 1.1.8 Effects on other organisms in the laboratory and field All chitin-synthesizing organisms show susceptibility to diflubenzuron. Bacteria were not affected by diflubenzuron at concentrations of 500 mg/kg soil; some stimulation of nitrogen fixation was seen. Diflubenzuron acetone solutions were degraded; the acetone was used as carbon source. Algal biomass increased at a diflubenzuron concentration of 1 g/litre. There were no adverse effects at concentration above the limit of diflubenzuron solubility. Fungi were temporarily affected at 0.1 g/litre in laboratory streams. Aquatic invertebrates show variable responses to diflubenzuron. Molluscs are insensitive, the LC50 being greater than 200 mg/litre. LC50 values for other invertebrates ranged from 1 to > 1000 g/litre, reflecting the effects of the compound on juvenile, moulting stages. A MATC for Daphnia has been estimated at > 40 and < 93 ng/litre; as expected, larval mayflies and other aquatic insect juveniles are highly susceptible. Overspray of water bodies would be expected to kill some aquatic invertebrates. In ecosystems and field experiments where diflubenzuron was applied directly to the water, the effects on most taxa were less severe than predictions from acute laboratory tests. No effects on aquatic organisms have been found after aerial applications to forests. The LC50 values for fish are > 150 mg/litre. In field experiments, fish kills have never been recorded. The oral and contact LD50 for honey-bees is greater than 30 g/bee. Honey-bee colonies were not affected after aerial application of 350 g diflubenzuron/ha. A 5-day dietary study on the mallard duck and bobwhite quail with levels of up to 4640 mg/kg feed revealed no observable signs of toxicity. Small songbirds in the forest ecosystem were not affected after aerial application of diflubenzuron at 350 g/ha. Small mammal species showed no reductions in numbers after application of diflubenzuron at 67 g/ha to a forest. 1.2 Evaluation 1.2.1 Evaluation of human health risks The primary manifestation of diflubenzuron toxicity is methaemoglobin induction. This toxicity occurs in a range of test animal species. It is attributable to the metabolite, 4-chloroaniline, which is known to induce methaemoglobin formation in several animal species and in humans. Diflubenzuron does not cause other toxicities on chronic dietary administration. It is not mutagenic or carcinogenic in mice or rats. However, its metabolite, 4-chloroaniline, is mutagenic in vitro and is carcinogenic in mice and male rats. Although 4-chloroaniline is a minor urinary metabolite of diflubenzuron in rats, the extent to which it is formed in vivo in various animal species remains unknown. Similarly, the comparative degree of absorption of its parent compound in various species is unknown. The sensitivity of human haemoglobin to methaemoglobin formation by 4-chloroaniline in vivo is not known. However, since induction of methaemoglobinaemia is consistently the most sensitive measure of diflubenzuron toxicity in the various animal species tested, it may be used as the basis to estimate the levels causing no toxicological effect. 1.2.2 Evaluation of effects on the environment Diflubenzuron adsorbs readily to soil with little subsequent desorption. Its mobility in soil is very low, practically all residues remaining within 15 cm of the top, even in sandy loam soils; diflubenzuron does not leach. It is only partly removed from foliage by heavy rainfall. Nevertheless, some diflubenzuron may be present in surface water shortly after application, due to flooding of treatment areas or agricultural run-off. Dissipation of diflubenzuron from water is rapid. Adsorption to sediment occurs within 4 days; both parent compound and 4-chloro- phenylurea metabolite may persist on sediment for at least 30 days. Uptake of diflubenzuron by plants through the leaves after aerial application does not occur. Some uptake of soil residues does occur in plants and this may be translocated. At the highest application rate (1 kg a.i./ha), following 1 month ageing of residues, up to 1 mg/kg residue may be found in various crops. Photolysis of diflubenzuron is slow with a calculated half-life of 40 days. Under environmental conditions abiotic degradation in water and soil represents a minimum route of break-down. Aerobic degradation in water is a microbial process with a half-life of a few days under both laboratory and field conditions. In the field, degradation of diflubenzuron applied at practical rates is influenced by pH, temperature, formulation, organic matter content and depth of the water. Degradation in soil through microbial hydrolysis is a rapid process, with a half-life of a few days, depending on diflubenzuron particle size. The major break-down products are 2,6-difluorobenzoic acid and 4-chlorophenylurea; a minor metabolite is parachloroaniline. All these are irreversibly bound to soil and/or further metabolized. The half-life of diflubenzuron residues on citrus fruits is significantly decreased by high temperature and humidity. Anaerobic degradation in water and sediment is slower than aerobic. Fish bioconcentrate diflubenzuron and some bioaccumulation takes place during extended exposure up to a plateau, depending on the water concentration, owing to fast degradation of diflubenzuron and excretion of metabolites; the depuration half-life is less than one day. The 4-chloroaniline metabolite has not been detected in fish. Fish are not sensitive to diflubenzuron, the LC50 values being > 150 mg/litre. Metabolites of diflubenzuron are also of low toxicity to fish. Chronic exposure has shown no effects on fish at recommended application rates; the compound does not persist in water and no chronic exposure is expected. Diflubenzuron is not phytotoxic to duckweed at the diflubenzuron solubility limit concentration. Honey-bees were not affected by topical applications of > 30 g/bee or dietary concentrations of up to 1000 mg/kg diet. Brood in hives was reduced when bees were fed syrup at 59 mg diflubenzuron/kg. Brood was also reduced following exposure of flying colonies. Earthworms were not affected at a concentration of 780 mg/kg soil, which is at least three orders of magnitude above reported soil residues. Diflubenzuron has low acute toxicity to birds, the oral and dietary LD(LC)50 values being greater than 3000 mg/kg diet. Following recommended application rates diflubenzuron is not expected to pose a hazard to birds. Extensive field studies have shown minimal or reversible effects on most aquatic invertebrates; daphnids were most seriously affected, with short-term reductions in populations of up to 75% following a single application of diflubenzuron. Fish were not affected by water overspraying. Neither bird nor mammal populations were adversely affected following forest spraying with diflubenzuron. A summary of risk quotients for birds, fish and aquatic invertebrates is given in Table 1. 1.2.3 Toxicological criteria for setting guidance values The toxicological studies on diflubenzuron of relevance for setting guidance values are shown in Table 2. Table 1. Toxicity/exposure ratios for birds, fish and aquatic invertebrates based on application rates of 2.5 kg a.i./ha of diflubenzuron to soybeans (worst case) Risk category LC50 (mg/litre Estimated exposure Toxicity/exposure or mg/kg diet) (mg/litre or ratio (TER)c mg/kg diet)a,b Acute bird 3762 73.7-535.7 51.0-7.0 Acute fish (stream) 150 0.0007 214 300 Acute fish (pond) 150 0.01 15 000 Acute aquatic invertebrate (stream) 0.005 0.0007 7.1 Acute aquatic invertebrate (pond) 0.005 0.01 0.5 a Estimated environmental concentration in the terrestrial environment (for bird exposure) is based on the stated application rate and the assumption of deposition on short grass using the US EPA nomogram. b Aquatic exposure concentrations were taken from the STREAM model based on a single application and estimated runoff into water; no direct overspray is included. c TER is the toxicity (as LC50) divided by the exposure; values at or below 1.0 indicate likely exposure to toxic concentrations by organisms in the different risk categories. Table 2. Toxicological criteria for estimating guidance values for diflubenzuron Exposure scenario Relevant route/effect/ Result/remarks (technical species diflubenzuron) Short-term dermal, irritation, rabbit non-irritant (1-7 days) ocular, irritation, rabbit marginal, high dose dermal, sensitization, non-sensitizing guinea-pig inhalational, toxicity, rat LC50 > 2.49 mg/litre (single exposure) Mid-term (1-26 weeks) 3 weeks; 5 days dermal, irritation, rabbit NOEL = 70 mg/kg body per week weight per day 3 weeks; 5 days inhalational, methaemoglobin NOAEL = < 0.12 mg/litre per week formation, rat Long-term dietary, methaemoglobin NOEL = 2 mg/kg body weight formation, rat per day dietary, methaemoglobin NOEL = 2.4 mg/kg body weight formation, mouse per day dietary, methaemoglobin NOEL = 2 mg/kg body weight formation, dog per day 1.3 Conclusions and recommendations Considering the toxicological characteristics of diflubenzuron, both qualitatively and quantitatively, it was concluded, on the basis of the NOEL of 2 mg/kg body weight per day derived in long-term toxicity studies on mice, rats and dogs and applying a 100-fold uncertainty factor, that 0.02 mg/kg body weight per day will probably not cause adverse effects in humans whatever the route of exposure. Biomonitoring of 4-chloroaniline during occupational exposures needs to be carried out. 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS 2.1 Identity Molecular structure Empirical formula C14H9ClF2N2O2 Common name Diflubenzuron Common trade names Dimilin; Micromite; Vigilante Common abbreviation DFB IUPAC name 1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)- urea CAS chemical name N-[[(4-chlorophenyl) amino] carbonyl]- 2,6-difluorobenzamide CAS registry number 35367-38-5 RTECS registry number YS6200000 Technical diflubenzuron contains > 95% pure compound. 2.2 Physical and chemical properties Diflubenzuron is an odourless white crystalline solid. It is almost insoluble in water and poorly soluble in apolar organic solvents. In polar to very polar solvents, the solubility is moderate to good, e.g., in acetone it is 6.5 g/litre at 20C. Diflubenzuron is highly soluble in N-methylpyrolidone (200 g/litre), dimethyl- sulfoxide and dimethylformamide (both 120 g/litre). Some physical and chemical properties of diflubenzuron are given in Table 3. Table 3. Physical and chemical properties of diflubenzuron Relative molecular mass 310.7 Melting point technical > 95% 210-230C > 99% pure 230-232C Vapour pressure at 25C 0.00012 mPa Volatility solid material < 4% from water pH 5.6 < 2% (virtually non-volatile) Specific gravity 1.56 n-Octanol/water partition coefficient (log Kow) 5000 Solubility in water (at 25C and pH 5.6) 8 x 10-5 g/litre Stability in water (0.0001 g/litre 4% decomposition after 3 weeks at pH 5 in the dark) 8% decomposition after 3 weeks at pH 7 26% decomposition after 3 weeks at pH 91 2.3 Conversion factor 1 ppm = 12.7 mg/m3 at 25C 1 mg/m3 = 0.079 ppm at 25C 2.4 Analytical methods Analytical methods for determining diflubenzuron in crops, soil, water and biological samples are summarized in Table 4. A review of the analytical methods has been presented by Rabenort et al. (1978). Two general types of assay procedures for diflubenzuron are available: high performance liquid chromatography (HPLC) and gas chromatography (GC). Table 4. Methods for the determination of diflubenzuron residues Sample type Extraction/clean-up Analytical Limit of Comments Reference method detection Crops, soil, water dichloromethane; clean-up HPLC 0.03 mg/kg Rabenort et al. (1978) on a Florisil column Milk ethyl acetate HPLC 0.1 mg/kg Corley et al. (1974) Crops acetone (n-hexane) HPLC 0.01 mg/kg Nakayama (1977a) Apples acetonitrile HPLC 0.008 mg/kg Goto (1977a) Tea acetone/dichloromethane HPLC 0.1 mg/kg Nakayama (1977b) Tea acetone or water HPLC 0.2 mg/kg Goto (1977b) Crops, soil, sediment; acetonitrile HPLC 0.05 mg/kg the procedures involve Celite Di Prima et al. (1978) aquatic and forest liquid-liquid partition, and foliage; fish and Florisil-aluminasilica gel shellfish; animal column chromatography; tissues 20 g sample Crops acetone-hexane GLC-ECD 0.20 mg/kg Lawrence & Sundaram (1+4) (1976); Di Prima (1976) Soybean acetonitrile for process GC-ECD 0.05 mg/kg after hydrolysis and Lawrence & Sundaram fractions, hulls and meal; derivatization (1976); Di Prima (1976) hexane-acetonitrile for oil Water dichloromethane TLC 0.1 mg/kg Singh & Kaira (1989) Table 4 (Con't) Sample type Extraction/clean-up Analytical Limit of Comments Reference method detection Water & soil hexane/ethyl acetate; GC/ECD 0.05 ng 100 ml sample of water Smith et al. (1983) evaporate to dryness; or 10 g sample of soil dissolve residue in benzene; derivatize with trifluoroacetic anhydride (with trimethylamine as catalyst); LC on Florisil/ hexane: ethylether (9:1 v/v) Water ethyl acetate, KCl; GC/ECD 20 g/litre % DEGS-LAC 728 on Cooke & Ober (1980) derivatize with Chromosorb W-AW at 165C trifluoroacetic anhydride; LC on Florisil Exposure pads methylene chloride or HPLC/UV 3 ng 103.2 cm2 pads Bogus et al. (1985) other solvents; clean-up (254 nm) on SEPPAC C18; elute with methanol The HPLC method is recommended by CIPAC as a method of choice (van Rossum et al., 1984). An alternative method for analysis of residues in crops, soil, mud and water using Celite column chromatography has been described by Di Prima et al. (1978). A gas chromatographic method used on the acetylated derivative of diflubenzuron was described by Worobey & Webster (1977) but has not been applied to crop samples. The formation of 4-chloroaniline from diflubenzuron under acidic conditions provides the basis for the GC method. Most of the recommended extraction procedures use acetonitrile or acetone followed by n-hexane or dichloromethane. Wie & Hammock (1982, 1984) developed three enzyme-linked immunosorbent assays (ELISA) for diflubenzuron. All three assays were based on antibodies raised against an N-carboxypropyl hapten of diflubenzuron, while a diflubenzuron phenylacetic acid derivative coupled to a carrier other than the immunizing antigen was used as the coating antigen. None of these assays demonstrated significant cross- reactivity with benzamide, urea, phenylurea or aniline components of diflubenzuron. Each of the three assays was shown to be as sensitive as the recommended HPLC methodology for the analysis of diflubenzuron in water. Using ELISA, DFB was detected in milk at a level of 1-2 g/litre without any sample extraction procedure. Wimmer et al. (1991) developed a gas chromatography/mass spectrometry (GC/MS) method using deuterated diflubenzuron as internal standard and claimed high sensitivity. The Joint FAO/WHO Codex Alimentarius Commission has given recommendations for the methods of analysis to be used in determining diflubenzuron residues (FAO/WHO, 1989). 3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1 Natural occurrence Diflubenzuron does not occur naturally in the environment. 3.2 Anthropogenic sources 3.2.1 Production levels and processes Diflubenzuron was first commercialized by Philips-Duphar BV, The Netherlands (now Solvay Duphar BV). Solvay Duphar BV produces diflubenzuron under the trade name Dimilin, but production figures are not available. Diflubenzuron is synthesized by the reaction of 2,6-difluoro- benzamide with p-chlorophenyl isocyanate. 3.2.2 Formulations Technical diflubenzuron is made into diflubenzuron 90% concentrate by air-milling with a grinding aid and sufficient kaolin to attain 90% active material. This is the product from which all other formulations are made; these are listed below. Dry products * Dimilin 25W: a 25% wettable powder (more or less the standard product) * Dimilin 5W: a local Italian formulation containing 5% active ingredient * Various granular formulations used locally in specific situations; these products are expected to be removed from the market within 2 or 3 years Water-based products * Dimilin SC-48: a suspension concentrate containing 48% active ingredient * Dimilin SC-15: a suspension concentrate containing 15% active ingredient for the French market * Dimilin 4L, a suspension concentrate (0.4 kg/litre) containing 48% active ingredient for the USA market Oil-based products * Dimilin ODC-45: an oil-based dispersible concentrate containing 45% active ingredient to be diluted with mineral or vegetable oil for spraying operations; this formulation may not be mixed with water * Dimilin OF-6: a dispersion in oil ready for direct spraying, containing 6% active ingredient; this product must not be diluted or mixed with water * Dimilin 2F: an oil-based suspension concentrate containing 24% active ingredient; it must not be diluted with water for spraying and is a local formulation development for the USA market The all-round formulations are Dimilin 25W, Dimilin 5W, Dimilin SC-48, Dimilin SC-15 and Dimilin 4L. Dimilin ODC-45 was developed specially for aerial spraying operations on non-food crops and forestry. Dimilin OF-6 was developed for broadcast aerial spraying operations to control locusts and grasshoppers. Dimilin 2F was developed for those purposes where oil must be added to improve spray deposit tenacity on crops such as cotton. 3.2.3 Uses Diflubenzuron was the first benzoylphenylurea to be discovered. Its insecticidal properties were first described by van Daalen et al. (1972). Diflubenzuron is effective as a stomach and contact insecticide, acting by inhibiting chitin synthesis and so interfering with the formation of the cuticle. Thus, all stages of insects that form new cuticles should be susceptible to diflubenzuron exposure. It has no systemic activity and does not penetrate plant tissue. Consequently, plant sucking insects are generally unaffected, and this forms the basis of its selectivity. The recommended application rates for diflubenzuron are given in Table 5. Diflubenzuron is effective at a concentration of 15-300 mg a.i./litre of water against leaf-feeding larvae and leaf miners in forestry (Lymantria dispar, Thaumethopoea pityocampa), top fruit ( Cydia pomonella, Psylla spp), citrus (Phyllocoptruta oleivora), field crops including cotton and soybeans (Anthonomus grandis, Anticarsia gemmatalis), and horticultural crops (Pieris brassicae). It is also effective against the larvae of Sciaridae and Phoridae in mushrooms (1 g/m2 casing at case mixing or as a drench in 2.5 litre of water to the finished casing), against mosquito larvae (20-45 g/ha water surface) and against fly larvae (Stomoxys calcitrans, Musca domestica) as a surface application in animal housings (0.5-1.0 g/m2 surface) (Worthing & Walker, 1987). Table 5. Recommended application rates for diflubenzuron on different cropsa Pest Crop Rate/concentration Apple rust mite apples/pears 0.01-0.015% a.i. Codling moth apples/pears 0.01-0.015% a.i. Leaf miners apples/pears 0.01-0.015% a.i. Leaf rollers 0.01-0.02% a.i. Pear suckers 0.01 (+0.3% crop oil)% a.i. 0.02-0.03% (without oil) a.i. Winter moth 0.02% a.i. Plum fruit moth plum 0.02% a.i. Olive moth plum 0.01-0.02% a.i. Citrus rust mite citrus fruit 0.0075-0.0125% a.i. Citrus weevil citrus fruit 0.015-0.03% a.i. Cotton ball weevil cotton 70 g/ha a.i. Army worms cotton 150-300 g/ha a.i. Army worms maize and Sorghum 70-150 g/ha a.i. Cotton leaf worms 75-150 g/ha a.i. Beet army worms peanuts 150-300 g/ha a.i. Rice water weevil rice 75-150 g/ha a.i. Fall army worms rice 70-100 g/ha a.i. Mosquitoes up to 100 g/ha a.i. Rice leaf rollers rice 75-250 g/ha a.i. Various pests peanuts up to 75 g/ha a.i. Various pests oil palm 50-150 g/ha a.i. Various pests soybean 20-150 g/ha a Solvay Duphar (1994) 4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, TRANSFORMATION AND FATE 4.1 Appraisal Diflubenzuron is hydrolysed and photolysed slowly (see section 2.2). Residues in the aquatic environment may decrease rapidly, due to adsorption by organic and inorganic matter. This process greatly reduces the availability of diflubenzuron to aquatic organisms. 4.2 Transport and distribution between media Diflubenzuron is generally applied either directly on plants or on water for mosquito control. 4.2.1 Soil mobility Diflubenzuron and its two formulations, Dimilin WP-25 and Dimilin SC-48, were applied separately at 17.23, 51.69 and 155.07 g a.i. (corresponding to 70, 210 and 630 g a.i./ha) to the top layers of columns (30 x 5.6 cm internal diameter) packed with either sandy or clay loam forest soils. Water (1.25 litre) equivalent to 50.8 cm of precipitation (representing an average annual rainfall) was allowed to leach through each column. After leaching, the columns were divided into five segments from bottom to top as follows: two 10-cm increments, one 5-cm increment and two 2.5 cm increments. Diflubenzuron residues in soils were extracted and analysed by HPLC. Diflubenzuron mobility was low and did not increase with dosage. At a deposit rate equivalent to 70 g a.i./ha, nearly all the residues were found within the top 2.5 cm of the column. Even at 630 g a.i./ha, only about 9% of the technical diflubenzuron, 7% of Dimilin SC-48 and 4% of Dimilin WP-25 moved below the 2.5 cm level in sandy loam. The mobility of diflubenzuron in clay loam was lower than in sandy loam. No residues were found below the 10 cm level or in the leachates in either soil type at any dosage levels. The mobility of diflubenzuron was also influenced by the additives present in the formulation, the mobilities being in the following order: technical diflubenzuron > Dimilin SC-48 > Dimilin WP-25 (Sundaram & Nott, 1989). Helling (1985) investigated the movement of 14C-labelled diflubenzuron in five soils and classified it as immobile in all of them. After six treatments of cotton fields with 14C-labelled diflubenzuron, most radioactivity was detected in the top 10 cm layer of soil (Bull & Ivie, 1978). Diflubenzuron was found to adsorb very rapidly to eight soil types (greater than 87% of the initial amount), and there was only limited desorption (Booth et al., 1987). Fourteen days after a single foliar application of 14C-labelled diflubenzuron to field-grown cotton, only just over 10% of the dose was absorbed into the plants. After 21 days and following a heavy rainfall, approximately 23% of the applied diflubenzuron remained on the treated leaf surfaces (Bull & Ivie, 1978). No leaching occurred when 14C-labelled diflubenzuron was applied to soil at the rate of 134.52 g/ha in an area with a normal rainfall of 32 cm (Danhaus et al., 1976). 4.2.2 Dissipation Diflubenzuron might enter an estuary either as a result of flooding of treated supra-tidal mosquito breeding lagoons during spring tides or from agricultural run-off after significant rainfall (Cunningham & Myers, 1986). Following aerial application at 67.26 g/ha to a watershed, diflubenzuron was found to reach the stream channel. It was also washed from the foliage as a result of several subsequent rainfalls (Jones & Konchenderfer, 1988). However, these discharges were very short-lived. No residues were found in sediments from a lake treated with diflubenzuron, suggesting rapid dissipation before or upon reaching the bottom sediment (Apperson et al., 1978). Pritchard & Bourquin (1981) demonstrated some affinity of diflubenzuron for sediments, i.e. a partition coefficient of 380 in simulated estuarine conditions. According to Cunningham & Myers (1986), sediment appeared to be a major site for diflubenzuron adsorption in a supra-tidal salt marsh. Carringer et al. (1975) found that the organic content of soil was the most important factor in determining adsorption and dissipation of diflubenzuron, and that adsorption was inversely related to the water solubility of diflubenzuron. 4.2.3 Evaporation When diflubenzuron was applied as Dimilin WP 80 at a concentration of 75 g/ha a.i. to bare soil (less than 1.5% organic matter) and red kidney bean leaves, no significant evaporation was measured under the following simulated climatological conditions: wind speed 1-2 m/s; temperature 20-21C; relative humidity 25-45% (van der Laan-Straathof & Thus, 1994). 