ENDOSULFAN Sinha 1997.

Endosulfan, a chlorinated cyclodiene compound is used worldwide as insecticide & acaricide for the control of various pests in agriculture. The effect of endosulfan on laboratory animals reported so far is restricted to immunological, neurological and genotoxic studies (Toxicological Profile 1993). 1.1 Identity CAS No.: 115-29-7 Molecular formula: C9H6Cl6O3S The chemical name of endosulfan is 6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9ahexahydro- 6,9-methano2,4,3-benzodioxathiepin-3-oxide. Technical endosulfan is a brown crystalline substance consisting of and -isomers in the ratio of approximately 70:30. Endosulfans chemical structure is shown below:

1.2 Physicochemical properties Technical endosulfan is usually sold in the form of brown crystalline flakes. Property Value Melting point 79100 C Vapour pressure 1.3 10-3 Pa at 25 C Solubility in water 60150 g/litre; increases with decreasing pH 1.3 Major uses

Endosulfan is a contact and stomach poison that has been used to control insects such as the Colorado potato beetle, flea beetle, cabbageworm, peach tree borer and tarnished plant bug, as well as several species of aphid and leafhopper. It is used in countries throughout the world to control pests on fruit, vegetables and tea and on non-food crops such as tobacco and cotton. In addition to its agricultural use and its use in the control of the tsetse fly, endosulfan is used as a wood preservative and for the control of home garden pests (IPCS, 1984). 1.4 Environmental fate Both endosulfan isomers undergo photolysis upon exposure to sunlight. The half-life is about 7 days. Endosulfan diol is the primary photolysis product; it is subsequently degraded to endosulfan -hydroxy ether (ATSDR, 2000). In water, endosulfan undergoes hydrolysis to endosulfan diol. The rate of hydrolysis is influenced by pH. Oxidative degradation also occurs. At pH 7, the half-lives for hydrolysis and oxidation were 23 and 25 days, respectively; at pH 5, the half-lives were 54 and 51 days, respectively (ATSDR, 2000). Endosulfan released to soil is subject to biodegradation. Biodegradation in soil and water is dependent on climatic conditions and on the type of microorganisms present. Endosulfan sulfate is the major degradation product in soil and is persistent in the soil (ATSDR, 2000).

ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 3.1 Air Residues of - and -endosulfan have been detected in ambient air samples in the USA (Kutz et al., 1976). Between 1970 and 1972, -endosulfan was found in 2.11% of samples tested in the USA at a mean concentration of 111.9 ng/m3 and a maximum of 2256 ng/m3. During the same period, -endosulfan was present in 0.32% of the samples at a mean concentration of 22.0 ng/m3 and a maximum of 54.5 ng/m3. This information suggests that the -isomer is more persistent in air. Both - and - endosulfan have been detected at levels up to 12 ng/litre in precipitation in the Great Lakes area of Canada and the USA (Strachan et al., 1980). 3.2 Water Endosulfan contamination does not appear to be widespread in the aquatic environment, but endosulfan has been found in agricultural runoff and rivers in industrialized areas where it is manufactured or formulated (IPCS, 1984). Endosulfan (one or both of its isomers) has been identified in 24 surface water

and 103 groundwater samples collected from 164 hazardous waste sites in the USA. Surface water samples in the USA generally contain less than 1 g/litre (ATSDR, 2000). 3.3 Food The main source of exposure of the general population is food, but residues have generally been found to be well below the FAO/WHO maximum residue limits (IPCS, 1984). These residue tolerances refer to the total residue of - and - endosulfan and endosulfan sulfate. Because of its use in tobacco farming, smoking may be an additional source of endosulfan exposure.

UNEP ; 2007 Excessive and improper application and handling of endosulfan have been linked to congenital physical disorders, mental retardations and deaths in farm workers and villagers in developing countries in Africa, southern Asia and Latin America. Endosulfan was found among the most frequently reported intoxication incidents, adding unintentionally further evidence to its high toxicity for humans29. In laboratory animals, endosulfan produces neurotoxicity effects, which are believed to result from overstimulation of the central nervous system. It can also cause haematological effects and nephrotoxicity. The -isomer was generally found more toxic than the -isomer30.

SIMONICH , 1995 Endosulfan is one of the last hexachlo-rocyclopentadiene insecticides remaining in widespread use (18), and it was found in high concentrations throughout the world (Fig. 1). Endosulfan is used on most crops, but the high concentrations of endosulfan measured in India and the Pacific Rim may result from its intensive use in rice cultiva-tion (18). It is clear from Fig. 1 that indus-trialized countries a remaking full use of this readily available insecticide

SYED ET AL., 2003 Endosulfan (6,7,8,9,10,10-hexachloro1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-

benzodioxathiepin-3-oxide) is a broad-spectrum insecticide and acaricide first regis-tered for use in the United States