4.2.4 Crop residue data When soybean and maize (corn) seedlings and potato tubers were planted into soil treated with 3H- or 14C-labelled diflubenzuron, only small amounts of radioactivity were taken up (Nimmo & de Wilde, 1976a). When 3H- or 14C-labelled diflubenzuron was applied to soil in which the seedlings of wheat and rice were already present, the 14C residues in rice and wheat leaves were between 0.1 and 0.5 g/kg. The residues consisted mainly of 4-chlorophenylurea and polar conjugates. The 14C residues in the wheat seeds were 0.02-0.04 mg/kg and 3H residues were lower (Nimmo & de Wilde, 1976b). The fate of diflubenzuron was studied following application to soybeans both in greenhouse and field conditions. It was found that 75 to 100% of the total residues in soybean plants consisted of unaltered diflubenzuron. There was no significant absorption or translocation of residues. Less than 0.05 mg/kg of the total residues was found in harvested soybean seed (Gustafson & Wargo, 1976). The diflubenzuron spray residue on aerial parts of plants is essentially stable. Leaf permeation does not occur and the compound is not translocated to other parts of the plant. It has been demonstrated that there is virtually no absorption, translocation or metabolism of foliar-applied diflubenzuron on greenhouse cotton plants (Nimmo & de Wilde, 1974; Nimmo, 1976a,b; Mansager et al., 1979). Plant metabolism studies in corn, soybean, cabbage and apples have demonstrated that no degradation products are found in plant tissues. The only residue component present was the parent compound diflubenzuron. Similar results were reported for cotton. Studies on citrus fruits, apples and soybeans have confirmed that the only residue component is the parent compound diflubenzuron. It can be concluded that plants do not metabolize diflubenzuron (Nimmo & de Wilde, 1974; Nimmo et al., 1978; Bull & Ivie, 1978; Nigg, 1989; Joustra et al., 1989; Serra & Joustra, 1990; van Kampen & Joustra, 1991; Thus & van der Laan, 1993). 4.3 Transformation 4.3.1 Abiotic degradation Under environmental conditions abiotic degradation of diflubenzuron represents a very minor route of breakdown, owing to the stability of the substance. 4.3.1.1 Photolysis On the basis of results from a 15-day photolysis experiment, a photolytic half-life of 40 days was calculated for diflubenzuron by regression analysis (Boelhouwers et al., 1988a,b). After one week of storage at 50C or after one day at 100C, there was no significant decomposition (< 2%). The solid is stable to sunlight. 4.3.1.2 Hydrolysis Abiotic hydrolysis of diflubenzuron in solution does not occur at normal pH values. At pH 9 the hydrolytic half-life is 32.5 days, 4-chlorophenyl urea (4-CPU) and 2,6-difluorobenzoic acid (2,6-DFBA) being the degradation products (Boelhouwers et al., 1988a). High temperature (121C) increases the degradation of diflubenzuron in aqueous media at levels greatly above its solubility in water and result in its rapid degradation to as many as seven identified products: 4-CPU, 2,6-DFBA, 2,6-difluorobenzamide, 4-chloroaniline, N,N'-bis (4-chlorophenyl) urea, 1-(4-chlorophenyl)- 5-fluoro-2,4 (1H,3H)-quinazolinedione and 2-[(4-chlorophenyl) amino]- 6-fluorobenzoic acid. 4-Chloroaniline, N,N'-bis (4-chlorophenyl) urea and 2[(4-chlorophenyl) amino]-6-fluorobenzoic acid were not detected at lower temperatures (0.1 mg [14C]-diflubenzuron/litre water or buffer at 36C). 4-Chloroaniline was a major degradation product of diflubenzuron in heat-treated samples, but it was not seen at lower temperatures (Ivie et al., 1980). The heat-induced degradation of diflubenzuron increased with increasing pH (Schaefer & Dupras, 1976). Nigg et al. (1986) found that high temperature and humidity significantly decreased the half- life of diflubenzuron residues on citrus fruit. 4.3.2 Biodegradation 4.3.2.1 Water a) Laboratory studies Degradation in water can also occur through microbial action, since in sterile water no breakdown or hydrolysis occurs (Boelhouwers et al., 1988a). In freshly sampled ditch water, Nimmo & De Wilde (1975a) demonstrated 50% degradation in 1-4 weeks. The breakdown products were the same as the primary soil metabolites (4-CPU and 2,6-DFBA). Ivie et al. (1980) reported the same metabolites. Anton et al. (1993) calculated the half-life of diflubenzuron in aerated and unaerated tap water to be less than half a day and less than one day, respectively. When diflubenzuron (1.3 mg/litre) was added to an anaerobic silt loam/water system, disappearance from the water phase showed a half- life of 18 days and from the total system a half-life of 34 days. The metabolites were 4-CPU and 2,6-DFBA, and almost no bound residue was formed (Thus et al., 1991). After 90 days less than 2% of added diflubenzuron remained in the system (Thus & van Dyk, 1991). In another study, van der Laan-Straathof & Thus (1993) calculated the half-life of diflubenzuron in water to be 2.5 days. Of the two degradation products, 4-CPU underwent no further degradation but 2,6-DFBA was mineralized. b) Outdoor models Schaefer et al. (1980) reported that, in pasture water with a pH of 8.2 and afternoon temperatures as high as 38-40C, there was a decline from an initial nominal concentration of 30 g/litre to a one-hour measured concentration of 20.3 g/litre and subsequently to 21.6, 13.6, 4.4, and 2.4 g/litre on days 1, 2, 3, and 4 respectively. Schaefer & Dupras (1976) applied two formulations of diflubenzuron (a wettable powder and a flowable formulation) to artificial ponds of 1 m2 surface area containing 318 litres of pond water. An initial concentration of 80 g/litre decreased to 50% within about 2 days. The diflubenzuron residue level after one week was 2-3 g/litre. The half-life of diflubenzuron (1 g/litre) in the aqueous fraction of sludge experiments was 4-15 h (Booth et al., 1987), and the half-life in sea water was reported to be less than 4 days (Schimmel et al., 1983). Cunningham & Myers (1986) estimated a half- life of less than 1 day for residues of diflubenzuron in water following three applications of 0.4% granules and three applications of 25% WP at a rate of 45 g a.i./ha to a supra-tidal salt marsh. Madder & Lockhart (1980) studied model ponds (20 m2) to which Dimilin WP-25 was applied at 56 g/ha (equivalent to 11.2 g/litre). For an unexplained reason, the measured concentration reached a maximum value of about 17.5 g/litre, 4 days after treatment. It decreased by around 50% during the next 5 days. A residue of 2 g/litre remained 2 weeks after application. On the basis of a bioassay, a diflubenzuron half-life of about 3 days was calculated. Collwell & Schaefer (1980) applied diflubenzuron to five experimental ponds (each 100 m2) at a mean concentration of 13 g/litre. The residue levels in water declined to an average of 7.2 g/litre after 24 h. In a study by Sarkar (1982), a 3 x 1 x 0.3 m open tank containing water was sprayed with a dispersion of Dimilin WP-25. Three subsequent applications were made, giving diflubenzuron concentrations of 25, 35 and 50 g/litre, respectively. These concentrations decreased to 50% in about 3-4 days. Pritchard & Bourquin (1981) studied the environmental fate of diflubenzuron under simulated estuarine conditions in a laboratory continuous-flow estuarine system and a static test system. The hydrolytic half-life of diflubenzuron was 17 days in the static test system, whereas the biological half-life was 5 days. 4-Chloroaniline was not detected in either of the systems. Thus & van der Laan-Straathof (1994) studied the fate of diflubenzuron in two model ditch systems. Diflubenzuron was added at a concentration of 0.94 mg/kg to two sediments (sandy loam and silt loam), both of which were covered with aerated surface water. It disappeared rapidly from the water phase through degradation and adsorption to the sediment, the half-lives being 1.9 and 1.1 days, respectively. Dissipation of diflubenzuron from the complete sandy loam and silt loam systems occurred with half-lives of 25 and 10 days, respectively. The metabolites (> 1% of the added diflubenzuron) consisted of CO2, 4-CPU and 2,6-DFBA. c) Field studies Apperson et al. (1978) described the treatment of three farm ponds with diflubenzuron levels of 2.5, 5 and 10 g/litre, and a lake with 5 g/litre. Shortly after the application, a rapid decline in diflubenzuron residues occurred, resulting in half-life values of only a few days. In the lake no residues were found in the sediment samples, suggesting that diflubenzuron was rapidly dissipated before, or upon reaching, the bottom sediment. Hester (1982) applied diflubenzuron at 0.045 kg a.i./ha to specially constructed estuarine ponds. The water residue levels decreased rapidly from 7.5 to 2 g/litre in 2-3 days (study II) and from 3.3 to 0.6 g/litre in 7 days (study I). d) Discussion and appraisal The rate of decrease in diflubenzuron concentration after application of the formulated product to natural waters depends on the combined action of many environmental factors. Factors affecting the degradation rate of diflubenzuron include the acidity (pH), the relative local abundance of soil and organic debris, and the water depth. Half-life values vary from less than 4 days to 4 weeks in laboratory experiments. The use of artificial ponds or basins, preferably outdoors, yields more relevant data and fairly consistent results. Dissipation half-life values vary from 1-5 days after diflubenzuron has been applied at recommended rates. The dissipation half-life of diflubenzuron in the aquatic environment is between one day and one week in most cases, depending on the properties of the applied formulation and on the characteristics of the application site. The presence of organic sediments (hydrosoil, plant debris) and a relatively high local temperature are factors that particularly accelerate the disappearance of diflubenzuron. 4.3.2.2 Soil a) Mobility in soil Diflubenzuron is immobile in soil, as demonstrated by Helling (1985) in column leaching experiments and Booth et al. (1987) in adsorption-desorption studies with eight soil types. The work of Carringer et al. (1975) suggests that soil organic matter is an important parameter in soil adsorption. Due to its immobility in soil, diflubenzuron is not likely to contaminate groundwater by vertical movement in soil or to contaminate open water by lateral movement in groundwater. This has been confirmed in studies carried out in field soils with growth of citrus fruits (Verhey, 1991a; Kramer, 1991), apple (Kramer, 1990, Verhey, 1991b), soybean (Kramer, 1992b) and cotton (Kramer, 1992a). After three applications of diflubenzuron (Dimilin 25W) at normal rates, most residue was found in the top 15 cm of soil and no residue was encountered below 30 cm. b) Degradation in soil The rates of disappearance of technical diflubenzuron applied at 10 mg/kg on quartz sand to natural sandy loam and muck soils were significantly greater than for the corresponding sterilized soils (e.g., 2-12% and 80-87% diflubenzuron, respectively, remaining at 12 weeks), demonstrating that soil microorganisms play a major role in their degradation (Chapman et al., 1985). Diflubenzuron is very rapidly hydrolysed in soil. The half-life time is 2 days to one week. The primary metabolites are 2,6-DFBA and 4-CPU. The process is microbial, since in sterilized soil no breakdown occurs. The rate of breakdown is strongly dependent on the particle size of diflubenzuron (see Fig. 1) (Nimmo et al., 1984, 1986). The half-life in water in alkaline pastures is 1 day and in neutral lake water it is from 10 to 15 days (Nimmo & de Wilde, 1975a). Metabolic routes other than 4-CPU and 2,6-DFBA are virtually irrelevant. Both primary metabolites are further metabolized, 2,6-DFBA with a half-life of about 4 weeks and 4-CPU with a dissipation time of 1 to 3 months. Radiolabelling of both primary metabolites and of a carbon atom in the ureido bridge shows carbon dioxide development from mineralization. However, both the benzoic acid ring carbon and the ureido bridge carbon are mineralized much faster than the aniline moiety carbon, suggesting that para-chloroaniline (PCA) is a major secondary metabolite that is virtually irreversibly bound to soil (Bollag et al., 1978; Mansager et al., 1979; Nimmo et al., 1984, 1986, 1990). Even as a bound residue PCA is metabolized. Apparently, the breakdown of 4-CPU in soil is a complex process in which PCA is a transient metabolite or intermediate. The breakdown process leads to products beyond the aniline structure. If PCA is applied to soil, 6 weeks incubation at 25C yields 60% breakdown products of a different nature (Bollag et al., 1978). The aniline itself is firmly bound to soil and immobilized (Hsu & Bartha, 1974; Moreale & van Bladel, 1976; Bollag et al., 1978; Simmons et al., 1989). Fig. 2 shows metabolic pathways in soil. The main metabolic pathway (over 90%) is hydrolysis, leading to 2,6-DFBA and 4-CPU. The second site of cleavage occurs at CN bonds 2 and 3. Both reactions lead to the formation of 2,6-difluorobenzamide (DFBAM), which readily hydrolyses to 2,6-DFBA (Verloop & Ferrell, 1977; Nimmo et al., 1984). The major metabolite in an activated sludge system is 4-CPU. This is the major metabolite reported in most soil metabolism experiments (Booth et al., 1987). 4-CPU was found to be converted into bound residues with a half-life of 5-10 weeks. In the bound residues, 4-CPU and PCA were present in roughly equal amounts after 2 months (Verloop & Ferrell, 1977). Free PCA was not found in soil (Nimmo et al., 1986). The soil type and characteristics appear to have no influence on the rate of degradation (Nimmo et al., 1984). Metcalf et al. (1975) found no significant degradation of diflubenzuron in a silty clay loam after incubation at 26.7C for periods of 1, 2 and 4 weeks. However, the authors did not take into account the particle size of the soil, and used techniques that have a negative influence on breakdown. The rate of degradation of 14C- or 3H-diflubenzuron applied to a mushroom growth medium (dose 2 g/m2) was between 30-50% in one month. The main degradation products, 4-CPU and 2,6-DFBA, were absorbed from the growth medium by the mushrooms, resulting in residue levels of 0.1-0.6 mg/kg and 1-3 mg/kg, respectively (Nimmo & de Wilde, 1977a). Free PCA or its further possible degradation products were not present in the extractable residues (Nimmo & de Wilde, 1975a; Verloop & Ferrell, 1977). Organic matter in soil significantly contributed to the adsorption of chloroaniline compounds and their immobilization (Hsu & Bartha, 1974; Moreale & van Bladel, 1976; Bollag et al., 1978). Nimmo & de Wilde (1975a) found a degradation half-life of 0.5-1 week at a diflubenzuron concentration of 1 mg/kg (corresponding to an application dose of approximately 300 g/ha). 2,6-DFBA was degraded with a half-life of approximately 4 weeks, and 4-CPU with a half-life of 2-3 months. Walstra & Joustra (1990) applied 0.69 mg diflubenzuron/kg to sandy loam. When incubated in the dark at 24C, they obtained a half- life for diflubenzuron of 50 h. Diflubenzuron was found to be rapidly degraded by four soil fungi ( Fusarium sp., Cephalosporium sp., Penicillium sp. and Rhodotorula sp.), the half-lives being 7, 13, 14 and 18 days, respectively (Seuferer et al., 1979). Several degradation studies on diflubenzuron (Dimilin 25 W) in field soils have been conducted (Kramer, 1990, 1991, 1992a,b; Verhey, 1991a,b). Most of the degradation half-lives were between one and two weeks, except in the case of the two Verhey studies, which yielded half-lives of more than two months. In all studies, the metabolites were 4-CPU and 2,6-DFBA. No degradation of diflubenzuron by the soil microorganism Pseudomonas putida was observed (Booth & Ferrell, 1977). 4.4 Bioaccumulation and biomagnification Metcalf et al. (1975) studied the fate of 14C-diflubenzuron in a laboratory model ecosystem. Diflubenzuron was clearly persistent in some organisms, such as algae (Oedogonium cardiacum), snails ( Physa sp.), caterpillars (Estigmene acrea) and mosquito larvae (Culex pipiens quinquefasciatus). The fish Gambusia affinis was able to degrade diflubenzuron more efficiently. Diflubenzuron did not biomagnify in the fish through food chain transfer. The biomagnifi- cation was about 40-fold greater in mosquito larvae than in Gambusia affinis. When the bluegill sunfish (Lepomis macrochirus) was exposed to 10 g diflubenzuron/litre for 24 h the tissues contained an average of 264 g/kg. After 24 to 48 h of exposure, fish degraded and eliminated the diflubenzuron. The excretory products were neither the parent compound nor 4-CPU. The amount of diflubenzuron remaining in fish tissues at various times was dependant on the reduction of residue concentration in water. However, the potential for degradation and elimination was very great (Schaefer et al., 1979). A dynamic 42-day study was conducted by Burgess (1989) in order to evaluate the bioconcentration of 14C-diflubenzuron by bluegill sunfish (Lepomis macrochirus). A flow-through proportional diluter system was used for a 28-day exposure period. Radioanalysis of fillet, whole fish and visceral portions was performed throughout the exposure period. Daily bioconcentration factors ranged from 34 to 200, 78 to 360, and 100 to 550 for fillet, whole fish and viscera, respectively. Uptake tissue concentrations of 14C-diflubenzuron ranged from 0.25 to 1.7 mg/kg for fillet, 0.58 to 3.3 mg/kg for whole fish, and 0.75 to 4.7 mg/kg for viscera. To measure the elimination of 14C-diflubenzuron, the test fish were placed in clean water for 14 days. Radioanalysis throughout the depuration period indicated 99% depuration for each of fillet, whole fish and viscera. The fillet concentration of 14C-diflubenzuron decreased from 1.6 mg/kg on day 28 of exposure to 0.012 mg/kg by day 14 of the depuration period. Whole fish levels decreased from 3.3 mg/kg on day 28 of exposure to 0.038 mg/kg by the end of the study; whereas, viscera concentrations dropped from 4.4 mg/kg on day 28 of exposure to 0.056 mg/kg by day 14 of depuration. BIOFAC modelling estimated the uptake rate constant (K1) to be 370 ( 57) mg/kg fish per mg/litre water per day, the depuration rate constant (K2) 1.2 ( 0.18) day-1, the time for 50% depuration 0.60 ( 0.09) days, the bioconcentration factor (BCF) 320 ( 70), and the time to reach 90% or steady state 2.0 ( 0.31) days. The BIOFAC-calculated BCF value was the same as the observed mean whole fish BCF of 320 for days 3, 7, 14, 21 and 28. Fig. 3 shows the accumulation, plateauing and depuration in this study. During the study, no mortality or abnormal behaviour was observed in the test fish. This appeared to indicate that the test fish were in good health and would provide acceptable data for defining the uptake/depuration potential of 14C-diflubenzuron. Analysis of fish revealed parent compound (80%), 2,6-difluorobenzamide (10-13%) and three other minor metabolites (one of which probably was 4-CPU). PCA was demonstrated to be absent (sensitivity limit 0.01 mg/kg). White crappies (Pomoxis annularis) contained residues from 355.1 to 62.2 g/kg at 4 and 21 days, respectively, following treatment of a lake with 5 g diflubenzuron/litre (Apperson et al., 1978). Channel catfish (Ictalurus punctatus) did not bioaccumulate diflubenzuron residues (less than 0.05 mg/kg) from treated soil in a simulated lake ecosystem (Booth & Ferrell, 1977). Assuming a biomagnification of 50-160, and that fish are capable of rapidly depleting residues from the body, the likelihood of fish accumulating significant residues of diflubenzuron is low (Apperson et al., 1978; Schaefer et al., 1980). 4.5 Interaction with other physical, chemical or biological factors Schaefer & Dupras (1976) reported that application of the technical grade compound in an ethanol carrier or as a flowable liquid formulation resulted in higher concentrations in the upper water levels of mosquito ponds for a period of 3 days following spray treatment than in the case of spray treatment with wettable powder formulation (the actual formulation used for mosquito control spraying). Seuferer et al. (1979) reported that the soil microorganisms Rhodotorula sp., Penicillium sp. and Cephalosporium sp. cannot utilize diflubenzuron as a sole carbon and energy source. However, accelerated breakdown of diflubenzuron occurred in the presence of these organisms. 4.6 Ultimate fate following use It appears that after direct spraying diflubenzuron is persistent on foliage, it remains almost completely at the site of application on the surface, and it does not penetrate the plants. Diflubenzuron is readily degraded in soils of various types and origin under aerobic or anaerobic conditions with a half-life in the range of 0.5 to 1 week. It is metabolized by microorganisms principally to 4-CPU and 2,6-DFBA. The latter is unstable with a half-life of 3-5 days (Nimmo et al., 1984) to 4 weeks (Verloop & Ferrell, 1977). The half-life of 4-CPU is about 6 weeks (Nimmo et al., 1984). Free PCA has not been detected in soil. In spite of rapid degradation in soil, small amounts of residue (up to 1 mg/kg, depending on ageing time and growth stage of plants) may be taken up by crops in treated soil (Thus et al., 1994). Field applications of diflubenzuron produce soil residues which might possibly lead to residues in rotational crops by re-uptake from soil. Studies with direct applications to field water show a moderate persistence of diflubenzuron in water. Half-life values average one week or less. This rapid rate of loss may be more dependent on adsorption to organic matter than on microbial degradation. 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1 Environmental levels 5.1.1 Air No information is available on air concentrations of diflubenzuron. 5.1.2 Water A total of 1160 ha of insect-infested forest in Finland was sprayed with diflubenzuron (25% WP) from a fixed-winged aircraft at an application rate of 75 g a.i. in 50 litres water per ha. The residues in "run-off" water (gathered in specially dug pits adjacent to the sprayed area) decreased from 5 g/litre one day after spraying to 0.1 g/litre after 2 months. The concentration in water in open pits was 0.1 g/litre 1 and 7 days after application and 0.2 g/litre 1 month after application. After 2 months no residues were detected. All water samples taken from outside the treated area contained less than 0.1 g/litre (the limit of sensitivity) (Mutanen et al., 1988). Diflubenzuron was found in the water of the Fraser River, Canada, up to 71 days following application with diflubenzuron (1% granular formulation) at a rate of 4.5 kg/ha (45 g a.i./ha). The peak value was 1.8 g/litre 8 days after treatment (Wan & Wilson, 1977). After aerial application of diflubenzuron (25% WP formulation) to two forest ponds in Canada, the maximum residue levels in water, sediment, aquatic plants and fish were 13.82 g/litre (at 1 h), 0.24 mg/kg (at 1 day), 0.36 mg/kg (at 1 day) and 0.11 mg/kg (at 1 day), respectively. The rate of dissipation was rapid, non- detectable levels being reached in 20 days for water, 5 days in aquatic plants and 3 days in fish (Kingsbury et al., 1987). A pond in Salt Lake County, Utah, USA, was treated with three applications of diflubenzuron at a rate of 280.25 g a.i./ha. Diflubenzuron was found at less than 0.05 mg/litre 4 days following treatment (Booth et at., 1987). Residues in three farm ponds in California treated with diflubenzuron (2.5, 5 and 10 g/litre) averaged 1.9, 4.6 and 9.8 g/litre, respectively, 1-4 h after the applications. They declined steadily averaging 0.5, 0.3 and 0.2 g/litre, respectively, 2 weeks later. Residues in a small lake treated at 5 g/litre averaged 3.3 g/litre following treatment and 0.4 g/litre after 35 days. No residues were found in sediment samples taken post- treatment (Apperson et al., 1978). One hour after a single application of 45 g diflubenzuron/ha to brackish water pools the residues in water and in sediment were 3.6 g/litre and 80 g/kg, respectively. The concentration in sediment increased to 520 g/kg after 1 day and reached its maximum of 780 g/kg 4 days following application (Hester et al., 1986). After 6 applications of diflubenzuron at a rate of 145.73 g/ha to Utah Lake, USA, the residues in sediments were less than 0.05 mg/kg (Booth et al., 1987). Other field studies with similar results have been reported by Anon (1980), Smith & Edmunds (1985), Van Den Berg (1986), Huber & Collins (1987), Jones & Kochenderfer (1988), Huber & Manchester (1988), Downey (1990) and Sundaram et al. (1991). It is clear that a variety of application scenarios will result in measurable residues of diflubenzuron in water (Table 6). The overall conclusion is that diflubenzuron residues in stagnant water dissipate rapidly within days. In flowing water, e.g., in wooded areas, diflubenzuron residues may peak shortly after rainfall but such peak concentrations are very transient in nature. 5.1.3 Food and feed Data on residues in food resulting from treatment with diflubenzuron have been summarized by FAO/WHO (1982a,b, 1985a,b, 1986a,b). Residue data obtained from various countries showed residues in apples below 1.0 mg diflubenzuron/kg at 2 weeks after the last application at recommended rates. Residues in whole citrus fruit were below 0.5 mg/kg 1 week after the last application at the recommended rate. Residues in soybean seed and cottonseed were generally below the limit of determination (0.05 mg/kg). Mushrooms have a residue pattern different from other plant material. In mushrooms growing on diflubenzuron-treated soil, high levels of the metabolite 2,6-DFBA are taken up from the soil. Diflubenzuron was found at a level of 0.1 mg/kg, while the 2,6-DFBA level was around 1 mg/kg (see chapter 4). Residues in wild mushrooms after aerial application to forests in Finland were on average 0.07 mg/kg 1 week after spraying with 75 g diflubenzuron in 50 litre water per ha. In bilberries the residues decreased on average from 0.2 mg/kg 1 day after spraying to 0.09 mg/kg after 1 month (Mutanen et al., 1988). Diflubenzuron applied as a wettable powder spray to growing alfalfa at 20-100 g/ha showed initial residue levels of 1.8-8.5 mg/kg. Residues of 0.3-1.5 mg/kg remained 22 days after applications (Lauren et al., 1984). Table 6. Summary and comparison of experimental parameters among key studies designed to measure environmental concentrations of diflubenzuron in water Medium Formulation a.i.% Method of Application Maximum Time for Minimum Time for References treated application rate a.i. concentration maximum concentrationa minimum concentration concentration Farm ponds 25 WP 25 hand sprayer 2.5-10 g/litre 1.9-9.8 g/litre 1-4 h 0.5-0.2 g/litre 14 days Apperson (0.06-0.2 ha) from boat et al. (1978) Small lake 25 WP 25 hand sprayer 5 g/litre 3.3 g/litre 4 h 0.4 g/litre 35 days Apperson (18.6 ha) from boat et al. (1978) Pond W-25 25 hand-operated 0.28 kg/ha 56 g/litre 96 h < 0.01 g/litre 40 days Booth spray et al. applicator (1987) Brackish 25 WP 25 clothes 0.045 kg/ha 7.5 g/litre 48-72 h < 0.3 g/litre 25-30 days Hester pools sprinkler (1986) Forest ponds 25 WP 25 aircraft 0.07 kg/ha 13.82 g/litre 1 h < DL 20 days Kingsbury (25 ha) (four et al. atomizers) (1987) Field plot 25 WP 25 fixed-wing 0.075 kg in 50 5.0 g/litre 24 h < DL 60 days Mutanen (1160 ha) aircraft litre water/ha et al. (1988) Fixed plots granular 1.0 aircraft 0.023 kg/ha, 1.8 g/litre 192 h < DL 60 days Wan & (3-40 ha) 0.46 kg/ha Wilson (1977) a DL = determination limit After two soil applications of 67.26 g/ha, the residues of diflubenzuron in the rotational crops (wheat, cabbage and onions) were less than 0.01 mg/kg (Danhaus & Sieck, 1976). Mian & Mulla (1983) studied the persistence of diflubenzuron in stored wheat after applications of 1, 5 and 10 mg/kg. The residue levels were 0.59, 2.75 and 5.00 mg/kg, respectively, 23 months after treatment. 5.1.4 Forest plants and litter The level of diflubenzuron residues in pine needles was on average 3.0 mg/kg 1 day after application to the forest in Finland at a rate of 75 g diflubenzuron in 50 litres water per ha. The level had decreased to 0.2-0.3 mg/kg or was not detectable 2 months later (Mutanen et al., 1988). Booth et al. (1987) found diflubenzuron residues of less than 0.05 mg/kg in the forest litter 1, 4, 10 and 21 days after treatment with 0.28 kg a.i./ha. Sundaram (1986) studied the residues in a forest in Canada after simulated aerial spraying of diflubenzuron in acetone and in fuel oil: Arotex 3470 mixture, each at 90 g a.i. in 18 litre/ha. The residue levels 1 h after application varied, respectively, from 23.8 to 30.6 g/g in foliage and from 3.08 to 4.60 g/g in litter. Forty-five days after spraying the residue levels in foliage were 0.80 and 3.9 g/g, respectively, for the above-mentioned formulations. Spray deposit patterns and persistence of diflubenzuron in white pine ( Pinus strobus L.) and sugar maple ( Acer saccharum Marsh.) canopies, forest litter and soil were studied after aerial application of a 250 g/kg wettable powder formulation (Dimilin WP-25) at 70 g a.i./ha, using three volume rates (2.5, 5 and 10 litres/ha), over three blocks in a mixed forest near Kaladar, Ontario, Canada, during 1986 (Sundaram, 1991). In the spray block that received 10 litres/ha, diflubenzuron persisted in foliage as long as 120 days after treatment, but it lasted for only about a week in forest litter and soil samples. At 2.5 and 5 litres/ha, diflubenzuron failed to persist in foliage as long, and residues in litter and soil, which were barely above the quantification limit, persisted only for a few days. 5.1.5 Aquatic organisms Residues in fish are given in section 4.4. 5.2 General population exposure Exposure of the general population to diflubenzuron via food and drinking-water may occur. Twelve volunteers with whole body dosimeters were exposed for 4 h to Dimilin 25 W after simulated indoor treatment of carpets at 0.16 g/m2. Average deposition was 5.3 2.3 g diflubenzuron/cm2 carpet. Total dermal exposure ranged from 0.053 to 0.25 mg/kg body weight per day to (average 0.15 0.066 mg/kg body weight per day). Assuming a dermal absorption of 0.2%, the total exposure via the dermal route was calculated to be 0.0003 mg/kg body weight per day. Air concentrations ranged from 10.2 to 32.4 g/m3 during the first 4 h and were < 1 g/m3 at 12-16 h. The total respiratory exposure was calculated to be 0.0011 mg/kg body weight per day. The total exposure, via the dermal and respiratory route, was calculated to be 0.0014 mg/kg body weight (Honeycutt, 1993). 5.3 Occupational exposure during manufacture, formulation or use In a US Department of Agriculture report, human exposure via a variety of exposure scenarios was estimated using standardized methods and assumptions. The exposure scenarios included mixing and loading by workers, via aircraft or truck spillage, and general public exposure via the diet or resulting from occupational aerial spraying. Dermal absorption of diflubenzuron was assumed to be 10%. Estimated realistic doses for humans were < 0.003 mg/kg body weight per day except where aircraft or truck spillages occurred, in which case exposures were significantly higher. Estimated worst-case doses for humans were < 0.01 mg/kg body weight per day, except where aircraft or truck spillages occurred (USDA, 1985). 6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS 6.1 Absorption Diflubenzuron is absorbed from the digestive tract but only poorly absorbed through the skin. Willems et al. (1980) found that in rats the relative intestinal absorption diminished greatly with increasing dose. Following a dose of 4 mg/kg body weight 42.5% was absorbed, but only 3.7% of a 900 mg/kg body weight dose was absorbed. Dermal absorption of 14C-diflubenzuron was only 0.2% when it was applied to the shaved skin of rabbits as an aqueous micro-suspension of 150 mg/kg (De Lange, 1979). When applied dermally to cattle 14C-diflubenzuron was not absorbed or degraded through the skin to any detectable degree (Ivie, 1978). 6.2 Distribution Body tissues show little tendency to retain diflubenzuron. Analysis of tissues for radiocarbon residues, 4 days (for sheep) or 7 days (for cows) after a single oral dose of 10 mg/kg body weight, indicated that only the liver contained appreciable levels of radioactivity, ranging from 2 to 4 mg/kg diflubenzuron equivalents (Ivie, 1977). More than one third of an oral diflubenzuron dose appeared in the bile of a cannulated sheep (Ivie, 1977). The highest 14C-diflubenzuron residue present in pig tissues after a single oral dose of 5 mg/kg body weight was 0.43 mg/kg in the gall bladder. All other tissue residue levels were found to be less than 0.30 mg/kg (Opdycke et al., 1982a). Twenty-two dairy cows were fed 14C-diflubenzuron (labelled in both phenyl moieties) in a diet at dose levels of 0.05, 0.5, 5, 25 and 250 mg/kg feed for 28 days. Residues in blood, fat and muscle were below the detection limit (0.0067-0.04 mg/kg) at all dose levels. They were only detected following a dose of 250 mg/kg in the liver and kidney where residues were 6.040 and 1.038 mg/kg, respectively. Residues in milk were found at dose levels of 5 and 250 mg/kg, where the highest levels of diflubenzuron were 0.009 and 0.20 mg/kg, respectively (Smith & Merricks, 1976a). In a study by Miller et al. (1976a), two dairy cows were fed diflubenzuron at 0.25 or 1 mg/kg body weight per day for 4 months. A third cow received an increased dosage of 8 to 16 mg/kg body weight per day, the highest value being maintained for three months. In the fat, liver and milk of the third cow, residues were 0.2, 0.13 and 0.02 mg/kg, respectively. When dairy bull calves (four treated and four controls) received diflubenzuron at 1.0 to 2.8 mg/kg body weight, residues were detected only in the tissue samples of one bull (0.02 mg/kg in liver and kidney, 0.04 mg/kg in the subcutaneous fat, and 0.08 mg/kg in the renal and omental fat (Miller et al., 1979). The maximum total residue in eggs 3 days after a single dose of 5 mg/kg 14C-diflubenzuron to hens was 0.248 mg/kg (Opdycke, 1976). When laying hens were administered 14C-diflubenzuron at dose levels 0.05, 0.5, and 5.0 mg/kg feed for 28 days, dose-related residues ranging from 0.007 mg/kg at the lowest to 1.2 mg/kg at the highest dose level were found in kidney, liver and fat. After 7 days of withdrawal, residues in all tissues and eggs were below the detection limit (0.0006-0.032 mg/kg) for all dose levels (Smith & Merricks, 1976b). When diflubenzuron was fed to white leghorn and black sex-linked cross hens at a level of 10 mg/kg feed for 15 weeks, detectable residues were found in eggs, liver and visceral fat. Residues were significantly higher in eggs from white leghorn hens than in eggs from black sex-linked cross hens, the average levels being 0.55 and 0.38 mg/kg, respectively (Miller et al., 1976b). 6.3 Metabolic transformation The metabolic fate of diflubenzuron has been studied in various species. Metabolic pathways of diflubenzuron are shown in Fig. 4 In rats and cows the major metabolic pathway involves hydroxylation of the phenyl moieties of the compound. About 80% of the metabolites in rat urine were identified as 2,6-difluoro-3- hydroxydiflubenzuron and 4-chloro-2-hydroxy- and 4-chloro-3- hydroxydiflubenzuron. About 20% underwent scission of the benzoyl ureido bridge. The major part was excreted as 2,6-DFBA and constituted more than half of the urinary metabolites. 4-CPU was not detected in bile or urine in a significant quantity (De Lange et al., 1975; Willems et al., 1980). The major metabolite in cow urine was 2,6-difluoro-3-hydroxy- diflubenzuron (45%). Relatively small quantities of 4-chloro-2- hydroxy- (1.6%) and 4-chloro-3-hydroxydiflubenzuron (3.7%) and the scission products 4-CPU (0.6%), 2,6-DFBA (6.0%) and 2,6-difluoro- hippuric acid (6.9%) were present (Ivie, 1978). The major metabolites (approximately 50%) in sheep urine were 2,6-DFBA and 2,6-difluorohippuric acid (Ivie, 1978). 14C-Diflubenzuron uniformly radiolabelled in both rings was administered to a pig as an oral dose of 5 mg/kg body weight. Of the administered dose, 82% was eliminated in faeces as parent compound and 5% was recovered in urine. Identification of the metabolic products in urine revealed 2,6-DFBA (0.28% of the dose), 4-CPU (0.82%), PCA (1.03%) and 2,6-difluorobenzamide (0.83%). Cleavage of the urea moiety between the benzoyl carbon and urea nitrogen was shown to be the primary degradation pathway in pigs (Opdycke et al., 1982a). In chickens only small quantities of the metabolites 2,6-DFBA, 4-CPU and PCA were found in excreta and tissues (Opdycke, 1976). Neither induction nor inhibition of mixed-function oxidase activity altered diflubenzuron metabolism in chickens (Opdycke et al., 1982b). After 4 days daily doses of 7.8 g diflubenzuron/kg body weight, De Bree et al. (1977) found PCA at a level of 30 ng/ml in rat plasma and 323 ng/g in erythrocytes. PCA, estimated by the concentration in the urine, represented at most 0.01% of the dose actually absorbed. 6.3.1 Metabolites - distribution, excretion, retention and turnover When 14C-PCA was administered orally as single doses of 0.3, 3.0 or 30.0 mg/kg to male Fischer-344 rats, approximately 75% of the administered radioactivity was excreted in the urine within 24 h, while approximately 10% appeared in the faeces. Excretion was virtually complete (92-97%) 7 days after dosing. The highest tissue levels of radioactivity following a single intravenous dose of 3.0 mg/kg were found in the liver, fat, muscle and skin. Tissue levels peaked within 5-60 min after dosing. By 3 days, concentrations in all tissues except the blood had declined to < 0.3% of the dose (Sipes & Carter, 1988). At this time, the only tissue containing more than 1% of the dose was the cellular compartment of blood, which contained 1-2% of the dose. The decline of PCA concentration in all tissues, except for urine, faeces and intestinal contents, was biexponential. The t alpha 1/2 for fat, muscle and skin was about 1.5 h, while the t1/2 was approx. 43-59 h. The t alpha 1/2 for liver was 3.5 h. Levels of unchanged PCA in all tissues peaked after 5 min following intravenous administration. The highest amount of unchanged PCA was attained in muscle (15% of radioactivity in the tissue) followed by skin (6%), fat (3%) and liver (2%). The decline of PCA in all tissues, except for the liver, followed biexponential kinetics with an estimated t alpha 1/2 of 8 min and a t1/2 of 3 to 5 h. PCA is rapidly metabolized to p-chloroacetanilide (PCAA) as the initial step in the metabolism and excretion of PCA. The decline of PCAA was monoexponential, the appearance half-life being approx. 6 min in the testes and 15 min in the brain. The elimination half-life in the brain, kidney, testes, muscle, skin and fat was around 1.0 to 2.0 h. The elimination of PCA does not depend on either the dose or route of administration. Approximately 4% of the urinary radioactivity in the 0-24 h urine sample was unchanged PCA; less than 1% was found in the faeces. PCAA was not detected in either urine or faeces over a 3-day period (Sipes & Carter, 1988). After a single intravenous dose of 14C-PCA (3 mg/kg), maximal tissue levels were reached within 15 min in most tissues. At this time, most of the radioactivity was located in muscle (34%), fat (14%), skin (12%), liver (8%) and blood (7%). Elimination half-lives from tissues ranged between 1.5 and 4 h. By 8 h, approximately 90% of the administered dose had been eliminated into urine and faeces. By 3 days, concentrations in all tissues, except blood, had declined to < 0.3% of the dose (US NTP, 1989). 6.4 Elimination and excretion After oral administration to rats of 5 mg diflubenzuron labelled with 3H in the benzoyl and with 14C in the aniline moiety, 95% of the 3H and 70-75% of the 14C radioactivity were retrieved in urine and faeces. 2,6-DFBA was shown to constitute more than half of the urinary metabolites (De Lange et al., 1977). Up to 1% of an oral dose of 5 mg 14C-diflubenzuron labelled at the benzoyl moiety was recovered in the expired air of rats (De Lange et al., 1974; Willems et al., 1980). When 14C-diflubenzuron, labelled in the aniline moiety, was administered by gavage (4, 16, 48, 128, 900 and 1000 mg/kg body weight) to rats, the urinary excretion was complete after 48-72 h. Urinary excretion after single oral administration of diflubenzuron relatively decreased with increasing dose level, being 27.6% of the dose at 4 mg/kg and 1% at 1000 mg/kg (De Lange et al., 1977). When 14C-diflubenzuron was administered at single oral doses of 12.5, 63.5, 202.5 and 925 mg/kg body weight to Swiss mice, the excretion was almost completed within 48 h. The cumulative percentage of the dose excreted in the urine decreased from 15% at the dose level of 12.5 mg/kg to approximately 2% at 925 mg/kg (De Lange & Post, 1978). Hawkins et al. (1980) studied the excretion of radioactivity in urine and faeces after oral administration of 3H/14C-diflubenzuron (7 mg/kg) to male cats. The radioactive dose was given on day 10 of a 15-day dosing regime of non-radioactive diflubenzuron (days 1-9 and days 11-15). The excretion of radioactivity in urine accounted for 9.5 and 9.6% of the 14C and 3H doses, respectively, during 6 days after dosing. The elimination of radioactivity in faeces accounted for 77.3 and 71.6% of the 14C and 3H doses, respectively, during 6 days after dosing. After an oral administration of 14C-diflubenzuron (5 mg/kg) to female pigs, 82% of the dose was eliminated via faeces and 5% via urine in 11 days (Opdycke, 1976). About 85% of a single oral dose of 14C-diflubenzuron (10 mg/kg body weight) administered to a cow was recovered in the faeces during the first 4 days after treatment. About 15% was recovered in urine and only about 0.2% was secreted in the milk (Ivie, 1977, 1978). Sheep excreted 41% of the dose (10 mg/kg) in the urine and 43% in the faeces during the 4 days after treatment. Bile-cannulated sheep eliminated 24% of the dose in the urine, 32% in the faeces and 36% in the bile. Sheep treated with 500 mg 14C-diflubenzuron/kg as a single oral dose eliminated a much smaller proportion of the 14C in urine and bile. This was probably due to reduced absorption from the gastrointestinal tract when the sheep were given an exaggerated dose (Ivie, 1977). An oral dose of 5 mg 14C-diflubenzuron/kg administered to white leghorn hens and Rhode Island red-barred Plymouth Rock buff cross hens was rapidly excreted unaltered within the first 8 h. Up to 91 and 82%, respectively, were excreted within 13 days (Opdycke, 1976). 6.5 Retention and turnover 6.5.1 Biological half-life From the studies of Willems et al. (1980) and Ivie (1978), the half-life of diflubenzuron appears to be 12 h in rat and sheep and 18-20 h in the cow. Diflubenzuron has been shown to pass intact through the intestinal tract and remained active in the manure (Nimmo & de Wilde, 1977b). 7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS 7.1 Single exposure The acute toxicity of diflubenzuron and its formulations to different species is summarized in Table 7. No signs of intoxication were observed during the 14-day period following a single administration of diflubenzuron. Van Eldik (1974) reported an intraperitoneal LD50 of > 2150 mg/kg in rats and mice. 7.2 Short-term exposure Rats (5 of each sex per group) were fed on a diet containing diflubenzuron at concentrations of 0, 800, 4000, 20 000 and 100 000 mg/kg feed for 4 weeks. Behaviour, body weight, food and water consumption were not affected by the treatment. There was a dose-related increase in the met- and sulfhaemoglobin content of the blood in all treated groups except for the methaemoglobin value for females in the 800-mg/kg dose group. Lower erythrocyte, packed cell volume (PCV) and haemoglobin values were observed in both sexes of the 100 000-mg/kg dose group. There was a dose-related increase in spleen and liver weights. Only the dose level of 800 mg/kg did not affect the liver weight (Palmer et al., 1977). Five male Swiss-albino rats were given 96.7 mg diflubenzuron/kg body weight per day, dissolved in corn oil, in their diet for 48 days. The total dose was 4640 mg/kg body weight. Five controls were given corn oil only in their diet. At the end of the study the treated groups showed a significantly lower mean haemoglobin concentration than the controls, and decreases in MCH and MCHC values (Berberian & Enan, 1989). Diflubenzuron was administered to Sprague-Dawley rats of the CD strain (20 of each sex per group) at dietary levels of 10 000 and 100 000 mg/kg feed for 9 weeks, followed by a 4-week withdrawal period. Lower values for red blood cell parameters were recorded at both dose levels. An increase in reticulocyte count and a pallor of the extremities and eyes were observed. The formation of methaemoglobin occurred in both males and females, with approximately 5-8% of the available haemoglobin being transformed to methaemoglobin. After a withdrawal period of 4 weeks methaemoglobin comprised less than 2% of the available haemoglobin in tested animals compared with approximately 0.6% in controls. Higher values for SGPT were recorded as well as heavier liver, spleen and adrenal weights. Minor enlargement of centrilobular hepatocytes in the highest dose group was observed, but this finding disappeared after the withdrawal period (Hunter et al., 1979). Table 7. Acute toxicity of diflubenzuron and its formulations Acute oral LD50 Acute dermal LD50 Acute inhalation Primary eye Primary skin Dermal LC50 irritation irritation sensitization Diflubenzuron mouse: > 4640 rat: > 10 000 rabbit: > 30 mg/ rabbit: 40 mg rabbit: non- guinea-pig: non- technical mg/kg body weight mg/kg body weight litre nominal instilled in eye; irritant (Taylor, sensitizing (Koopman, 1977c) marginal irritant 1973a) (Prinsen, 1992) (Davies & rat: > 4640 mg/kg rabbit: > 4 ml/kg > 3.75 mg/litre Ligget, 1973) body weight body weight of 50% actual (Berczy et (van Eldik, 1973a; in gum tragacanth al., 1975a) Koopman, 1977a) (Davies & Halliday, 1974) Diflubenzuron mouse: > 5000 rat: > 2000 mg/kg rat: > 13.8 mg/ rabbit: 100 mg rabbit: 500 mg guinea-pig: 25% 90% concentrate mg/kg body weight body weight litre nominal instilled in eye; non-irritant w/w in paraffin (Koopman & Pot, (Koopman, 1984a) very slight (OECD Guideline (Grade 1); weak 1986) > 2.49 mg/litre irritant (Koopman, 404) (Koopman, sensitizer (OECD actual (Greenough 1984c) 1984d) Guideline 406) rat: > 5000 mg/kg & McDonald, (Kynoch & Smith, body weight 1986) 1986) (Koopman, 1984b) Dimilin WP 25% rat: > 40 000 rat: > 20 000 rat: > 150 mg/ rabbit: 100 mg rabbit: 500 mg guinea-pig: non- mg/kg body weight mg/kg body weight litre nominal instilled in eye; non-irritant to sensitizing (Janssen & Pot, slight to only minimally (Kynoch & Elliott, mice: > 40 000 1987e) > 3.5 mg/litre moderate, irritant 1978,a,b) mg/kg body weight actual (Arts, transientsient eye (Taylor, 1973b; (van Eldik, 1973b; 1991) irritation (Snoeij Chandran, 1981; Koopman, 1977b) & Bus-Pot, 1991) Snoeij & Bus-Pot, 1990) Table 7. (Con't) Acute oral LD50 Acute dermal LD50 Acute inhalation Primary eye Primary skin Dermal LC50 irritation irritation sensitization Dimilin SC-48 rat: > 5000 mg/kg rat: > 2000 mg/kg rabbit: 0.1 ml rabbit: non- guinea-pig: weak body weight body weight - instilled in eye; irritant (Janssen sensitizer (Janssen & Pot, (Janssen & Pot, slight irritant & Pot, 1987b) (Kynoch & 1987c) 1987d) (Janssen & Pot, Parcell, 1987) 1987a) Dimilin SC-15 albino rats: 0.1 rabbit: minimally - - - ml instilled in irritant - eye; non-irritant (Prinsen, 1989a) (Prinsen, 1989b) Dimilin 4F rat: > 5000 mg/kg rat: > 2000 mg/kg rat: > 1.9 mg/litre rabbit: 0.1 ml rabbit: 0.5 ml guinea-pig: non- body weight body weight actual (Jackson instilled in eye; minimally irritant sensitizing (Spanjers, 1988a) (Spanjers, 1988b) et al., 1990) non-irritant (Prinsen, 1988a) (Prinsen, 1989c) (Prinsen, 1988b) Dimilin 2F rat: > 5000 mg/kg rat: > 2000 mg/kg rat: > 4.4 mg/litre rabbit: 0.1 ml rabbit: 0.5 ml guinea-pig: body weight body weight actual very instilled in eye; moderate irritant moderate (Grade (Koopman, 1985d) (Koopman, 1985c) slightly irritant moderate irritant OECD Guideline III) (Kynoch & (Zwart, 1985) OECD Guideline 404 (Koopman, Parcell, 1987) 405 (Koopman, 1985a) 1985b) Dimilin ODC 45 rat: > 37 300 mg/kg rat: > 37 300 mg/kg rabbit: 0.1 ml rabbit: 0.5 ml body weight body weight instilled in eye; moderate irritant (Koopman & (Koopman, 1980b) marginally (Koopman, Jongeling, 1979) irritant (Koopman, 1980c) 1980a) Table 7. (Con't) Acute oral LD50 Acute dermal LD50 Acute inhalation Primary eye Primary skin Dermal LC50 irritation irritation sensitization Dimilin OF 6 rat: > 5000 mg/kg rat: > 2000 mg/kg rat: > 95.7 mg/ rabbit: 0.1 ml rabbit: 0.5 ml guinea-pig: non- body weight body weight litre nominal: instilled in eye; topical; mild sensitizing (Besten et al., (Besten et al., > 2.17 mg/litre non-irritant irritant (Prinsen, 1993) 1993a) 1993b) actual (Janssen & (Janssen & van (Janssen & van van Doorn, 1993a) Doorn, 1993c) Doorn, 1993b) Wistar rats (10 of each sex per group) were fed diflubenzuron in the diet for 13 weeks at concentrations of 0, 3.125, 12.5, 50 or 200 mg/kg feed. Behaviour, growth and food intake were unaffected by the treatment. At the highest dose level the PCV value, the haemoglobin concentration and the number of erythrocytes were decreased. There was an increase in the SGPT and SGOT activities in the males of the highest dose group at the end of the experiment. A slight increase in the number of normally occurring scattered small foci of necrotic parenchymal cells was observed, accompanied by mononuclear inflammatory cell infiltration and proliferation of reticuloendothelial system cells in the liver of both males end females of the 50 and 200 mg/kg groups (Kemp et al., 1973a,b). Absence of toxic effects in chronic/oncogenicity studies at low dose levels necessitated re-evaluation of liver histopathology in the 90-day feeding study; "piece meal" liver cell necrosis was reported at the two highest dose levels, i.e. 50 and 200 mg/kg feed. The original slides of the study were re-evaluated by four histopathologists in four different laboratories. They independently and unanimously agreed that the lesions in the livers of treated rats were found to the same extent in the livers of untreated rats. This showed that the high dose levels in the study did not demonstrate a treatment-related necrotic effect in the liver (Offringa, 1977). Technical grade diflubenzuron was administered in the diet to male and female 21- to 28-day-old Sprague-Dawley rats (40 of each sex per group) at dose levels of 0, 160, 400, 2000, 10 000 and 50 000 mg/kg feed for 13 weeks. No apparent treatment-related effects were noted on mortality, clinical observations, body weight gain, food consumption, clinical chemistry or urinalysis. A treatment-related significant increase in methaemoglobin concentration was noted in all treated groups. Sulfhaemoglobin values showed increases at dose levels of 2000 mg/kg or more. A significant treatment-related decrease in haemoglobin, PCV and erythrocyte count was observed in males and females at all dose levels by the end of the study. An increase was noted in the reticulocyte count at all dose levels except 160 mg/kg, and the number of the Heinz bodies was higher in the 10 000 and 50 000 mg/kg groups. After 7 weeks, spleen weights were increased in the females at all dose levels, but after 13 weeks no effect was found at 160 mg/kg. With the exception of the lowest dose level, all treated groups showed a higher liver weight. The administration of diflubenzuron resulted in a dose-related increase in the incidence of chronic hepatitis and haemosiderosis of the liver. It was also associated at all dose levels with haemosiderosis and congestion of the spleen and mild erythroid hyperplasia of the bone marrow. The severity of the lesions tended to increase with the dose. Liver lesions were more severe in males than in females and were more severe at 13 weeks than at 7 weeks. A no-observed-effect level (NOEL) was not established (Burdock et al., 1980b; Goodman, 1980b). Diflubenzuron was given to CFLP mice for 6 weeks at levels of 16 and 50 mg/kg feed. There were no clinical signs and no effects on food consumption, body weight, blood chemistry or macroscopic pathology. In three of the eight animals given 50 mg/kg, foci of liver cell necrosis, with or without inflammatory cell filtration, were noted. Other organs were not examined microscopically (Hunter et al., 1974). Diflubenzuron was administered to Swiss Webster mice in a 30-day oral intubation study. Groups of five mice each received either no treatment, vehicle control (Polyethylene glycol 400) or diflubenzuron suspensions at dose levels of 125, 500 or 2000 mg/kg body weight. Hepatic glutathione- S-transferase activity and morphological characteristics were studied. Diflubenzuron was shown to elicit hepatocellular changes at all dose levels. The activities of three glutathione- S-transferases ( S-aryl, S-aralkyl and S-epoxide) were irregularly altered in a non-dose-related manner. Light microscopy revealed radial arrays of hepatocellular vacuolization between the portal and central vein areas. There was evidence of an increase in the amount of endoplasmic reticulum (Young et al., 1986). Male and female mice of the B6C3F1 strain (40 of each sex per group) received diflubenzuron (97.2% a.i.) in the diet at dose levels of 0, 16, 50, 400, 2000, 10 000 or 50 000 mg/kg feed for 13 weeks. An additional group of 100 of each sex served as a control. No compound-related effects were apparent with respect to clinical signs, survival, growth rates, total food consumption or gross pathology. Significant treatment-related increases in met- and sulfhaemoglobin concentrations were noted in all treated groups, except in the group fed 16 mg/kg. At the higher dose levels, there was a decrease in haematocrit and erythrocyte counts and an increase in reticulocyte, platelet and Heinz body counts. Significantly higher alkaline phosphatase activity was noted in the 10 000 and 50 000 mg/kg groups. Compound-related effects on the weights of liver and spleen were noted. In the females, adrenal weight was decreased (but not in a dose-related fashion) at all dose levels after 7 weeks and increased after 13 weeks in higher dose levels. Higher adrenal weight was observed in treated males than in the controls. Histopathological examination revealed treatment-related centrilobular hypertrophy of hepatocytes, with or without cell necrosis, haemosiderosis of the liver and spleen, extramedullary haematopoiesis and mild chronic hepatitis in treated animals of both sexes, some of which effects were observed at the lowest dose level (16 mg/kg). The liver lesions were more severe in males than in females, being most severe in the high- dose males. The NOEL for methaemoglobin formation was 16 mg/kg feed (Burdock et al., 1980a; Goodman, 1980a). HC/CFLP mice (40 of each sex per group) were fed diflubenzuron (97.2% purity) for 14 weeks at levels of 0, 80, 400, 2000, 10 000 and 50 000 mg/kg feed. On the second day of treatment, the majority of mice treated with 10 000 or 50 000 mg/kg showed dark eyes and/or prominent caudal blood vessels. On day 5, blue/grey discoloration of the extremities was noted for the majority of mice treated with 50 000 mg/kg. Mice in the lowest dose group exhibited no clinical signs. Mortality, food consumption, water consumption and body weight changes were not significantly affected by the treatment. Lower PCV and red blood cell counts were found at all dose levels except 80 mg/kg. The total white blood cell count, lymphocyte count, haemoglobin concentration, incidence of Heinz bodies and red blood cell count were increased in all treated groups. At week 7, there was an increase in the number of reticulocytes in treated mice, particularly in males treated at 10 000 or 50 000 mg/kg. At week 14, the reticulocyte counts were similar to those of the controls. A treatment-related increase in both met- and sulfhaemoglobin was recorded in all treated groups at weeks 7 and 14 of the investigation. Plasma glutamic-pyruvic transaminase values were increased at all dose levels, with the exception of 80 mg/kg feed. Lower blood cholesterol levels were noted in the 2000, 10 000 and 50 000 mg/kg groups. Macroscopic examination showed dark discoloration and/or enlargement of the spleen and pale subcapsular areas of the liver in all dose groups after both 7 and 14 weeks. Histopathological examination of the spleen revealed increased haemosiderosis at all dose levels except 80 mg/kg. In the liver, areas of focal necrosis and/or fibrosis in the parenchyma, with or without associated inflammatory cells, fibroblasts or pigment-laden macrophages, were observed. At higher dose levels necrotic and fatty hepatocytes and brown pigment-laden Kupffer cells were found. A NOEL was not established (Colley et al., 1981a,b). Diflubenzuron was fed to groups of three male and three female beagle dogs for 13 weeks at concentrations of 0, 10, 20, 40 and 160 mg/kg diet. No effect of the treatment on behaviour, body weight or food and water consumption was observed. Elevated SAP and SGPT values were recorded for some dogs receiving 40 or 160 mg diflubenzuron/kg feed. After 6 weeks, methaemoglobin and another abnormal pigment, probably sulfhaemoglobin, were demonstrated in dogs given 160 mg/kg. After 12 weeks of administration, some recovery was observed. Organ weights and gross and microscopic evaluation did not reveal any treatment-related effects. The NOEL for methaemoglobin formation was 40 mg/kg feed (Chesterman et al., 1974). Diflubenzuron was administered daily in gelatin capsules to male and female beagle dogs (6 of each sex per group) at dose levels of 2, 10, 50 or 250 mg/kg body weight per day for 52 weeks. There were no treatment-related effects on mortality, food consumption or body weight gain. Dose-related marginal increases in methaemoglobin and sulfhaemoglobin were recorded from 10 mg/kg upwards. At 50 and 250 mg/kg, haemoglobin concentration and MCHC were decreased whereas reticulocyte and platelet counts were increased. Heinz bodies were also detected in several animals receiving 50 and 250 mg/kg. Dose- related increases in liver and spleen weights were found in the 50 and 250 mg/kg males. Histopathological evaluation of the liver revealed an increase in the incidence of pigmented macrophages and Kupffer cell siderosis at 50 and 250 mg/kg in both males and females. The NOEL based on the increase in met- and sulfhaemoglobin was 2 mg/kg body weight (Greenough et al., 1985). In a study by Berczy et al. (1975c), rats were exposed daily for a one-hour period to technical diflubenzuron dust at nominal concentrations of 0, 0.5, 5.0 and 50 mg/litre air, respectively (the actual concentrations were 0, 0.12, 0.87 and 1.85 mg/litre, respectively). Exposures were repeated over a period of 3 weeks, 5 days per week. The methaemoglobin levels in male rats at the two lower concentrations and female rats of all test groups were significantly higher than those of controls. Rabbits were exposed daily for one-hour periods to technical diflubenzuron dust at concentrations of 0.5, 5.0 and 25 mg/litre air (the measured concentrations were 0.15, 0.75 and 1.99 mg/litre, respectively). Exposures were repeated over a period of 3 weeks, 5 days each week. There were no signs of irritation in animals exposed to 0.5 mg/litre, but mild transient respiratory irritation was seen at the two higher concentrations. Haematological examination, biochemistry tests and macroscopic inspection revealed no treatment- related abnormalities (Berczy et al., 1975b). Diflubenzuron, at levels of 4.64, 10 and 21.5% weight/volume was applied daily to the intact and abraded skin of rabbits at a dosage level of 1.5 ml/kg body weight, 5 days a week, for 3 consecutive weeks (equivalent to 69.6, 150 and 322.5 mg diflubenzuron/kg body weight per day). The sulfhaemoglobin level was increased in one rabbit out of 20 in the 10% group, and in 5 rabbits out of 20 at the high treatment level. The 10% treatment level was considered to be the NOEL based on sulfhaemoglobin formation (Davies et al., 1975). 7.3 Long-term exposure Diflubenzuron was administered to Sprague-Dawley rats (60 of each sex per group) in their diet at levels 0, 10, 20, 40 and 160 mg/kg feed for 104 weeks. There were no treatment-related effects on body weight gain, food intake, renal function or on macroscopic and microscopic pathology. In rats treated with 160 mg/kg, significantly higher methaemoglobin levels were recorded. The tumour profile of treated rats was similar to that of the controls. The NOEL based on methaemoglobin was 40 mg/kg, equivalent to a mean intake of 1.43 and 1.73 mg/kg per day for males and females, respectively (Hunter et al.,1976). When diflubenzuron was administered in the diet to male and female Sprague-Dawley rats (50 of each sex per group) at dosage levels of 0, 156, 625, 2500 and 10 000 mg/kg feed for 104 weeks, there was no evidence of an effect on mortality or treatment-related clinical signs. Significantly increased absolute methaemoglobin and sulfhaemoglobin values were observed in all male treatment groups. However, increases in relative methaemoglobin values (% of total haemoglobin) were only noted in the 156, 2500 and 10 000 mg/kg groups, while sulfhaemoglobin increases were noted in the 156, 625 and 10 000 mg/kg females. There was a significant increase in absolute and relative spleen weights in the two highest dose groups of both sexes, together with haemosiderosis in spleen and liver. There was no evidence of carcinogenicity after 2 years of feeding diflubenzuron (Burdock et al., 1984). In a study by Hunter et al. (1975), diflubenzuron was administered to CFLP mice for 80 weeks at dietary levels of 0, 4, 8, 16 and 50 mg/kg feed. There were no overt signs of reaction to treatment. Behaviour, mortality, food and water consumption and body weight were unaffected by the treatment. The macroscopic changes observed were those commonly seen in mice of this strain and age. No histopathological changes were seen that were considered to be related to the administration of diflubenzuron. The incidence of liver cell tumours was higher in this study than it was in other studies performed in these laboratories using this strain of mouse. The increase was seen in both treated and control groups of either sex and showed no evidence of a treatment-related effect. There was no evidence of a treatment-related effect on tumour incidence in the CFLP mouse. Male and female HC/CFLP mice (88 of each sex per group) were fed diflubenzuron at dietary levels of 0, 16, 80, 400, 2000 and 10 000 mg/kg feed for 91 weeks. There was no indication of a treatment-related effect on survival, food consumption or body weight gain. Treatment-related elevations of MCH and MCHC values were recorded from week 26 onwards in mice given 10 000 mg/kg. The incidence of Heinz bodies increased in a dose-related manner among mice given 400, 2000 or 10 000 mg/kg from week 52 onwards. Dose- related increases in methaemoglobin levels were found from week 26 onwards and in sulfhaemoglobin levels from week 52 onwards in the mice fed 80 mg/kg or more. Elevated alkaline phosphatase (AP) activities were seen at weeks 24, 76 and 89 among male mice receiving 2000 and 10 000 mg/kg and at week 84 among mice of the 400 mg/kg dose group. An increased incidence of splenic and/or hepatic enlargement was seen among mice treated with 10 000 mg/kg. Cyanosis of the skin was noted among mice at 400, 2000 and 10 000 mg/kg. Increased liver and spleen weights were found among mice given 2000 and 10 000 mg/kg. An increased incidence of hepatocyte enlargement and increased extramedullary haematopoiesis in the liver and spleen were seen in mice at high dose levels. In the 400, 2000 and 10 000 mg/kg groups, there was an increased incidence of siderocytosis in the spleen and of pigmented Kupffer cells in the liver. There was no treatment-related effect on tumour incidence (Colley et al., 1984). 7.4 Skin and eye irritation; sensitization Relevant data are given in Table 7. Administration of technical diflubenzuron to the intact and abraded skin of albino rabbits did not produce irritation of the skin after exposure times of 24 and 72 h (Taylor, 1973a,b). Diflubenzuron was found to be a moderate irritant to the rabbit skin after application of 0.5 ml of 45% oil dispersible concentrate for 24 h (Koopman, 1980a; Prinsen, 1990). Diflubenzuron (both technical and 45% oil dispersible concentrate) was considered to be marginally irritant to the rabbit eye (Davies & Ligget, 1973; Koopman, 1980b). Diflubenzuron (48% water-based paste) was not found to be a dermal sensitizer in guinea-pigs (Kynoch & Parcell, 1987). Technical diflubenzuron was studied for skin sensitization in a maximization test on guinea-pigs and was found to be non-sensitizing (Prinsen, 1992). However, some formulations are mild sensitizers (Table 7). 7.5 Reproductive toxicity, embryotoxicity and teratogenicity Diflubenzuron was fed to pathogen-free rats of the CFY strain (20 of each sex per group) at dietary levels of 0, 1000 and 100 000 mg/kg for one generation and one litter. The animals were maintained on their respective diets for 9 weeks prior to mating. There were no clear effects on mating performance, pregnancy rate, duration of gestation, litter size, offspring mortality, litter weight or the type and distribution of abnormalities. Dose-related effects of diflubenzuron were demonstrated at 17 weeks in adults and consisted of reduced values for PCV, haemoglobin, total red cells count and MCHC, increased values for methaemoglobin, MCV, spleen weight and siderocyte incidence in the spleen, and the occurrence of iron- pigment-containing Kupffer cells in the liver. A dose-related effect on the liver was also shown by increased weight and SGPT activity and centrilobular hepatocyte enlargement. Reduced blood glucose concentrations were recorded in both treated groups. At the highest dosed level, the offspring showed increased liver and spleen weights for both sexes (Palmer et al., 1978). In a three-generation reproductive study with rats fed diflubenzuron at concentrations of 10, 20, 40 and 160 mg/kg feed, no adverse treatment-related effects on mating performance, pregnancy rate, duration of gestation or litter parameters (total loss, size, mean pup weight, mortality, abnormalities) were found (Palmer & Hill, 1975a). After gavage administration of diflubenzuron at doses of 1, 2 and 4 mg/kg body weight per day to pregnant rats during days 6-15 of gestation, no effects were observed on embryonic or fetal development (Palmer & Hill, 1975b). Treatment of pregnant New Zealand white rabbits at oral doses of 1, 2 and 4 mg/kg body weight per day during days 6-18 of gestation did not affect embryonic or fetal development as assessed by the incidence of major malformations, minor anomalies and skeletal variants (Palmer & Hill, 1975c). In a study by Booth (1977), pregnant Swiss mice were fed a diet containing 50 mg/kg of diflubenzuron (partly 14C) for a period of 17 days. Some of these mice were killed at day 17 after conception, while the others were allowed to give birth. The results of this study showed that mucopolysaccharide synthesis in embryonic mouse-limb cartilage was normal. Diflubenzuron did not pass through embryonic membranes nor was it passed from mother to suckling young mice. Analysis of 226 embryos showed no gross teratogenic effects. Two groups of 24 timed-mated female rats of the Crl:CD (SD) BR strain were dosed once daily by the oral route between days 6 and 15 of pregnancy. One group was dosed with diflubenzuron at 1000 mg/kg per day, while the other group was given the vehicle (1.0% gum tragacanth) only. Clinical signs, body weights and food consumption were recorded. The females were killed on day 20 of pregnancy and a necropsy was performed. The fetuses were subjected to detailed external, visceral and skeletal examinations. There were no maternal deaths or treatment-related changes in clinical condition. Treatment did not affect maternal growth or food consumption. There were no maternal abnormalities at necropsy that were considered to be related to treatment. The mean numbers of corpora lutea, implantations and live fetuses were similar in all groups. Both pre- and post- implantation losses were unaffected by treatment. Fetal weights and sex ratio were unaffected by diflubenzuron treatment. The incidences of major external/visceral and skeletal abnormalities, and minor external/visceral abnormalities were not affected by diflubenzuron treatment. The incidence of fetuses with minor skeletal abnormalities was slightly higher in the treated group, but was within the usual background range. There were intergroup differences in the proportions of fetuses with specific minor abnormalities and variants of skeletal ossification. For some bones, the differences achieved statistical significance. However, on balance, treated group fetuses were considered to be similarly ossified to the control fetuses. Oral administration of diflubenzuron at a dose level of 1000 mg/kg per day did not elicit maternal toxicity or any evidence of embryotoxicity (Kavanagh, 1988a). Two groups of 16 timed-mated female New Zealand White rabbits were dosed once daily by the oral route from days 7 to 19 of pregnancy, inclusive. One group was dosed with diflubenzuron at 1000 mg/kg per day and the other group with the vehicle (1.0% gum tragacanth) only. Clinical signs, body weights and food consumption were recorded. The females were killed on day 28 of pregnancy and a necropsy was performed. The fetuses were subjected to detailed external, visceral and skeletal examinations. There were no maternal deaths, changes in clinical condition or abnormalities at maternal necropsy considered to be related to diflubenzuron treatment. Two animals were killed prematurely during the study, one from the control group and one from the treated group. The changes in clinical condition and abnormalities observed at necropsy in these animals were considered to be unrelated to treatment. Treatment did not affect maternal growth or food consumption. The mean numbers of corpora lutea, implantations and live fetuses were similar in all groups. Both pre- and post-implantation losses were unaffected by treatment. Fetal weights and sex ratio were unaffected by diflubenzuron treatment. The incidence of both major and minor external/visceral and skeletal abnormalities, as well as the numbers of skeletal variants, was unaffected by diflubenzuron treatment. Oral administration of diflubenzuron at a dose level of 1000 mg/kg per day did not elicit maternal toxicity or any evidence of embryotoxicity (Kavanagh, 1988b). In a study by Kubena (1982), diflubenzuron was fed at levels of 0, 2.5, 25 and 250 mg/kg feed to male and female layer-breed chickens from 1 day of age through a laying cycle. Characteristics measured were egg production, egg weight, eggshell weight, fertility, hatchability and effects on the progeny. Feeding diflubenzuron at levels up to 250 mg/kg feed did not affect these characteristics. Groups of chicken eggs were injected near the embryonic coelom with a suspension of 10 mg diflubenzuron in 0.1 ml of peanut oil. Diflubenzuron did not cause significant malformations in the embryos (Seegmiller & Booth, 1976). 7.6 Mutagenicity and related end-points Diflubenzuron was examined by in vitro and in vivo mutagenicity tests. The results are summarized in Table 8. Mutagenicity tests have also been carried out with the major metabolites of diflubenzuron (Table 9). Table 8. Mutagenicity tests with diflubenzuron End-point Organisms, Dose level, Metabolic activationa Results References cells, strains concentration (presence/absence = +/-) Microorganisms Reverse Salmonella typhimurium mutation TA98, TA100, 10-1000 g/plate +/- negative Bryant (1976) TA98, TA100, TA1537, TA1978 1000 g/spot +/- negative Bryant (1976) Reverse S. typhimurium 0.1-500 g/plate +/- negative Brusick & Weir mutation TA98, TA100, TA1535, (1977a) TA1537, TA1538 Reverse S. typhimurium 10, 100 or 1000 g/plate; +/- negative McGregor et al. mutation TA98, TA100, 19, 186, 1860 g DFB/plate (1979) TA1535, TA1537 (as Dimilin W-25) Reverse S. typhimurium up to 5000 g/plate +/-b negative Moriya et al. mutation TA98, TA100, TA1535, (1983) TA1537, TA1538 Escherichia coli. WP2 hcr Reverse S. typhimurium up to 1000 g/plate +/- negative Koorn (1990) mutation TA98, TA100, TA1535, TA1537, TA1538 Table 8. (Con't) End-point Organisms, Dose level, Metabolic activationa Results References cells, strains concentration (presence/absence = +/-) Mammalian cells in vitro Forward L5178Y mouse lymphoma 1.17-300 g/ml +/- negative McGregor et al. mutation cells (1979) Chromosomal Chinese hamster ovary up to 200 g/ml +/- negative Taalman & Hoorn aberrations cells (1986) Unscheduled human diploid WI-38 cells 50-1000 g/ml +/- negative Brusick & Weir DNA synthesis (blocked in the G1 phase) (1977c) DNA repair rat hepatocytes up to 333 g/ml negative Enninga (1990) (unscheduled DNA synthesis) Cell transformation BALB-3T3 cells, 0.02-0.312 g/ml - negative Brusick & Weir in vitro (1977b) Cell transformation pregnant hamster, ip 10, 200 and 500 mg/kg negative Quarles et al. (transplacental injection on 10th day of body weight (1980) transformation) gestation, fetal cell culture 3 days after injection Table 8. (Con't) End-point Organisms, Dose level, Metabolic activationa Results References cells, strains concentration (presence/absence = +/-) Mammals Micronucleus mouse bone marrow 15, 150, 1500 mg/kg body negative McGregor et al. weight 30 and 6 h before (1979) necropsy Dominant male mice mated to 1000 and 2000 mg/kg body negative Arnold et al. lethal 3 females weekly for 6 weeks weight intraperitoneal (1974) a Unless indicated otherwise, S9 was obtained from livers of rats treated with Arochlor-1254 b Source of S9 not indicated Table 9. Mutagenicity tests with diflubenzuron metabolites End-point Test system Dose level Metabolic activationa Results References (presence/absence = +/-) A. 4-chlorophenylurea Microorganisms Reverse mutation Salmonella typhimurium and differential TA98, TA100, TA1535, 1000 g/spot +/- negativeb Dorough (1977) killing TA1537, TA1538, TA1978 Reverse mutation TA98, TA100 10, 100, 500, negative 1000 g/plate Reverse mutation S. typhimurium 0.1-500 g/plate +/- negative Jagannath & Brusick TA98, TA100, TA1535, (1977a) TA1537, TA1538 Mammalian cells Unscheduled human WI-38 cells 6.25-50 g/ml +/- negative Matheson & Brusick DNA synthesis (blocked in the G1 phase) (1978b) Cell BALB-3T3 cells, 0.019-0.312 mg/ml not stated weak Matheson & Brusick transformation in vitro positive at (1977a) 0.312 mg/ml Table 9. (Con't) End-point Test system Dose level Metabolic activationa Results References (presence/absence = +/-) B. 2,6-Difluorobenzoic acid Microorganisms Reverse mutation S. typhimurium 1000 g/spot +/- negative Dorough (1977) and differential TA98, TA100, TA1535, killing TA1537, TA1538, TA1978 Reverse mutation S. typhimurium 10, 100, 500, TA98, TA100 1000 g/plate Reverse mutation S. typhimurium 0.1-500 g/plate +/- negative Jagannath & Brusick TA98, TA100, TA1535, (1977b) TA1537, TA1538 Mammalian cells Unscheduled human WI-38 cells 75-500 g/ml +/- positive Matheson & Brusick DNA synthesis (blocked in the G1 phase) with (1978a) activation Cell BALB-3T3 cells, 0.156-2.5 mg/ml not stated weak Matheson & Brusick transformation in vitro positive at (1977b) 2.5 mg/ml C. 4-Chloroaniline (PCA) Microorganisms Reverse mutation S. typhimurium 1000 g/spot +/- negativeb Dorough (1977) and differential TA98, TA100, TA1535, killing TA1537, TA1538, TA1978 Table 9. (Con't) End-point Test system Dose level Metabolic activationa Results References (presence/absence = +/-) Reverse mutation TA98, TA100 10, 100, 500, positive in 1000 g/plate TA98 at 500 and 1000 g with activation Reverse mutation S. typhimurium 0.1-500 g/plate +/- negative Jagannath & Brusick TA98, TA100, TA1535, (1977c) TA1537, TA1538 Reverse mutation S. typhimurium TA98, TA100, TA1530, 0-1500 g/plate +/- negative Gilbert et al. (1980) TA1535, TA1537, TA1538 Reverse S. typhimurium C3076, 1000 g/plate +/- negative Thompson et al. mutation D3052, G46, TA98, TA100, (1983) TA1535, TA1537, TA1538, Escherichia coli WP2, WP2 uvrA Reverse S. typhimurium 3333 g/plate +/- positivec Dunkel et al. (1985) mutation TA98, TA100, TA1535, TA1537, TA1538, Escherichia coli Reverse S. typhimurium 1666 g/plate +/- positived Mortelmans et al. mutation TA97, TA98, TA100, TA1535 negative (1986) DNA damage Escherichia coli polA+/polA- 250 g/plate - positive Rosenkranz & Poirier (1979) Table 9. (Con't) End-point Test system Dose level Metabolic activationa Results References (presence/absence = +/-) Mutation Aspergillus nidulans 200 g/ml - positive Prasad (1970) Mammalian cells Forward mutation L5178Y mouse lymphoma +/- positived Caspary et al. (1988) tk+/- cells Unscheduled human WI-38 cells 250-1000 g/ml +/- negative Matheson & Brusick DNA synthesis (blocked in the G1 phase) (1978b) Unscheduled rat primary hepatocytes 5-50 g/ml - positive Williams et al. DNA synthesis (1982) Unscheduled rat primary hepatocytes 50 nmol/ml - negative Thompson et al. DNA synthesis (1983) Sister chromatid Chinese hamster ovary 1600 g/ml +/- positived US NTP (1989) exchange cells Chromosomal Chinese hamster ovary cells 1000 g/ml +/- positived US NTP (1989) aberrations and negative Cell BALB-3T3 cells, 0.039-0.625 mg/ml not stated negative Matheson & Brusick transformation in vitro (1978c) a Unless indicated otherwise, S9 was obtained from livers of rats treated with Arochlor-1254 b No increase in revertants, strains TA1538/TA1978 positive for differential killing c Tested in three independent laboratories d Tested in two independent laboratories According to the findings presented, neither diflubenzuron nor its major metabolites may be considered to have mutagenic effect. Several positive effects were, however, obtained with PCA. 7.7 Carcinogenicity Long-term carcinogenicity studies were described in section 7.3. In both mouse and rat oncogenicity studies, diflubenzuron at dose levels up to 10 000 mg/kg feed caused no change in tumour profile or onset of tumours. In the rat oncogenicity study, the incidence of sarcoma in the spleen and phaeochromocytomas was not increased. In the mouse oncogenicity study, the incidence of hepatocellular neoplasms or haemangiosarcomas in spleen and liver was not increased. Therefore, diflubenzuron, in combination with its metabolites as generated in the animal metabolic system, is not oncogenic. Significantly, there were no non-neoplastic or neoplastic lesions of the vasculature, including that of liver and spleen, in B6C3F1 male mice treated with diflubenzuron. Similarly, there were no fibrotic or carcinomatous lesions in the spleen of treated male F-344/N rats. 7.8 Other special studies Diflubenzuron has been studied in mice for its growth-inhibiting activity in serially transplanted B16 malignant melanoma and CA1025 skin carcinoma. A single 800 mg/kg intraperitoneal injection of diflubenzuron induced rapid (24 h) decreases in tumour volume in 78% and 66% of the tumours, respectively, while in control mice 85% of the melanomas and 91% of the skin carcinomas increased in volume over the same time period (Jenkins et al., 1984). These observations were later confirmed, and it was suggested that the activity was due to derivatives of a hydroxylated metabolite (Jenkins et al., 1986). Studies of nucleoside uptake by Harding Passey melanoma cells in vitro indicated a rapid (< 5 min) inhibition of the uptake of uridine, adenosine and cytidine, but not of thymidine, by diflubenzuron; this could not be reversed by washing. De novo nucleic acid synthesis was not impaired and in vitro cell growth was unaffected (Mayer et al., 1984). El-Sebae et al. (1988) tested the effect of diflubenzuron on protein and RNA biosynthesis by rabbit liver and muscle tissues kept in an incubation medium. The synthesis of protein and RNA was significantly stimulated in the liver and inhibited in the muscle by graded doses. The maximum effect on both tissues was reached at 5 g diflubenzuron/ml for protein synthesis and at 0.2 g/ml for RNA synthesis, the effect on protein synthesis being more pronounced than that on RNA synthesis in both tissues. 7.8.1 Special studies on met- and sulfhaemoglobin formation The ability of diflubenzuron to induce methaemoglobin and sulfhaemoglobin formation has been recognized since the initial toxicity studies on the compound. Methaemoglobinaemia has been demonstrated after oral, dermal and inhalatory exposure to diflubenzuron in various species (see section 7.2 and 7.3). It is the most sensitive parameter in this case. Fifteen male Wistar rats received diflubenzuron (technical) by gastric intubation at a dose level of 5000 mg/kg body weight per day for 8 days. No effect was observed on Heinz body formation, whereas met- and sulfhaemoglobin levels were significantly increased when compared with the control group. The increase in the methaemoglobin level was about 6% (Keet, 1977a). When diflubenzuron was administered to male Wistar rats for 28 days at oral doses of 100 and 500 mg/kg body weight, it induced elevation of methaemoglobin concentration and reticulocytes count in both of the treated groups. However, there was no dose-response relationship at the dose levels investigated (Tasheva & Hristeva, 1991, 1993). Technical diflubenzuron was administered by gastric intubation to male Swiss mice daily for a period of 14 days at dose levels of 0, 8, 40, 200, 1000 and 5000 mg/kg body weight. Body weight measurement and macroscopic evaluation did not reveal any effect of the treatment. At dose levels of 1000 and 5000 mg/kg the percentages of methaemoglobin and erythrocytes containing Heinz bodies were increased. The sulfhaemoglobin level was statistically significantly increased at 200, 1000 and 5000 mg/kg in comparison to the control group. The NOEL was considered to be 40 mg/kg body weight based on sulfhaemoglobin (Keet, 1977b). When female mice were fed 0, 50, 200, 400, 1000 and 2000 mg diflubenzuron/kg feed for 30 days, sulfhaemoglobin was demonstrated in the blood from 200 mg/kg onwards (being 13% of total haemoglobin at 2000 mg/kg). Mice fed 1000 and 2000 mg/kg showed signs of cyanosis after 3 weeks. Recovery was completed after a 3-week withdrawal period (Bentley et al., 1979). When 15 male New Zealand White rabbits were fed technical or analytically pure diflubenzuron (640 mg/kg feed) for 21 or 18 days, respectively, the methaemoglobin and sulfhaemoglobin levels were significantly increased (Keet, 1977c). Male and female cats (24 of each sex per group) received diflubenzuron orally for 21 days at dose levels of 0, 30, 70, 100, 300 and 1000 mg/kg body weight and were observed for the subsequent 14-day period. A dose-related elevation of methaemoglobin level was observed with ceiling values at 300 and 1000 mg/kg. For male cats the NOEL was 30 mg/kg, but no NOEL was achieved for females. Increased sulfhaemoglobin and Heinz bodies were observed in all treated groups. NOEL values for sulfhaemoglobin were not achieved. The haemoglobin concentration, reticulocyte number and organ weights were not affected by the treatment (Schwartz & Borzelleca, 1981). After dermal application of technical diflubenzuron at a dose level of 1.5 ml/kg body weight for 18 days to rabbits, the methaemoglobin level was increased (Keet, 1977c). The available data demonstrate that dose-response relationship for production of methaemoglobin exists. This is considered to be the most sensitive end-point after repeated exposure in experimental animals. Table 10 summarizes the effects on methaemoglobinaemia as determined in various studies. 7.9 Toxicity of metabolites In rat metabolic studies it was shown that about 20% of absorbed diflubenzuron is metabolized to 2,6-DFBA and its counterpart 4-CPU. Only a small fraction of the 4-CPU is metabolized to PCA (see section 6). The acute oral toxicity of the major metabolites of diflubenzuron is summarized in Table 11. Loss of activity, catatony, paralysis and severe bradypnoea were observed in rats treated with the metabolite 4-CPU. The minimum symptomatic dose level was 100 mg/kg body weight. At autopsy, the animals showed congested blood vessels and haemorrhage in the gastrointestinal tract (Koelman-Klaus, 1978a). Rats dosed with 2,6-DFBA showed slight increase in startle response, activity, abdominal and limb tone, slight decrease in grooming activity, slightly abnormal gait and body posture, mild restlessness, irritation and aggressivity, pilo-erection and increased alertness. The minimum symptomatic dose level was 464 mg/kg body weight (Koelman-Klaus, 1978b). Mutagenicity tests have been carried out with 2,6-DFBA, 4-CPU and PCA (see section 7.6 and Table 9). Table 10. Summary of the effects on methaemoglobinaemia in various species Species Route Duration No-observed-effect level Reference Rat diet 4 weeks male: not achieved; female: 800 mg/kg feed Palmer et al. (1977) (equivalent to 45 mg/kg per day) Rat diet 13 weeks not established - lowest dose tested 160 mg/kg feed Burdock et al. (1980b) Rat diet 104 weeks 40 mg/kg feed (equivalent to 2 mg/kg Hunter et al. (1976) per day body weight) Mouse oral 2 weeks 200 mg/kg body weight Keet (1977b) Mouse diet 13 weeks male/female: 16 mg/kg feed (equivalent to Burdock et al. (1980a) 2.4 mg/kg body weight per day) Mouse diet 14 weeks not established - lowest dose tested 80 mg/kg feed Colley et al. (1981a,b) Mouse diet 91 weeks male/female: 16 mg/kg feed (equivalent to Colley et al. (1984) 2.4 mg/kg body weight per day) Cat oral 3 weeks male: 30 mg/kg body weight; female: not achieved Schwartz & Borzelleca (1981) Dog diet 13 weeks male/female: 40 mg/kg feed Chesterman et al. (1974) Dog oral (capsules) 52 weeks male/female: 2 mg/kg body weight Greenough et al. (1985) Table 11. Acute toxicity of diflubenzuron metabolites Metabolite Species Sex Route LD50 Reference (mg/kg) 4-Chlorophenylurea rat male oral 1080 Koelman-Klaus female oral 1210 (1978a) 2,6-Difluorobenzoic rat male, oral 4640 Koelman-Klaus acid female (1978b) 7.9.1 Carcinogenicity studies with 4-chloroaniline The diflubenzuron metabolite, 4-chloroaniline (PCA), has been assayed for carcinogenicity by the US NCI (1979) and by the US NTP (1989) using Fischer-344 rats and B6C3F1 mice on both occasions. In the earlier of these studies, technical-grade PCA was administered at dietary concentrations of 250 and 500 mg/kg to rats and 2500 and 5000 mg/kg to mice. Groups of 50 male and 50 female animals of each species were randomized to the treatment groups at approximately six weeks of age. The control groups consisted of 20 animals of each sex and species. All animals which survived were treated for 78 weeks and observed, untreated, for a further 24 weeks (rats) or 13 weeks (mice). Survival in all groups was good and it was judged that there were adequate numbers at risk for late-developing tumours. In rats, the most significant findings were treatment-related proliferative splenic capsular and parenchymal lesions in males and females and, in male rats of the high-dose group, the occurrence of several types of unusual splenic neoplasms (i.e. fibroma, fibrosarcoma, sarcoma NOS, haemangiosarcoma and osteosarcoma) which appeared to arise from areas of capsular or parenchymal fibrosis. These neoplasms were combined for analysis because it was considered that the fibromas were a benign form of sarcoma and that the neoplasms had a common cellular origin. The combined incidences were 0/20 control, 0/49 low-dose and 10/49 high-dose rats. This result indicated a carcinogenic effect of treatment in male rats. There was no similar effect in the spleen of females. In mice of each sex, there was increased incidence of haemangiomas and haemangiosarcomas in various organs. The combined incidences were 2/20 in control males, 10/50 in low-dose males and 14/50 in high-dose males, and 0/18 in control females, 3/49 in low- dose females and 8/42 in high-dose females. In addition, there was, in female mice only, a non-significant increase in hepatocellular carcinomas and adenomas combined (0/18 control, 1/49 low-dose and 6/41 high-dose). The smaller number of control group animals in these experiments significantly reduced the statistical power. It was recognized that PCA is unstable in feed and so the animals in the early experiments may have received lower doses than intended. Consequently, PCA was administered by gavage with an aqueous vehicle containing hydrochloric acid in the later experiments (US NTP, 1989). Groups of 49 or 50 rats and mice of each sex (7 to 8 weeks old) were administered PCA at dose levels of 2, 6 or 18 mg/kg (rats) or 3, 10, or 30 mg/kg (mice) on 5 days per week for 103 weeks. Vehicle control groups of 50 males and 50 females received deionized water by gavage. Survival was adequate for analysis, although variable. It was lower, for example, in the vehicle control groups of rats, which was attributable to the higher incidence of mononuclear cell leukaemia in these groups. Significant, non-neoplastic findings in rats included treatment-related increases in the incidence of splenic fibrosis (males: 3/49 control, 11/50 low-dose, 12/50 mid-dose, 41/50 high-dose; females: 1/50 control, 2/50 low-dose, 3/50 mid-dose, 42/50 high-dose), lipocytic infiltration of the spleen (males: 0/49, 0/50, 0/50, 24/50; females: 0/50, 0/50, 0/50, 11/50) and adrenal medullary hyperplasia in female rats (4/50, 4/50, 7/50, 24/50). In addition, the methaemoglobin level was consistently increased in the mid- and high- dose groups of male rats at 6, 12, 18 and 24 months. There was no NOEL for this parameter in male rats. Bone-marrow hyperplasia was also observed. The combined incidences of uncommon splenic sarcomas (fibrosarcomas, osteosarcomas or haemangiosarcomas) were increased in male rats but not in females (males: 0/49, 1/50, 3/50, 38/50; females: 0/50, 0/50, 1/50, 1/50). There was also a small increase in phaeochromocytomas or malignant phaeochromocytomas in male rats (13/49, 14/48, 15/48, 26/49). The incidences of mononuclear cell leukaemia were reduced in male and female rats of the treatment groups (males: 21/49, 3/50, 2/50, 3/50; females: 10/50, 2/50, 1/50, 1/50). This reduction may be related to splenic toxicity, since splenectomy of Fischer rats at one to two months of age markedly reduces the incidence of mononuclear cell leukaemia later in life (Boorman et al., 1990). However, similar reductions in the incidence of this neoplasm are not seen with several other aniline-like chemicals that also cause splenic toxicity. In male mice, but not in females, the incidence of haemangiosarcomas of the liver and spleen was slightly greater in the high-dose group than in the controls (males: 4/50, 4/49, 1/50, 10/50) and the incidences of hepatocellular carcinomas or adenomas (combined) were also increased in treated male mice (males: 11/50, 21/49, 20/50, 21/50). The incidences of malignant lymphomas were slightly reduced in treated male and female mice (males: 10/50, 3/49, 9/50, 3/50; females: 19/50, 12/50, 5/50, 10/50). There are various points of similarity between the two studies described above: (a) in male and female rats there was splenic toxicity; (b) in male rats, there was a treatment-related increase in uncommon splenic sarcomas; and (c) in male mice, there was a treatment-related increase in haemangiosarcomas and haemangiomas combined. The reductions in the incidence of mononuclear cell leukaemia in rats and malignant lymphomas in mice seen in the latter study were not observable in the earlier study because their incidence in the control groups of that study was already low. It is concluded that PCA is carcinogenic in both mice and rats. 8. EFFECTS ON HUMANS No data concerning the effects of diflubenzuron on human health are available. 9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD 9.1 Laboratory experiments 9.1.1 Microorganisms Diflubenzuron was tested for its effects on morphogenesis in Streptomyces spp. Exposure to 400 mg diflubenzuron/litre resulted in reduced dominance of spore hairs and reduced width of the outer wall, and prevented formation of the inner spore wall in S. babergiensis. In S. coelicolor, 1600 mg diflubenzuron/litre altered the structure of the fibrillar pattern of spore envelopes. Exposure to diflubenzuron resulted in small increases in exported protein and in an approximately 20% increase in chitinase in both Streptomyces spp (Smucker & Simon, 1986). 9.1.1.1 Water Aquatic bacterial biomass and density were not affected by diflubenzuron (1.0 g/litre), although after three months of continuous exposure some decline in species diversity was found. Owing to detrimental effects on aquatic arthropods, e.g., filter feeders that may use bacteria as a food source, such effects may very well be secondary in nature and not a direct effect of diflubenzuron (Hansen & Garton, 1982b). In a test on the alga Selenastrum capricornutum, a nominal concentration of 0.20 mg diflubenzuron/litre did not reduce the biomass and can be considered a no-observed-effect concentration (NOEC) (Berends & Thus, 1992b). 9.1.1.2 Soil Soil microorganisms were able to use diflubenzuron as carbon source when added as an acetone solution (Seuferer et al., 1979; see section 4.3.2.2). The effect of diflubenzuron (100, 200, 300, 400 and 500 mg/kg soil) was studied in non-sterile soil incubated under aerobic conditions and in sterilized soil inoculated with Azotobacter vinelandii. The presence of diflubenzuron had a stimulatory effect on nitrogen fixation in both non-sterile and sterile soil (Martinez- Toledo et al., 1988a). At similar concentrations, diflubenzuron did not affect the growth of Azotobacter vinelandii in culture media, either with or without a nitrogen source (Martinez-Toledo et al., 1988b). 9.1.2 Aquatic organisms 9.1.2.1 Microorganisms Cyanobacteria (the blue-green alga Plectonema boryanum) grew rapidly in the presence of diflubenzuron (initial concentration 0.1 mg/litre) with no visible signs of inhibited growth (Booth & Ferrell, 1977). Concentrations of 1, 10, 50 and 100 mg/litre did not affect the growth of six species of fungi: Rizopus arrhizus, Aspergillus niger, Fusarium oxysporum, Mycorrhizae-Rhizopogan vinicolor, Pythum debaranum and Trichoderma viride (Booth et al., 1987). However, these authors autoclaved the medium containing diflubenzuron, which led to extensive breakdown of the pesticide (Willems et al., 1977). Five-day lethality tests on the algae Selenastrum capricornutum and Anabaena flos-aquae resulted in NOAEC values above the exposure concentrations of 300 and 330 g/litre, respectively (Thompson & Swigert, 1993b,c). Similarly, tests on the diatoms Navicula pelliculosa and Skeletonema costatum led to NOAEC values of 380 and 270 g/litre, respectively (Thompson & Swigert, 1993d,e). Algae were affected at 1.0 g/litre by technical diflubenzuron in dimethylformamide added to laboratory stream channels: the alga biomass increased and chlorophyll and phaeocitin levels were elevated. Fungi were only affected temporarily by 0.1 g/litre under the same circumstances. Changes in species diversity had disappeared after 2 months of continuous dosing (Hansen & Garton, 1982 b). 9.1.2.2 Plants In a 14-day toxicity test on diflubenzuron in duckweed (Lemna gibba), the NOAEC was higher than the tested concentration (190 g/litre) (Thompson & Swigert, 1993a). 9.1.2.3 Invertebrates The acute toxicity of diflubenzuron to a number of non-target aquatic invertebrates is presented in Table 12. Comprehensive eviews on the subject have been published recently, e.g., by Fischer & Hall (1992) and by Cunningham (1986) on the effects of diflubenzuron on estuarine crustaceans. The acute LC50 for insects ranges from 1 g/litre (Diptera) to 250 g/litre (Coleoptera), and mayflies have an LC90 of 1 to 10 g/litre. Other aquatic arthropods such as water fleas, scuds and sow bugs have LC50 values of 5 to 15 g/litre. Table 12. Acute toxicity of diflubenzuron for non-target aquatic invertebrates Species Size/age Stat/flowa Temperature Hardness pH Parameter Concentration Reference (C) (mg/litre) (g/litre) Daphnia magna 1st instar stat 22 40 7.2 48-h EC50 15 Julin & Sanders (1978) (Water flea) Daphnia magna 24 h stat 20 50 48-h LC50 4.55 Hansen & Garton (1982a) (Water flea) stat 20 100 48-h LC50 6.89 Hansen & Garton (1982a) stat 20 200 48-h LC50 4.42 Hansen & Garton (1982a) Daphnia magna 0-24 h 20 294 8.1 48-h EC50 7.1 Kuijpers (1988) (Water flea) 20 294 8.1 24-h EC50 68 Kuijpers (1988) Gammarus pulex mature stat 12 40 7.2 96-h LC50 30 Julin & Sanders (1978) (Scud) Hyallela azteca 2-4 mm flow 20 25 96-h LC50 1.84 Hansen & Garton (1982a) (Amphipod) Chironomus plumosus 4th instar stat 22 40 7.2 48-h EC50 560 Julin & Sanders (1978) (Midge) Cricotopus sp. 4th instar flow 20 25 EC50 (moulting 1.72 Hansen & Garton (1982a) (Midge) success) Tanytarsus dissimilis 2nd instar flow 20 25 EC50 (moulting 1.02 Hansen & Garton (1982a) (Midge) success) Acartia tonsa adult constant 20 10b 5-day LC50 > 1000 Tester & Costlow (Copepod) daily (1981) replenishment Table 12 (Con't) Species Size/age Stat/flowa Temperature Hardness pH Parameter Concentration Reference (C) (mg/litre) (g/litre) Mysidopsis bahia adult intermittent 24-25 24-27b 96-h LC50 2.06 Nimmo et al. (1980) (Mysid shrimp) flow adult continuous 24-26 23-29b 21-day LC50 1.24 Nimmo et al. (1980) flow Palaemonetes pugio larvae stat 22 20b 96-h LC50 1.44 Wilson & Costlow (Grass Shrimp) (1987) post larval renewal 22 20 96-h LC50 1.62 Wilson & Costlow (1987) a Stat = static conditions (water unchanged for duration of test); flow = flow-through conditions (diflubenzuron concentration in water continuously maintained) b Salinity (expressed as parts per thousand) Concentrations of diflubenzuron causing significant mortality of several freshwater invertebrates are presented in Table 13. Table 13. Concentrations of diflubenzuron causing significant mortality of freshwater invertebratesa Organisms Concentration (g/litre) Water flea (Daphnia magna) 2.0 Amphipod (Hyalella azteca) 2.0 Snail (Juga plicifera) > 36 Snail (Physa spp.) > 36 Caddis fly (Clisforonia magnifica) 0.1 Midge (Tanytarsus dissimilis) 4.9 Midge (Cricotopus spp.) 1.6 a From: Nebeker et al. (1983) The 96-h LC50 for the snails Juga plicifera and Physa sp. was > 45 g/litre (Hansen & Garton, 1982a). The 48-h EC50 values of diflubenzuron metabolites for midge larvae were > 100 mg/litre for 4-CPU and 2,6-DFBA and 43 mg/litre for PCA (Julin & Sanders, 1978). Diflubenzuron suspended in water at a concentration of 200 mg/litre was not directly toxic to the freshwater clam Anodonta cygnea during 3 months of treatment. Diflubenzuron produced disturbances in the calcification process in the lamellar layer of the shell. Positive PAS reaction of the secretory cells on the outer mantle epithelium has been observed (Machado et al., 1990). Daphnia magna was continuously exposed to 14C-diflubenzuron at concentrations of 5.6, 14, 23, 40 and 93 ng/litre. After 21 days of exposure daphnid survival at the highest concentration (93 ng/litre) was 50%. In the remaining concentrations it ranged from 93 to 98%, which was comparable to the survival (99%) of the control organisms. Reproduction and body length were affected only at the highest concentration. The maximum acceptable toxicant concentration (MATC) of 14C-diflubenzuron for Daphnia magna was > 40 and < 93 ng/litre (Surprenant, 1988). Technical diflubenzuron in dimethylformamide at 0.1, 1, 10 and 50 g/litre was added continuously to complex laboratory stream channels supplied periodically with field-collected microorganisms for 5 months. LC50 values ranging from 1.0 to 1.8 g/litre were determined for four insect and crustacean species ( Tanytarsus dissimilis, Cricotopus sp. and Hyalella azteca). A chronic effect level was obtained only for Daphnia magna (0.06 g/litre). Mayflies and stoneflies were the most sensitive. They were severely affected at 1 g/litre within one month (the sampling interval) and numbers were two to three orders of magnitude lower, almost leading to their elimination. The survival of chironomids was reduced by 10 g/litre (Hansen & Garton, 1982a,b) (see also section 9.1.1.1). Benthic communities in outdoor experimental streams were exposed to 1 or 10 mg/litre of diflubenzuron for 30 min. The effect was assessed daily by examining drifting pupal exuviae over a period of one month following the treatment. No drift of macrobenthos was induced at the time of application. However, diflubenzuron affected the emergence of all species examined. High larval mortality for a species of chironomid was observed directly in the stream treated with diflubenzuron, where numbers of mayfly nymphs and caddisfly larvae were also decreased (Yasuno & Satake, 1990). Technical diflubenzuron in acetone was applied at 5 g/litre to two aquaria in a simulated field test outdoors. Daphnid numbers were markedly reduced three days after treatment, but recovered slowly. Copepods numbers were moderately reduced, exceeding the control numbers after 18 days. The seed shrimp population showed no harmful effect (Miura & Takahashi, 1974a,b). In a study by Collwell & Schaefer (1980), diflubenzuron was applied to experimental ponds (mean concentration of 13.2 g/litre) in California. An hour after treatment, cladoceran ( Ceriodaphnia sp., Diaphanosoma sp., Chydorus sp., Bosmina sp. and Daphnia sp.) numbers were strongly reduced and did not return to pretreatment levels until more than 5 weeks after treatment. Copepod ( Diaptomus sp. and Cyclops sp.) numbers were also reduced but to a lesser extent and for a shorter period than for the cladocerans. The rotifers increased in abundance in both the control and treated ponds during the first 8 days following treatment. Using laboratory tests to study mortality, Miura & Takahashi (1974a) found that crustaceans, especially the tadpole shrimp (T. longicaudatus), clam shrimp ( Eulimnadia spp.) and water fleas ( Daphnia and Moina spp.), were highly susceptible to diflubenzuron at levels below 0.01 mg/litre. Copepods, Cyclops and Diaptomus spp. showed some tolerance, whereas seed shrimp ( Cypricerus and Cypridopsis spp.) tolerated as much as 0.5 mg/litre. Among aquatic insects tested, mayfly nymphs ( Callibaetis spp.) were most susceptible. Aquatic midge larvae (G. holoprasinus) also showed susceptibility. However, dytiscid ( T. bassillaris and Laccorhilus spp.) and hydrophilid beetles ( H. triangularis and T. lateralis (mosquito predators)) demonstrated a strong tolerance. Mosquito fish (Gambusia affinis) showed no effect at a relatively high dose of 1 mg/litre. When larvae of the crab Rhithropanopeus harrisii were exposed to sublethal concentrations of diflubenzuron (0.05, 0.1, 0.3 and 0.5 g/litre), swimming speed increased in stage I, II and III zoeae, 0.3 g/litre being the lowest effective concentration. Phototaxis was altered in stage IV only at concentrations as low as 0.1 g/litre (Forward & Costlow, 1978). Christiansen et al. (1978) showed that nearly 100% of Rhithropanopeus harrisii larvae at each of the four zoeal stages died when moulting to the succeeding stage after only 3 days of exposure to 10 g diflubenzuron/litre. This concentration was also lethal for larvae of Sesarma reticulatum (Say). Christiansen & Costlow (1980) exposed larvae of the estuarine crab Rhithropanopeus harrisii in laboratory conditions to 10 g diflubenzuron/litre as an indicator of persistence of diflubenzuron in brackish water. Disturbance of endocuticle deposition seemed to occur as soon as newly hatched larvae (less than 12 h old) were exposed to diflubenzuron. Diflubenzuron not only affected endocuticle deposition in the larvae, but also exocuticle deposition. The only part of the crab larval exoskeleton that did not seem to be affected by diflubenzuron was the epicuticle (Cristiansen et al., 1978; Cristiansen & Costlow, 1982). Diflubenzuron, at concentrations of 0.02 and 0.2 mg/litre, enhanced mortality during moulting of the crab Carcinus mediterraneus (Czerniavsky) (Cardinal et al., 1979). Cirripede crustaceans, (barnacles, Balanus eburneus) exposed to concentrations of 1 to 1000 g/litre over a 28-day period showed a dose-dependent mortality. Heavy mortality occurred during the second week of exposure. Lethal and sublethal effects were observed at concentrations as low as 50 g/litre (Gulka et al., 1980). Disruption of the exoskeleton of B. eburneus caused by diflubenzuron was similar to that observed in insects. Development of barnacles exposed to diflubenzuron for 10 days or more at 750 and 1000 g/litre was delayed in the premoult phase of cuticle secretion (Gulka et al., 1982). Wilson & Costlow (1986) found that diflubenzuron concentrations of 2.5 and 5 g/litre were lethal to larvae of the grass shrimp (Palaemonetes pugio), causing 100% mortality on days 14 and 6, respectively. Wilson et al. (1985) studied the effects of diflubenzuron upon phototaxis of larvae of P. pugio. The depression in positive phototaxis and elevation in negative phototaxis were most pronounced at 0.5 g/litre, and the lowest test concentration to affect phototaxis was 0.3 g/litre. The alterations in photo responses varied with the embryonic stage at which exposure to diflubenzuron commenced. This study was carried out at optimal salinity and temperature, but in the estuary these conditions fluctuate daily, which may amplify the observed effects. Nimmo et al. (1980) observed that chronic exposure of the mysid shrimp Mysidopsis bahia to 0.075 g diflubenzuron/litre reduced the reproductive success of both parents and progeny even after they were transferred to uncontaminated sea water. Diflubenzuron reduced the reproductive life span of adult brine shrimps (Artemia salina) at levels of 2-10 g/litre and caused death of immature shrimps within 3 days at concentrations above 10 g/litre (Cunningham, 1976). Fiddler crabs (Uca pugilator) were exposed to diflubenzuron (Dimilin WP-25%) at 0.5, 5 and 50 g/litre for 1, 2, 3 and 4 weeks after multiple autonomy of one chela and five walking legs. Exposure to diflubenzuron retarded the rate of limb regeneration in a dose- dependent fashion. The effects of diflubenzuron were seen even in crabs exposed for only one week. Significant retardation was evident by day 21 at 5.0 g/litre but was not statistically significant at 0.5 g/litre. At 5 and 50 g/litre moult-associated mortality was seen. The number of setae on regenerated limbs was less than the number on the intact limbs. The effects were reduced in experiments in which sediment was present (Weis et al., 1987). The burrowing activity of Uca pugilator in sand under laboratory conditions was not altered when the sand was contaminated with 1 mg diflubenzuron/litre, indicating a lack of avoidance of diflubenzuron-contaminated sand. However, exposure for 1 week to 0.5, 5.0 or 50 g/litre led to a decrease in the amount of burrowing activity. The behavioral response was not changed after exposure for 1, 2 or 3 weeks and was not concentration-related (Weis & Perlmutter, 1987). Survival, moulting and behaviour of juvenile fiddler crabs were significantly affected by exposure to diflubenzuron (0.2, 2, 20 and 200 g/litre) for 24 h weekly during 10 weeks. All crabs in the 200 and 20 g/litre groups died after 8 and 23 weeks, respectively. The no-observed-effect concentrations (NOEC) for moulting (time to the first moult), survival (time until death), and behaviour (ability to escape from the test container) were 20, 2 and 0.2 g/litre, respectively (Cunningham & Myers, 1987). Larvae of the stone crab (Menippe mercenaria) were exposed to 0.5, 1.0, 3.0, and 6.0 g diflubenzuron/litre in combination with different temperature and salinity. All of these concentrations were lethal to the larvae. Evidence for synergistic effects of diflubenzuron and temperature or salinity was observed. Tolerance of the megalopa of the blue crab Callinectes sapidus to diflubenzuron at concentrations of 0.5, 1.0, 3.0 and 6.0 g/litre was slightly higher than for Menippe mercenaria but was also dependent upon temperature and salinity. At 20C the percentage survival at a concentration of 1 g/litre was similar to that observed for the controls (Costlow, 1979). Tester & Costlow (1981) reported that the marine copepod Acartia tonsa exposed to 1 and 10 g diflubenzuron/litre for 36 h failed to produce viable nauplii even after they had been placed in clean sea water. No viable nauplii were produced by these females for at least 30 h after treatment ended. Adult crustaceans were more resistant to exposure than their larvae at high concentrations (100-200 g/litre) of technical grade diflubenzuron. Adults also exhibited significant mortality associated with moulting (Cunningham, 1976; Cardinal et al., 1979; Gulka et al., 1980). When larvae of horseshoe crabs (Limulus polyphemus) were exposed to 5 and 50 g diflubenzuron/litre, the crabs in the 50 g/litre group exhibited severe mortality immediately after ecdysis. The larval stages were shown to be quite resistant to diflubenzuron, compared with other crustacean larvae (Weis & Ma, 1987). 9.1.2.4 Vertebrates Data on the acute toxicity of diflubenzuron and its metabolites for fish are presented in Tables 14 and 15, respectively. A 96-h acute toxicity test on juvenile sheepshead minnow (Cyprinodon variegatus) in a flow-through system resulted in no mortality at the nominal exposure concentration of 130 g diflubenzuron/litre (Graves & Swigert, 1993). A similar test under semi-static conditions on zebra fish (Brachydanio rerio) and rainbow trout (Oncorhynchus mykiss) at a nominal diflubenzuron concentration of 200 g/litre also showed no mortality, nor changes in the appearance Table 14. Acute toxity of diflubenzuron to fish Organism Size/age Stat/flowa Temperature Hardness pH Parameter Concentration Reference (C) (mg/litre) (mg/litre) Coho salmon 1 g stat 11 4.55 6.5 96-h LC50 > 150 McKague & (Oncorhynchus kisutch) Pridmore (1978) Rainbow trout 1 g stat 11 4.55 6.5 96-h LC50 > 150 McKague & (Oncorhynchus mykiss) Pridmore (1978) Rainbow trout 1.2 g stat 12 40 7.2 96-h LC50 240 Julin & Sanders (Oncorhynchus mykiss) (1978) Rainbow trout not flow-through 96-h LC50 140 Marshall & Hieb (Oncorhynchus mykiss) reported (1973) Rainbow troutb not flow-through 96-h LC50 195 Marshall & Hieb (Oncorhynchus mykiss) reported (1973) Fathead minnow 0.87 g stat 22 40 7.2 96-h LC50 430 Julin & Sanders (Pimephales promelas) (1978) Channel catfish 2.2 g stat 22 40 7.2 96-h LC50 370 Julin & Sanders (Ictalurus punctatus) (1978) Bluegill 0.5 g stat 22 40 7.2 96-h LC50 660 Julin & Sanders (Lepomis macrochirus) (1978) Bluegill not flow-through 96-h LC50 135 Marshall & Hieb (Lepomis macrochirus) reported (1973) Bluegillb not flow-through 96-h LC50 230 Marshall & Hieb (Lepomis macrochirus) reported (1973) Table 14. (Con't) Organism Size/age Stat/flowa Temperature Hardness pH Parameter Concentration Reference (C) (mg/litre) (mg/litre) Mummichog adult 2.7 g stat 24 22 ppt 8.0 96-h LC50 32 990 Lee & Scott (Fundulus heteroclitus) renewal salinity (1989) Mummichogb not flow-through 96-h LC50 255 Marshall & Hieb (Fundulus heteroclitus) reported (1973) a Stat = static conditions (water unchanged for duration of test); flow = flow-through conditions (diflubenzuron concentration in water continuously maintained) b Formulated product 25 WP Table 15. Acute toxicity of metabolites of diflubenzuron to fish (from: Julin & Sanders, 1978) Concentrations (mg/litre) Organism Water Effect temperature measured (C) 4-Chlorophenyl 2,6-Difluorobenzoic 4-Chloroaniline urea acid Rainbow trout 12 96-h LC50 72 (57-90) > 100 14 (11-16) (Oncorhynchus mykiss) Channel catfish 22 96-h LC50 > 100 > 100 23 (18-29) (Ictalurus punctatus) Fathead minnow 22 96-h LC50 > 100 69 (55-87) 12 (7-18) (Pimephales promelas) Bluegill 22 96-h LC50 > 100 > 100 2.4 (1.8-3.2) (Lepomis macrochirus) or behaviour of the fish (Berends & van der Laan-Straathof, 1994a,b). All these test concentrations were above the diflubenzuron solubility level of 80 g/litre. Nebeker et al. (1983) found no significant reduction in survival of fathead minnow (Pimephales promelas) or guppy (Poecilia reticulata) as a result of exposure to diflubenzuron at concentrations below 36 g/litre during acute (96-h) and chronic tests. No acute response resulted from exposure of fish to a diflubenzuron concentration of 45 g/litre, and no chronic effects were observed at this concentration, the highest one tested (Hansen & Garton, 1982a,b). Diflubenzuron was not toxic to either rainbow trout or coho salmon exposed at concentrations up to 150 mg/litre for a 96-h period. A 15-min exposure to 1 g/litre did not result in any fish mortality (McKague & Pridmore, 1978). Madder & Lockhart (1978) found a dose-related decrease of glutamic-oxaloacetic transaminase activity in rainbow trout (Oncorhynchus mykiss) exposed to diflubenzuron at concentrations of 0.625, 1.25, 2.5, 5 and 10 mg/litre. At a concentration of 0.01 mg/litre, diflubenzuron had a repellent effect on precocious male Atlantic salmon parr (Granett et al., 1978). 9.1.3 Terrestrial organisms 9.1.3.1 Plants Photosynthesis, respiration and leaf ultrastructure of soybeans were unaffected by diflubenzuron at doses up to a level of 0.269 kg a.i./ha (Hatzios & Penner, 1978). Diflubenzuron is used as an insecticide in forestry, agriculture and horticulture. No phytotoxicity was reported in the field studies cited in section 9.2. 9.1.3.2 Invertebrates The oral and contact LD50 values of diflubenzuron for honey-bees are greater than 30 g/bee (Stevenson, 1978). Diflubenzuron did not show any toxicity to bees at concentrations up to 1000 mg/kg in the diet (Yu et al., 1984). Barker & Taber (1977) found that diflubenzuron reduced brood production when fed for 10 days to honey-bees at 59 mg/kg in sugar syrup, but not at 5.9 or 0.59 mg/kg. Barker & Waller (1978) confirmed that 60 mg/kg in sugar syrup or 100 mg/litre in water caused colonies to produce less brood. Stoner & Wilson (1982) found that diflubenzuron fed to flying colonies at 1 or 10 mg/kg for a year significantly reduced the amount of sealed brood. Nation et al. (1986) reported that diflubenzuron did not cause reduction in pollen consumption or brood production when fed for 10 weeks to caged colonies of honey-bees at 10 mg/kg, but it caused more than 50% reduction in the amount of syrup stored. Gordon & Cornect (1986) showed that, at concentrations that are effective in suppressing egg hatching and larva development of the cabbage maggot Delia radicum, diflubenzuron did not adversely affect eggs, first-instar larvae or adults of the rove beetle Aleochara bilineata, an important predator and parasitoid of the cabbage maggot. When the acute toxicity of diflubenzuron to the earthworm Eisenia fetida was tested in artificial soil to which diflubenzuron had been added, 780 mg/kg dry soil was the no-observed-effect concentration (NOEC) for the 14-day test period (Berends et al., 1992). For the WP-25 formulation, the NOEC was 1.0 g/kg (Berends & Thus, 1992a). 9.1.3.3 Vertebrates a) Birds The acute oral LD50 of technical diflubenzuron for red-winged blackbirds (Agelaius phoeniceus) is 3762 mg/kg body weight. In an 8-day dietary LD50 study on mallard duck and bobwhite quail using technical diflubenzuron, levels up to 4640 mg/kg in the feed gave no observable signs of toxicity (Maas et al., 1980). When diflubenzuron was fed to mature White leghorn hens at dietary levels of 10, 50, 100, and 500 mg/kg for 8 weeks, there were no adverse effects on feed consumption, body weight, egg production, egg weight, eggshell thickness, fertility, hatchability or progeny performance. The highest dose used was 50 times higher than the efficacy level for fly control (Cecil et al., 1981). In chronic toxicity tests, groups consisting of 10 male and 10 female one-day-old chicks of barred Plymouth rocks and white leghorn hens, Nicholas white turkeys, mallard ducks and ring-necked pheasants were given diets containing diflubenzuron levels of 0.25, 1.25, 25 and 250 mg/kg for 91 days after hatching. No differences between control and treatment groups were observed in mortality, food consumption, body weight, comb and wattle development, weight of inner organs, serum hormone levels or general behaviour (Maas et al., 1980). There was no effect of diflubenzuron on the content of hyaluronic acid in the skin of Hubbard broiler chickens when they were fed at levels of 2.5 and 250 mg/kg in the diet for 98 days after hatching (Deul & Jong, 1977). In an experiment with the same dose levels and duration, diflubenzuron had no effect on hyaluronic acid synthesis or comb deposition in either growing broilers or layers (Crookshank et al., 1978). White leghorn and black sex-linked cross hens were fed diflubenzuron at a level of 10 mg/kg in the ration for 15 weeks. Diflubenzuron had no effect on body weight gain, egg production, fertility or hatchability (Miller et al., 1976b). In a feeding study with 2.5, 25 and 250 mg/kg, diflubenzuron did not affect bobwhite quail reproduction (Booth et al., 1987). Diflubenzuron did not show significant teratogenic activity on chick embryos over time when injected at 10 mg/egg (Booth et al., 1987). A one-generation bobwhite quail (Colinus virginianus) reproduction study was conducted in which a diflubenzuron-containing diet was administered ad libitum to young adults (24 weeks old at test initiation) approaching their first breeding season. Dietary concentrations of 250, 500 or 1000 mg/kg did not result in treatment- related mortality, overt signs of toxicity or effects upon adult body weight or feed consumption during the 21-week exposure period. There were no apparent treatment-related effects upon reproductive parameters at 250 or 500 mg/kg. There may have been a slight reduction in the number of eggs laid, although this was not statistically significant or dose-related at 1000 mg/kg. On the basis of a possible effect on egg production at 1000 mg/kg, the NOEC for diflubenzuron in this study was above 500 mg/kg (Beavers et al., 1990a). In a one-generation reproduction study on the mallard duck (Anas platyrhynchos), diets containing diflubenzuron were administered ad libitum to young adults (27 weeks old at test initiation) approaching their first breeding season. Dietary diflubenzuron concentrations of 250, 500 and 1000 mg/kg did not result in treatment-related mortality, overt signs of toxicity or effects upon adult body weight or feed consumption during the 20-week exposure period. There were no apparent treatment-related effects upon reproductive performance at any of the concentration tested. At 1000 mg/kg there was a slight, but statistically significant, reduction in mean eggshell thickness. On the basis of the effect upon eggshell thickness at 1000 mg/kg, the NOEC for diflubenzuron in this study was 500 mg/kg (Beavers et al., 1990b). b) Mammals In studies by Ross et al. (1977a,b), diflubenzuron was administered in the feed to sheep (3 of each sex per group) as a model for ruminant wildlife at concentrations of 500, 2500 and 10 000 mg/kg feed for 13 weeks. No treatment-related effects were observed on food consumption, body weight gain, haematological parameters or urinalysis. Increase in met- and sulfhaemoglobin levels were observed at 13 weeks and there was a reduction in the weight of the thyroid. No histopathological abnormalities were observed. Both the plasma and the erythrocyte cholinesterase activities were unaffected by the treatment after 6 weeks. 9.2 Field observations 9.2.1 Microorganisms 9.2.1.1 Water The laboratory data available (see section 9.1.1.1) make it unlikely that detrimental effects will occur. Rotifers were unaffected by diflubenzuron (28 and 56 g a.i./ha) in both experimental and naturally treated ponds (Ali & Lord, 1980). 9.2.1.2 Soil One aerial application of diflubenzuron at 67.26 g/ha had no adverse effects upon populations of bacteria, actynomycetes or fungi in leaf litter and forest soil (Wang, 1975; Kurczewski et al., 1975). 9.2.2 Aquatic organisms 9.2.2.1 Plant The laboratory data available (see section 9.1.2.2) make it unlikely that detrimental effects will occur. 9.2.2.2 Invertebrates Recent field studies have demonstrated that effects on aquatic fauna are limited and transient, and that recovery is evident after 3 months (Huber & Collins, 1987; Kingsbury et al., 1987; Huber & Manchester, 1988; Ali et al., 1988; Ali & Kok-Yokomi, 1989; Sundaram et al., 1991). Diflubenzuron (25% WP) applied at 33.63 and 134.52 g a.i./ha, 4 times at 2-week intervals, had no adverse effect on freshwater clams 10 days after final treatment (Jackson, 1976). When diflubenzuron (25% WP) was applied at 1.121-280.25 g a.i./ha to flooded rice fields in Louisiana, USA, significant reduction of Tropisternus spp. and Libellulidae was found 80 days after treatment. Significantly more chironomid and baetid immatures occurred due to reduction in the number of predators (Steelman et al., 1975). Mulla et al. (1975) found that at an application rate of 28 and 56 g a.i./ha diflubenzuron reduced slightly, and only for a short time, the number of mayfly ( Beatis sp.) in the treated ponds. The numbers, however, appeared to be within natural fluctuation limits equal to the check ponds or the pretreatment levels during all other sampling periods. At these application rates, diflubenzuron caused a short-term reduction in copepod populations, but they started to increase on the 11th or 15th day of post-treatment sampling. There was little or no effect on the ostracod population. In a forestry spraying programme at 0.0672 kg a.i./ha, aquatic arthropods were studied in a water shed (White Deer Creek). Sampling at three test stations and one control station in the watershed revealed a rich fauna, dominated numerically by Ephemeroptera, Chironomida, Trichoptera and Plecoptera in descending order of abundance. Significant differences in larval abundance in the surber samples were found to occur from one sampling day to the next, or between pre-spray and post-spray periods, for most of the organisms examined. In all cases, control and test stations showed similar patterns of variation, so treatment effects were discussed as a probable cause. The number of organisms tended to be higher at the upstream stations. Chironomida and Trichoptera pupal abundance in the surber samples was examined for decreases after spraying but numbers were too low to permit statistical analysis. Pupal abundance showed some direct relationship to larval abundances. Nymphal drift rates were examined for possible increases after spraying, but most of the significant changes were decreases during the post-spray period. These decreases also occurred at the control station. The drift rates of nymphal and pupal exuviae also did not indicate a treatment effect. Individual species abundances in the surber samples were examined for possible selective effects of diflubenzuron. Comparisons were made between pre-spray and post-spray periods at both test and control stations, but no evidence of a treatment-related decrease in the abundance of any of these organisms was found. The net effect was usually an increase in the density of these organisms during the post- spray period. No organisms showing abnormal ecdysis or pupation were found in any of the samples. It was concluded that spraying with diflubenzuron had no adverse effect on the macrobenthic community in White Deer Creek (White, 1975). In a study by Booth (1975), diflubenzuron (25% WP) was applied at 44.84 g a.i./ha to small ponds in Utah. Biosamples taken 30 and 80 days later showed that, although larva and immature aquatic insect populations were decreased at 30 days, the total number of adults was not significantly different from the control number at 80 days. Booth & Ferrell (1977) examined the effect of diflubenzuron on over 20 different species (corixidae and collembolans) after multiple pond applications at 45 g a.i./ha. Only immature corixidae (water boatmen) and collembolans (springtails) were significantly affected. Segmented worms (Oligochaeta) and midges (Chironomidae) were unaffected by six applications of diflubenzuron at a rate of 145.73 g a.i./ha to Utah Lake (Booth et al., 1987). Diflubenzuron (28 and 56 g a.i./ha) reduced numbers of Chaoborus sp. and Baetis sp. in both experimental and natural treated ponds. Chaoborus sp. recovered within 1-3 weeks (Ali & Lord, 1980). The application of granular diflubenzuron at 0.11 kg a.i./ha (about 3.7 g/litre) to residential-recreational lakes caused a 62-75% reduction of Daphnia pulex and Daphnia galeata during 7 days following treatment. The populations recovered in the second week after treatment. A 30% reduction in Diaptomus spp. was noted 2 days after the treatment. Hyalella azteca was affected markedly, with a maximum reduction of 97% after 3 weeks of treatment. No detectable effects on Cyprinotus sp., Cyclops sp. or Bosmina longirostris were observed (Ali & Mulla, 1978a). Ali & Mulla (1978b) also studied the impact of diflubenzuron on invertebrates in a residential-recreational lake after two treatments with 25% WP formulation at 156 g a.i./ha (about 12 g/litre). Diflubenzuron concentrations in water were not measured but were based on nominal initial concentrations. The results from this study are summarized in Table 16. Diflubenzuron (25% WP) at rates of 0.02, 0.025, 0.03, 0.035, 0.04, 0.045 and 0.05 lb a.i./acre was applied to 19 irrigated pastures and spring ponds (in some cases up to 4 times) at 2- to 3-week intervals in California. Cladoceran and nymphal mayfly populations were temporarily and slightly reduced, but recovered within a short period of time. Even repeated treatment of the same pastures did not eliminate the populations. The impact on copepod populations was inconclusive. Adult beetles demonstrated high tolerance to Table 16. Effect of diflubenzuron on invertebrates in Vallage Grove Lake, Californiaa Species First application (April) Second application (August) Effect Recovery Effect Recovery Daphnia leavis elimination within no recovery 6 months Birge 1 week after treatment Ceriodaphnia sp. elimination within no recovery 6 months 1 week after treatment Bosmina longirostris elimination within recovery after elimination after reappearance in small numbers (O.F. Muller) 1 week 11 weeks 1 week 8-9 weeks after treatment Cyclops spp. elimination within recovery within elimination within recovery after 4 weeks 1 week 6-7 weeks 1-2 weeks Diaptomus sp. elimination within recovery after absent prior to reappearance in small numbers 1 week 4 months treatment 1-2 months later Hyalella azteca elimination within no recovery 6 months (Saussure) 4 weeks after treatment Caenis sp. elimination within recovery within elimination within recovery in 4-5 weeks 3 weeks 6-7 weeks 2-3 weeks Physa sp. no adverse effects no adverse effects Cypridopsis sp. no adverse effects no adverse effects a From: Ali & Mulla (1978b) diflubenzuron, but few dead larvae ( Laccophilus spp., Hydrophilus triangularis and Tropisternus lateralis) were observed. Spiders ( Pardosa spp. and Lycosa spp.) showed no apparent effects. There were no deleterious effects on planarian, rotifer, seed shrimp and fresh water flagellate populations (Miura, 1974). Diflubenzuron (25 WP formulation) was applied 3 times at 2.5-week intervals to man-made pools in Manitoba, Canada at 56 g/a.i. ha (this produced initial concentrations of 0.02 mg/litre). The treatment produced no detectable effect on chironomid larvae in terms of numbers or composition, although dead larvae were noted after treatment. Daphnid populations were significantly reduced on most sampling dates. Repetitive treatments prevented the recovery of cladocerans. Despite these reductions, daphnids were not annihilated. At a higher rate (0.22 kg a.i./ha, about 7.4 g/litre), both species of Daphnia were eliminated for 3 months after treatment. Populations of Diaptomus spp. declined to zero 7 days after treatment, but recovered during the second week following treatment. Diflubenzuron caused reductions in the number of H. azteca (32-100%) during the 2 months following treatment. Seed shrimp populations were stressed for only 2 weeks, and there were no observable effects on oligochaetes at a treatment level of 0.22 kg a.i./ha (about 7.4 g/litre). Copepods only occasionally showed significant reduction, and recovered within 10 days after treatment. Of the ten other non-target invertebrates (i.e. Chaoboridae, Tipulidae, Ephemeroptera, Corixidae, Nonectidae, Gerridae, Dystiscidae, Hydrophilidae, Hydrarina and Gastropoda), only mayfly nymphs (Ephemeroptera) were significantly reduced (Madder, 1977). When diflubenzuron at a rate of 34.2 g/ha was applied aerially to a forested area in the USA, there were no significant effects on the population structure of the benthic macro-invertebrate community. Some decreases in the number of mayflies Heptagenia sp. and Rhithrogenia sp. were noted in treatment streams, but the total taxa richness values remained high. An increase in abundance of the caddisfly Lepidostoma sp. was found. Allonarcys sp. was not affected by the treatment (Huber & Collins, 1987; Huber & Manchester, 1988). After aerial application of diflubenzuron to two forest ponds in Canada the greatest effect was on crustacean zooplankton, especially cladocerans, with limited effect on pond benthos. Recovery of population of even the most severely affected organism such as Daphnids was well established by 3 months after treatment. The maximum residue found in water was 13.82 g/litre 1 h after application (Kingsbury et al., 1987). Diflubenzuron WP-25 was applied at a rate of 70 g active ingredient in 10, 5 or 2.5 litre/ha to three spray blocks in a mixed boreal forest near Kaladar, Ontario, Canada. Water, sediment and aquatic plants were collected from two ponds and a stream at intervals up to 30 days after treatment for analysis of diflubenzuron residues. The duration of detectable residues was different for each substrate, but in all cases it was less than 2 weeks. Zooplankton and benthic invertebrate populations were monitored for up to 110 days after spraying in two ponds in the high-volume rate block and in control ponds. Significant mortality occurred in two groups of caged macroinvertebrates (amphipoda and immature corixidae) 1 to 6 days after the ponds were treated with diflubenzuron. Three taxa of littoral insects ( Caenis, Celithemis and Coenagrion) were significantly reduced in abundance in the treated ponds 21 to 34 days after treatment but recovered to pre-treatment levels by the end of the season. Of the six remaining groups studied, only one (immature corixidae) may have been slightly affected by treatment. Zooplankton (cladocera and copepoda) population numbers were reduced 3 days after treatment and remained suppressed for 2-3 months (Sundaram et al., 1991). Blumberg (1986) found no effect on population numbers in a forested ecosystem after aerial application of diflubenzuron (25 WP) at rate 140 g a.i./ha. When diflubenzuron (33.63 and 134.52 g a.i./ha) was applied 4 times at 2-week intervals to small ponds in Virginia, numbers of daphnids were markedly reduced at both treatment levels. There was no appreciable effect on two other invertebrate species ( Chironomidae or Chaoborus) (Birdsong, 1977). In a study by Rodrigues (1982), diflubenzuron (25% WP) was applied at a rate of 1 mg/litre per 30 min to three forest streams in New York. The reduction of chironomid larvae 15 days after treatment ranged from 35 to 91% at different sites in the three streams. Of the two stonefly families present in the three streams, Nemouridae were affected more than Leuctridae, with decreases of 75-94% and 70%, respectively. Ephemeroptera were reduced, but Trichoptera and Coleoptera were unaffected. Following aerial application at 67.26 g a.i./ha to a watershed, diflubenzuron reached the stream channel (also as a result of wash-off from foliage following several subsequent rainfalls), but the invertebrate populations were not affected (Jones & Konchenderfer, 1988). After two applications of 44.8, 112 and 224 g a.i./ha, grass shrimp showed mortality of 86.6, 100, and 100%, respectively. At the two lowest application rates, fiddler crabs ( Uca spp.) showed mortalities of 53.3 and 66.6%, respectively (McAlonan, 1976). Diflubenzuron (25% WP) at 33.63 and 134.52 g/ha was applied 4 times at 2-week intervals to man-made ponds in North Carolina, Arkansas and Texas, USA. In North Carolina no apparent effect on phytoplankton was found. There was a marked decrease in the numbers of crustaceans ( Cladocera and Copepoda), as well as of certain species from benthic communities such as Hexagenia and Chaoborus in Arkansas. A relative decrease in copepod numbers was observed, but the authors attributed this partly to natural mortality during winter. In Texas there was a marked decrease in the numbers of all crustaceans and a corresponding increase in rotifer populations, particularly Asplanchina (Aquatic Environmental sciences, 1976a,b,c). When diflubenzuron (25% WP) at 33.63 and 134.52 g/ha was applied 4 times at 2-week intervals to man-made ponds in Alabama, USA, daphnid populations were reduced to 50% of the pre-treatment level at a rate of 134.52 g/ha but increased at 33.63 g/ha to a level of 15% of that in a control pond. Gastropod numbers decreased in the benthic samples but increased in the periphyton samples from the suspended plate samplers in the treated ponds (Jackson, 1976). Diflubenzuron at 33.63 and 134.52 g/ha was applied 4 times at 2-week intervals to a tidally influenced salt marsh in Virginia, which was sampled biologically 14 times over a 70-day period. Three species of arthropods, Cyathura polita, Ceratopogonidae sp., and Psychodidae sp., showed significant reductions in numbers. Oligochaetes, Hanynkia speciosa and the molluscs were not significantly affected (Matta, 1976). Six applications of diflubenzuron (28 g a.i./ha) over an 18-month period caused statistically significant differences in the population density of aquatic organisms in a Louisiana coastal marsh. None of the organisms affected were completely eliminated from the ecosystem. Populations of five taxa (nymphs of Trichocorixa louisianae Jaczewski and Buenoa spp., Coenagrionidae naiad spp., Berosus infuscatus Le Conte adults, and Hyalella azteca Saussure) were significantly reduced. Population of 15 taxa, i.e. Physa sp., Ceanis sp. and Callibaetis sp. naiads, Noteridae larvae, Hydrovatus cuspidatus, Kunze adults, Hydrovatus sp. larvae, Dytiscidae larvae, Mesovelia mulsanti Jaczewski adults, Trichocorixa louisiana adults, larvae of Chironomidae, Ephudridae, Dolichopodiae and Tabanidae, and the fish Gambusia affinis (Baird and Griard) and Jordanella floridae (Goode and Bean), showed significant increases after exposure to diflubenzuron. The 27 remaining aquatic organisms (members of the Hemiptera, Coleoptera, Mysidacea, Decapoda, Diptera and Odonata) showed no statistically significant differences when compared with untreated populations (Farlow et al., 1978). After treatment with diflubenzuron (as 1% granular formulation) at rate of 4.5 g a.i./ha to a marsh habitat on the Fraser River, Canada, the population of cladocerans appeared to be depressed for about 5 days but they recovered 2 weeks after treatment. There was no effect on copepods or ostracods. Significant reduction in the numbers of water beetles and zooplankton occurred (Wan & Wilson, 1977). In a field study by Hester et al. (1986), WP-25 formulation was evaluated in natural salt water pools to determine its toxicity to juvenile stages of three estuarine crustaceans exposed to a single application of 45 g diflubenzuron/ha. One hour after application, water residues of diflubenzuron averaged 3.6 g/litre and mortality was 46.5, 60.7 and 40.6% for Callinectes sp., Palaemonetes pugio and Uca pugilator, respectively, over the 10-day observation period. When these species were introduced into the pools 7 days after treatment, mortality was not significantly greater in the treated group than in the controls over the 21-day observation period. The maximum concentration after organisms were introduced was 0.69 g/litre, and mortalities of 22.2, 1.5 and 4.2% for Callinectes sp., Palaemonetes pugio and Uca pugilator, respectively, were reported. In a study by Hester (1982), diflubenzuron (25% WP) at the rate of 0.045 kg a.i./ha was applied to tidal ponds. Residue analysis showed 2 to 5 g/litre after 1 h. The mortality of three estuarine decapods: blue crabs ( Callinectes sp.), grass shrimp (Palaemonetes pugio) and fiddler crabs (Uca pugilator) was 44, 61 and 41%, respectively. When these decapods were introduced to the ponds 7 days after application, the residues in water were 0.4-0.7 g/litre and mortality was 22, 1.5 and 4%, respectively. 9.2.2.3 Vertebrates During a forestry spraying programme at 0.067 kg diflu- benzuron/ha, a survey on fish was performed in the watershed. Observations made on caged brown trout in the stream from day -7 to day +6 revealed no immediate mortality or erratic behaviour attributable to treatment. Barrier seines placed at the upstream and downstream boundaries of the spray area did not collect any dead or dying fish during the 1-week period they were in the stream. Visual observations made by walking along the stream during this same period also revealed no dead or dying fish. Post-spray population estimations revealed increases in both test and control areas. However, statistical analysis indicated no significant differences pre- and post-spray between stations. The treatment had no observable effect on the development of fish larvae. Immediate mortality was not observed in caged fish or stream fish collected by barrier seines. Delayed mortality attributable to spraying was not shown by population estimations taken 6 weeks following spray, nor were any dead or dying fish seen during this period. There appeared to be no adverse effect of diflubenzuron sprayed at 0.067 kg a.i./ha on the fish species observed in this study (White, 1975). No effect on fish populations ( Micropterus salmoides, Lepomis macrochirus and Gambusia affinis) was observed after four applications of diflubenzuron (33.69 and 134.52 g a.i./ha) at 2-week intervals to small ponds in Virginia (Birdsong, 1977). Killifish (Fundulus heteroclitus) showed no effect after three applications of diflubenzuron at rates from 11.12 to 224.2 g a.i./ha to replicated semi-natural pools (McAlonan, 1976). Diflubenzuron revealed no toxic effect on bullheads or sunfish after aerial application of 35 g/ha in Canada (Buckner et al., 1975). Growth of bluegill sunfish ( Lepomis macrochirus Rafinesque) was not affected by diflubenzuron applications at rates of 2.5-10 g/litre to ponds and lakes in California (Apperson et al., 1978). Ten days after the fourth treatment to man-made ponds with diflubenzuron at 33.63 and 134.52 g a.i./ha no apparent adverse effect on largemouth bass was observed (Jackson, 1976). No death of fish ( Pomoxis nigromaculatus and Ictalurus nebulosis) occurred after application of diflubenzuron (mean concentration of 13.2 g/litre one hour after treatment) to experimental ponds in California. For 1 month following treatment, stomach content analyses showed alteration in the diet of the fish (Collwell & Schaefer, 1980). 9.2.3 Terrestrial organisms 9.2.3.1 Invertebrates Honey-bee colonies remained normal after an aerial application of 350 g diflubenzuron/ha in Canada (Buckner et al., 1975). A comparative study of the effect of diflubenzuron on carabid fauna in oak woods in Westphalia in 1987-1988 was reported. The total number of captured beetles in treated areas was lower in 1989. Evaluation of the 12 beetle species most frequently caught indicated lower numbers than in control areas. The decrease coincided with the diflubenzuron treatment in the middle of May. No higher active density of species reproducing in late summer or autumn was observed in the untreated areas (Klenner, 1990). In a forestry spraying programme (0.0672 kg diflubenzuron per ha), a survey of microarthropods collected from leaf litter and soil in control and sprayed areas of the Bald Eagle State Forest, Pennsylvania, USA, showed little or no effect of diflubenzuron on the spray plot fauna. Increases and decreases in both the control and spray population sizes during the 6-week study period could seemingly be explained on the basis of seasonal population cycles, increased soil moisture condition and differential extraction efficiencies (White, 1975). 9.2.3.2 Vertebrates Small song birds in a forest ecosystem were unaffected after an aerial application of 350 g diflubenzuron/ha in Canada (Buckner et al., 1975). In a forestry spraying programme (0.0672 kg diflubenzuron/ha) surveys were made on birds and small mammals. Effects on birds were assessed using single male mapping surveys. The survey methodology was designed to minimize bias due to differences between observers, plots, times of day and abundance of birds. Vegetation and defoliation types were identified and mapped and defoliation was sampled at 136 sites. Four hundred and sixteen surveys were conducted on treated plots, defoliated plots, and undisturbed plots. No differences were observed in the number of individuals singing, the amount of time each individual spent singing or the song repetition rate during song bouts. The results show that diflubenzuron did not lead to the death or emigration of singing birds and probably had no direct adverse physiological effects on birds since their vocal behaviour changed little, if at all. The singing male surveys did not detect a presumed food shortage among canopy feeders caused by the defoliation. Small mammals studied in this project were censused as thoroughly as possible. The short pre-spray sampling period for the treated plot was a handicap in evaluating the pre-spray population levels; however, post-spray sampling compensated for this to some degree. Peromyscus, Clethrionomys and Sorex consume arthropods to varying degrees and may be exposed to diflubenzuron present as residues. Diflubenzuron had no demonstrable effects on any of these small mammals sampled in this study. The application of diflubenzuron at the rate of 0.067 kg/ha provided good foliage protection within the spray sites and apparently made the dying gypsy moth larvae unpalatable. Other contact and stomach pesticides generally do not have this effect on caterpillars and are consequently consumed by insectivorous mammals. The reduced exposure by non-ingestion is no doubt a beneficial aspect. None of the species of small mammals showed reductions in numbers from pre-spray to post-spray periods that could be attributed to the compound. Stomach analysis did not indicate any change in the feeding habits of any of the small mammals from either plot in the study. Changes in reproduction could not be detected. However, of the Peromyscus and Clethrionomys animals that were dissected and examined, 7 out of 12 females were in a healthy condition or had gravid reproductive organs. Others were either virgins or placental scars could not easily be seen, as in Sorex. The body weight of all the small mammals in the study was normal for each species. Juveniles were easily distinguished by weight and gonad development. A regression comparing gonad weights and body weights of Clethrionomys indicated a strong correlation. Since the body weight of small mammals increases with age, this was to be expected (White, 1975). Diflubenzuron sprayed at 0.14 and 0.28 kg/ha over forests in north-eastern Oregon, USA, did not have any detectable adverse effect on bird population numbers, nesting or bird behaviour (Richmond et al., 1979). 10. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES Diflubenzuron was evaluated by the Joint FAO/WHO Meeting on Pesticide Residues (JMPR) in 1981, 1984, and 1985 (FAO/WHO, 1982a,b; 1985a,b; 1986a,b). In 1985, JMPR established an Acceptable Daily Intake (ADI) for human beings of 0-0.02 mg/kg body weight per day, based on the fact that the following levels produced no toxicological effects: rat: 2 mg/kg body weight (40 mg/kg diet) mouse: 2.4 mg/kg body weight (16 mg/kg diet) dog: 2 mg/kg body weight per day Diflubenzuron has been classified as "a product unlikely to present an acute hazard in normal use", on the basis of an acute oral LD50 for the rat that is greater than 4640 mg/kg body weight (WHO, 1994). REFERENCES Ali A & Kok-Yokomi ML (1989) Field studies on the impact of a new benzoylphenylurea insect growth regulator (UC-84572) on selected aquatic nontarget invertebrates. Bull Environ Contam Toxicol, 42: 134-141. 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(Unpublished proprietary report submitted to WHO by Solvay Duphar BV, Weesp, The Netherlands). Burdock GA, Serota DG, Purvis D, & Alsaker RD (1980b) Subchronic dietary toxicity study in rats - Diflubenzuron. Final report: Volume 1 (Project No. 533-119). Vienna, Virginia, Hazleton Laboratories America Inc. (Unpublished proprietary report submitted to WHO by Solvay Duphar BV, Weesp, The Netherlands). Burdock GA, Wolfe GW, Hepner KE, Alsaker RD, Koka M, & Phipps RB (1984) Oncogenicity study in rats on diflubenzuron. Final report. Vienna, Virginia, Hazleton Laboratories America Inc. (Unpublished proprietary report No. 56645/08/1984, submitted to WHO by Solvay Duphar BV, Weesp, The Netherlands). Burgess D (1989) Uptake, depuration and bioconcentration of 14C-diflubenzuron by Bluegill sunfish ( Lepomis macrochirus. Columbia, Missouri, Analytical Bio-Chemistry Laboratories, Inc. (Proprietary report No. 56635/16/1989, submitted to WHO by Solvay Duphar BV, 'sGraveland, The Netherlands). 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Christiansen ME & Costlow JD (1982) Ultrastructural study of the exoskeleton of the estuarine crab Rhithropanopeus harrsii Effect of the insect growth regulator Dimilin (diflubenzuron) on the formation of the larval cuticle. Mar Biol, 66: 217-226. Christiansen ME, Costlow JD, & Monroe RJ (1978) Effects of the insect growth regulator Dimilin (TH6040) on larval development of two estuarine crabs. Mar Biol, 50: 29-36. Colley JC, Batham P, Heywood R, Street AE, Gopinath C, Cherry CP, Gibson WA, & Prentice DE (1981a) The effects of dietary administration of diflubenzuron to male and female HC/CFLP mice for 14 weeks: Volume I. Huntingdon, England, Huntingdon Research Centre (Unpublished proprietary report No. PDR/294/80185, submitted to WHO by Solvay Duphar BV, Weesp, The Netherlands). Colley JC, Batham P, Heywood R, Street AE, Gopinath C, Cherry CP, Gibson WA, & Prentice DE (1981b) The effects of dietary administration of diflubenzuron to male and female HC/CFLP mice for 14 weeks: Volume II. Huntingdon, England, Huntingdon Research Centre (Unpublished proprietary report No. PDR/294/80185, submitted to WHO by Solvay Duphar BV, Weesp, The Netherlands). Colley J, Heywood R, Street AE, & Gopinath C (1984) The effect of diflubenzuron given by oral administration with the feed on toxicity and tumour development in male and female HC/CFLP mice. Final report. Huntingdon, England, Huntingdon Research Centre (Unpublished proprietary report No. 56645/32/1984, submitted to WHO by Solvay Duphar BV, Weesp, The Netherlands). Collwell AE & Schaefer CH (1980) Diets of Ictalurus nebulosus and Pomoxis nigromaculatus altered by diflubenzuron. Can J Fish Aquat Sci 37(4): 632-639. Cooke M & Ober AG (1980) OV-17-QF-1 Capillary column for organochlorine pesticide analysis. J Chromatogr, 195: 265-269. Corley C, Miller RW, & Hill K (1974) Determination of N-(4-chlorophenyl)-N-(2,6-difluorobenzoyl)-urea in milk by high-speed liquid chromatography. J AOAC, 57(6): 1269-1271. Costlow YD (1979) Effect of Dimilin on development of larvae of the stone crab Menippe mercenaria and the blue crab Callinectes sapidus In: Vernberg WB, Calabrese A, Thurburg FP, & Vernberg FJ ed. Marine pollution: Functional responses. New York, London, Academic Press, pp 355-363. Crookshank HR, Sowa BA, Kubena L, Holman GM, Smalley HE, & Morison R (1978) Effect of diflubenzuron (Dimilin) on the hyaluronic acid concentration in the chicken combs. Poult Sci, 57: 804-806. Cunningham PA (I976) Effects of Dimilin (TH-6040) on reproduction in the brine shrimp, Artemia salina Environ Entomol, 5: 701-706. Cunningham PA (1986) A review of toxicity testing and degradation studies used to predict the effects of diflubenzuron (Dimilin) in estuarine crustaceans. Environ Pollut, A40: 63-86. 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Zeist, The Netherlands, TNO-CIVO Toxicology and Nutrition Institute (Proprietary report No. V85.347/250925, submitted to WHO by Solvay Duphar BV, 'sGraveland, The Netherlands). RESUME 1. Identit, proprits physiques et chimiques et mthodes d'analyse Le diflubenzuron appartient au groupe des insecticides drivs de la benzoylphnylure. Son activit insecticide est due une interaction avec la synthse et le dpt de la chitine. Il forme des cristaux blancs inodores dont le point de fusion est de 230-232C. Il est lgrement soluble dans l'eau (0,2 mg/litre 20C) et pratiquement non volatil. Il est relativement stable en milieu acide ou neutre mais s'hydrolyse en milieu alcalin. Le diflubenzuron s'obtient par raction du 2,6-difluoro- benzamide sur l'isocyanate de 4-chlorophnyle. Le dosage des rsidus de diflubenzuron prsents dans l'eau, les chantillons biologiques,et le sol peut s'effectuer par chromatographie liquide haute performance avec dtection par UV ou encore par chromatographie en phase gazeuse avec dtection par capture d'lectrons, soit directement sur la molcule initiale, soit sur un driv (libration de 4-chloroaniline et action de l'anhydride trifluoractique). 2. Sources d'exposition humaine et environnementale Le diflubenzuron est un produit de synthse utilis en agriculture, en foresterie et dans les programmes de sant publique pour dtruire les ravageurs et les vecteurs de maladies. Diffrentes formulations existent cet usage. On ne possde pas de renseignements au sujet de cas d'exposition humaine au diflubenzuron qui auraient pu se produire. 3. Transport, distribution et transformation dans l'environnement En gnral, le diflubenzuron est appliqu directement sur les vgtaux et les eaux traiter. Le feuillage ne constitue pas une porte d'entre dans la plante. Le diflubenzuron est rapidement adsorb sur les particules du sol. Il reste fix dans les 10 premiers cm du sol sur lequel il est pandu. Il est peu probable qu'il subisse un lessivage. Dans divers types de sol, il subit une dcomposition arobie ou anarobie avec une demi-vie de quelques jours. La vitesse de dcomposition dpend largement de la taille des particules de diflubenzuron. La principale voie mtabolique (plus de 90%) esst l'hydrolyse qui conduit la formation d'acide 2,6-difluorobenzoque et de 4-chlorophnylure; ces deux composs sont dgrads leur tour, respectivement en 4 et 6 semaines. On n'a pas dcel la prsence de 4-chloroaniline libre dans le sol. Le diflubenzuron se dcompose rapidement dans les eaux neutres ou alcalines. On constate qu'une fois pandu sur l'eau, il se rpartit rapidement entre celle-ci et les sdiments.Le compos initial et la 4-chlorophnylure peuvent persister plus de 30 jours dans les sdiments. Le diflubenzuron ne s'accumule pas chez les poissons. 4. Concentrations dans l'environnement et exposition humaine L'utilisation en agriculture, en foresterie ou pour la dmoustication n'entrane qu'une exposition ngligeable de la population gnrale par l'intermdiaire de la nourriture ou de l'eau. 5. Cintique et mtabolisme chez les animaux de laboratoire Chez l'animal de laboratoire, le diflubenzuron est absorb au niveau des voies digestives et, un moindre degr, au niveau cutan. Chez le rat, il existe un mcanisme d'absorption saturable. Dans ces conditions, une forte proportion du diflubenzuron administr par voie orale se retrouve dans les matires fcales. Le diflubenzuron se rpartit largement dans les tissus, mais il ne s'y accumule pas. Le mtabolisme du diflubenzuron a t tudi chez diverses espces animales. Chez les mammifres, la principale voie mtabolique comporte une hydroxylation. L'hydrolyse peut se produire au niveau de l'une quelconque des trois liaisons carbone-azote. Chez le porc et le poulet, l'hydrolyse s'effectue principalement au niveau du pont urido. Chez le rat et la vache, l'hydroxylation constitue la principale voie mtabolique. Chez le mouton, le porc et le poulet, les principaux mtabolites sont le 2,6-difluorobenzamide et la 4-chlorophnylure; on trouve aussi, en moindre proportion, du 2,6-difluorobenzamide et de la 4-chloroaniline. Chez le rat et les bovins, 80% des mtabolites sont constitus de 2,6-difluoro-3- hydroxydiflubenzuron, de 4-chloro-2-hydroxydifluorobenzuron et de 4-chloro-3-hydroxydiflubenzuron. Les tudes de mtabolisme indiquent qu'il ne se forme pratiquement pas de 4-chloroaniline chez le rat et les bovins. Chez les chats, les porcs et les bovins, l'limination s'effectue principalement par la voie fcale, hauteur de 70 85%. Chez les ovins, la voie urinaire et la voie fcale ont peu prs la mme importance de ce point de vue. Chez le rat et la souris, l'excrtion urinaire dcrot proportionnellement l'augmentation de la dose. Moins de 1% de la dose administre par la voie orale se retrouve dans l'air expir. Le diflubenzuron n'est prsent qu' l'tat de rsidus dans le lait. Il n'existe pas d'tude sur la cintique et le mtabolisme du diflubenzuron chez l'homme et notamment, sur son degr de biotransformation en 4-chloroaniline. 6. Effets sur les mammifres de laboratoire et les systmes d'preuve in vitro Quel que soit le mode d'exposition, le diflubenzuron prsente une faible toxicit aigu. En se basant sur le fait que sa DL50 aigu par voie orale est suprieure 4640 mg/kg de poids corporel chez le rat, l'OMS estime qu'il s'agit d'un produit qui ne prsente vraisemblablement pas de risque d'intoxication aigu en utilisation normale. Chez ce mme animal, la DL50 aigu par voie percutane est suprieure 10 000 mg/kg de poids corporel et la CL50 dpasse 2,49 mg/litre par la voie respiratoire. Au cours d'une priode de deux semaines pendant laquelle diverses espces animales avaient reu du diflubenzuron en une seule prise et selon divers modes d'administration, on n'a constat aucun signe d'intoxication. Le diflubenzuron n'est pas irritant pour la peau (chez le lapin) et ne provoque pas non plus de sensibilisation cutane (chez le cobaye). Il est lgrement irritant pour la muqueuse oculaire chez le lapin. Le diflubenzuron provoque une mthmoglobinmie et une sulfhmoglobinmie. Une mthmoglobinmie lie la dose a t mise en vidence aprs exposition d'animaux de diverses espces au diflubenzuron par la voie orale, percutane ou respiratoire. Cet effet constitue le point d'aboutissement toxicologique le plus sensible chez les animaux de laboratoire. En prenant comme critre la mthmoglobinmie, la dose sans effet observable est de 2 mg/kg de poids corporel par jour chez les rats et les chiens et de 2,4 mg/kg de poids corporel par jour chez les souris. Les tudes de toxicit long terme effectues sur des souris et des rats ont montr que les modifications imputables au traitement correspondaient principalement l'oxydation de l'hmoglobine et une altration des hpatocytes. Des tudes de cancrognicit effectues sur des rats et des souris des doses allant jusqu' 10 000 mg/kg de nourriture, n'ont pas rvl de modification de l'incidence tumorale qui soit imputable au traitement. En particulier, on n'a pas observ de noplasmes au niveau du msenchyme splnique ou hpatique lors d'tudes de cancrognicit utilisant de la 4-chloroaniline. Plusieurs tudes toxicologiques portant sur la fonction de reproduction ont t menes sur des rats, des souris, des lapins et trois espces d'oiseaux; elles n'ont pas mis en vidence d'effets pathognes et le produit ne s'est pas rvl embryotoxique. Les tudes de tratognicit effectues sur des rats et des lapins se sont galement rvles ngatives. Le diflubenzuron et ses principaux mtabolites ont galement t soumis diverses preuves de mutagnicit in vivo e in vitro Ni le diflubenzuron ni ses principaux mtabolites n'ont donn de rsultat positif dans ces preuves. Le mtabolite secondaire, c'est--dire la 4-chloroaniline, a donn des rsultats positifs dans plusieurs preuves de mutagnicit in vitro portant sur divers points d'aboutissement toxicologiques. Il est cancrogne pour le rat et la souris. Les tumeurs imputables l'administration de 4-chloroaniline se sont rvles bnignes; quant aux tumeurs malignes observes, il s'agissait de tumeurs du msenchyme splnique chez les rats mles ainsi que d'hmangiomes et d'hmangiosarcomes splniques ou hpatiques chez les souris mles. 7. Effets sur l'homme On a signal des cas de mthmoglobinmie chez des travailleurs exposs de par leur profession et des nouveau-ns exposs par inadvertance de la 4-chloroaniline, un mtabolite secondaire du diflubenzuron. Les sujets qui prsentent un dficit en NADH- mthmoglobine-rductase peuvent tre particulirement sensibles la 4-chloroaniline et par consquent une exposition au diflubenzuron. 8. Effets sur les autres tres vivants au laboratoire et dans leur milieu naturel Tous les organismes qui synthtisent la chitine sont sensibles au diflubenzuron. A la concentration de 500 mg/kg de terre, les bactries n'ont pas souffert d'une exposition au diflubenzuron. Il y a eu une certaine stimulation de la fixation d'azote. Les bactries dcomposent les solutions de diflubenzuron dans l'actone, solvant qu'elles utilisent comme source de carbone. Une concentration de diflubenzuron de 1 g/litre a provoqu un accroissement de la biomasse algaire. Aucun effet nocif n'a t observ des concentration suprieures la limite de solubilit du diflubenzuron. Des champignons placs dans un courant cr en laboratoire ont t temporairement affects la concentration de 0,1 g/litre. Les invertbrs aquatiques prsentent des ractions varies au diflubenzuron. Les mollusques n'y sont pas sensibles, avec une CL50 suprieure 200 mg/litre. Chez les autres invertbrs, la CL50 peut aller de 1 > 1000 g/litre, ce qui peut reflter la sensibilit de ces organismes au moment de la mue. On estime que pour la daphnie, la concentration tolrable maximale en produit toxique est suprieure 40 ng/litre et infrieure 93 ng/litre. Comme prvu, les larves d'phmres et autres formes pr-imaginales d'insectes divers, sont trs sesnsibles au diflubenzuron. Le traitement des eaux de surface par le diflubenzuron est donc probablement susceptible de causer une certaine mortalit parmi les insectes aquatiques. Lors de traitements exprimentaux effectus sur le terrain et dans divers cosystmes, on a constat que la plupart des organismes avaient moins souffert que ne le laissaient prvoir les tudes toxicologiques en laboratoire. Aucun effet n'a t constat sur les organismes aquatiques aprs traitement de forts par voie arienne. Pour les poissons, la CL50 est suprieure 150 mg/litre. Les essais effectus sur le terrain n'ont enregistr aucune mortalit chez les poissons. La DL50 par voie orale et par contact est suprieure 30 g par insecte chez l'abeille mellifique. Aprs pandage de diflubenzuron par voie arienne raison de 350 g/ha, on n'a pas constat de dommage parmi les colonies d'abeilles des alentours. Une tude alimentaire de 5 jours sur des colverts et des gallinacs du genre colin avec des doses allant jusqu' 4640 mg/kg de nourriture, n'a pas rvl de signes de toxicit. Aprs pandage de diflubenzuron par voie arienne sur des forts raison de 350 g/ha, on n'a pas constat de dommages parmi les oiseaux chanteurs de l'cosystme forestier. Aprs pandage de diflubenzuron raison de 67 g/ha sur une fort, on n'a pas observ de rduction dans l'effectif des populations de petits mammifres. RESUMEN 1. Identidad, propiedades fsicas y qumicas y mtodos analticos El diflubenzurn pertenece al grupo de los insecticidas derivados de la benzoilfenilurea. Su accin insecticida se debe a la interaccin con la sntesis y/o deposicin de quitina. Forma cristales blancos inodoros con un punto de fusin de 230-232C. Es bastante soluble en agua (0,2 mg/litro a 20C) y prcticamente involtil. Es relativamente estable en medios cidos y neutros, pero se hidroliza en condiciones alcalinas. El diflubenzurn se produce haciendo reaccionar 2,6-difluoro- benzamida con 4-clorofenilisocianato. Los residuos de diflubenzurn pueden medirse en el agua, en muestras biolgicas y en el suelo mediante cromatografa lquida de alta resolucin con deteccin de radiacin ultravioleta o mediante cromatografa de gases con detector de captura de electrones para el anlisis de la molcula intacta o tras la derivatizacin de la 4-cloroanilina liberada con anhdrido trifluoractico. 2. Fuentes de exposicin humana y ambiental El diflubenzurn es un compuesto sinttico utilizado en la agricultura, en la silvicultura y en programas de salud pblica para combatir plagas de insectos y vectores. Para esos usos existen diferentes formulaciones de diflubenzurn. No se dispone de informacin pertinente sobre la exposicin humana a este producto. 3. Transporte, distribucin y transformacin en el medio ambiente El diflubenzurn suele aplicarse directamente a las plantas y al agua. No se produce absorcin a travs de las hojas. La adsorcin del diflubenzurn en el suelo es rpida. El producto se inmoviliza en la capa superior de 10 cm del suelo al que se aplica, y las probabilidades de lixiviacin son escasas. El diflubenzurn se degrada en suelos de diversos tipos y orgenes en condiciones aerobias o anaerobias, con una semivida de pocos das. La velocidad de degradacin depende en gran medida del tamao de las partculas de diflubenzurn. La principal ruta metablica (ms del 90%) es la hidrlisis, que produce 2,6-cido difluorobenzoico y 4-clorofenilurea; estos productos se degradan con semividas del orden de cuatro y seis semanas, respectivamente. No se ha detectado 4-cloroanilina libre en los suelos. El diflubenzurn se degrada rpidamente en aguas neutras o alcalinas. Estudios de aplicacin al agua revelan que el diflubenzurn se concentra rpidamente en el sedimento; el compuesto de origen y la 4-clorofenilurea pueden persistir en el sedimento por ms de 30 das. El diflubenzurn no es objeto de bioacumulacin en los peces. 4. Niveles ambientales y exposicin humana La exposicin de la poblacin general al diflubenzurn por medio del agua o los alimentos de resultas de su utilizacin en la agricultura, contra insectos forestales o en la lucha contra los mosquitos, es insignificante. 5. Cintica y metabolismo en animales de laboratorio En animales de experimentacin, el diflubenzurn se absorbe en el tubo digestivo y, en menor medida, a travs de la piel. En el tubo digestivo de la rata existe un mecanismo de absorcin saturable, por lo que una gran proporcin del diflubenzurn administrado oralmente aparece en las heces. El diflubenzurn tiene una amplia distribucin en los tejidos, pero no se acumula. El destino metablico del diflubenzurn ha sido estudiado en diversas especies. La principal va metablica en los mamferos es la hidroxilacin. La hidrlisis del diflubenzurn puede producirse en cualquiera de los tres enlaces carbono-nitrgeno. En los cerdos y los pollos, la principal ruta de hidrlisis es el puente ureido. En las ratas y las vacas, la principal va metablica es la hidroxilacin. En las ovejas, los cerdos y los pollos, los metabolitos ms importantes son el 2,6-cido difluorobenzoico y la 4-clorofenilurea; los metabolitos secundarios son la 2,6-difluorobenzamida y la 4-cloroanilina. En las ratas y el ganado vacuno, el 80% de los metabolitos est constituido por 2,6-difluoro-3-hidroxidiflubenzurn, 4-cloro-2-hidroxi-diflubenzurn y 4-cloro-3-hidroxidiflubenzurn. Los estudios metablicos indican que en las ratas o el ganado vacuno se forman cantidades mnimas o nulas de 4-cloroanilina. La principal ruta de eliminacin es a travs de las heces, con porcentajes de entre el 70 y el 85% en los gatos, los cerdos y el ganado vacuno. En el ganado ovino, la eliminacin se distribuye aproximadamente por igual entre la orina y las heces. En las ratas y ratones, la excrecin urinaria disminuye proporcionalmente al aumento de la dosis. Menos del 1% de una dosis oral se recupera en el aire exhalado. En la leche slo se han hallado residuos nfimos. No se dispone de ningn estudio humano de la cintica y el metabolismo del diflubenzurn, incluido el alcance de la biotransformacin en 4-cloroanilina. 6. Efectos en mamferos de laboratorio y en sistemas de pruebas in vitro El diflubenzurn tiene una toxicidad aguda baja por cualquier va de exposicin. La OMS lo ha clasificado como producto con pocas probabilidades de presentar un riesgo agudo en el uso normal, sobre la base de una DL50 aguda por va oral de ms de 4640 mg/kg de peso corporal en las ratas. La DL50 aguda por va cutnea en las ratas es superior a 10 000 mg/kg de peso corporal, mientras que la CL50 aguda por inhalacin en las ratas excede de 2,49 mg/litro. No se han observado signos de intoxicacin en los 14 das siguientes a una administracin nica de diflubenzurn por diversas rutas a una variedad de especies animales. El diflubenzurn no provoca irritacin cutnea (en el conejo) ni sensibilizacin de la piel (en el cobayo). Produce una ligera irritacin a los ojos en el conejo. El diflubenzurn causa metahemoglobinemia y sulfohemoglobinemia. Tras la exposicin oral, cutnea o por inhalacin de diversas especies al diflubenzurn se ha demostrado la presencia de metahemoglobinemia dosisdependiente. Este efecto es la variable de evaluacin toxicolgica ms sensible en los animales de experimentacin. El NOEL basado en la formacin de metahemoglobina es de 2 mg/kg de peso corporal por da en las ratas y los perros, y de 2,4 mg/kg de peso corporal por da en los ratones. En estudios de toxicidad a largo plazo realizados con ratones y ratas, los cambios relacionados con el tratamiento se han asociado principalmente a la oxidacin de la hemoglobina o a alteraciones de los hepatocitos. En estudios de la carcinogenicidad en ratones y ratas con niveles de hasta 10 000 mg/kg en la alimentacin, no se observaron efectos relacionados con el tratamiento en la incidencia de tumores. Especficamente, no se registraron neoplasias mesenquimatosas del bazo o el hgado como las observadas en los estudios de carcinogenicidad con 4-cloroanilina. En varios estudios de la toxicidad reproductiva en ratas, ratones, conejos y tres especies aviarias no se observ ningn efecto en la reproduccin, ni tampoco embriotoxicidad. Los estudios de teratogenicidad en ratas y conejos no revelaron ningn efecto teratognico. El diflubenzurn y sus principales metabolitos han sido sometidos a una serie de ensayos de mutagenicidad in vitro e in vivo Ni el diflubenzurn ni sus principales metabolitos tienen efecto mutagnico. El metabolito secundario 4-cloroanilina ha dado un resultado positivo en varios ensayos de mutagenicidad in vitro con diversas variables de valoracin. Es carcingeno en las ratas y los ratones. Las lesiones neoplsicas relacionadas con la administracin de 4-cloroanilina fueron tumores mesenquimatosos benignos y malignos en el bazo de ratas macho, y hemangiomas y hemangiosarcomas, principalmente en el bazo y el hgado de ratones macho. 7. Efectos en el ser humano Se ha notificado que el metabolito 4-cloroanilina del diflubenzurn ha causado metahemoglobinemia en trabajadores sometidos a exposicin y en neonatos expuestos por inadvertencia. Algunas personas con carencia de NADH metahemoglobina reductasa pueden ser particularmente sensibles a la 4-cloroanilina y, por lo tanto, a la exposicin al diflubenzurn. 8. Efectos sobre otros organismos en el laboratorio y en el terreno Todos los organismos que sintetizan quitina presentan sensibilidad al diflubenzurn. Las bacterias no resultaron afectadas por el diflubenzurn a concentraciones de 500 mg/kg de suelo; se observ cierta estimulacin de la fijacin de nitrgeno. Las soluciones de diflubenzurn-acetona se degradaron; la acetona se utiliz como fuente de carbono. La biomasa de algas aument a una concentracin de diflubenzurn de 1 g/litro. No se observaron efectos adversos a concentraciones superiores al lmite de solubilidad del diflubenzurn. Los hongos resultaron temporalmente afectados a 0,1 g/litro en condiciones de laboratorio. Los invertebrados acuticos presentan respuestas variables al diflubenzurn. Los moluscos son insensibles, con una CL50 superior a 200 mg/litro. Los valores de la CL50 de otros invertebrados van desde 1 hasta ms de 1000 g/litro, en funcin de los efectos del compuesto en las fases juvenil y de muda. Para Daphnia se ha estimado una MATC de > 40 y < 93 ng/litro; como era de prever, las larvas de mosca de mayo y otros insectos acuticos juveniles son sumamente sensibles. El rociamiento de masas de agua matar probablemente algunos invertebrados acuticos. En los ecosistemas y experimentos de campo en que se aplic diflubenzurn directamente al agua, los efectos en la mayora de los grupos taxonmicos fueron menos graves de lo previsto a partir de las pruebas de laboratorio sobre efectos agudos. No se han observado efectos en los organismos acuticos despus de aplicaciones areas a los bosques. Los valores de la CL50 para los peces son de > 150 mg/litro. En los experimentos prcticos no se ha registrado nunca la muerte de peces. La DL50 oral y por contacto en la abeja de miel es superior a 30 g/individuo. Las colonias de abejas no resultaron afectadas tras la aplicacin area de 350 g de diflubenzurn/hectrea. Un estudio de alimentacin de cinco das de duracin en patos silvestres y codornices con niveles de hasta 4640 mg/kg de pienso no revel ningn signo observable de toxicidad. Las pequeas aves canoras del ecosistema forestal no resultaron afectadas por la aplicacin area de diflubenzurn a razn de 350 g/hectrea. Las especies mamferas pequeas no sufrieron mermas de las poblaciones tras la aplicacin de diflubenzurn en un bosque a razn de 67 g/hectrea.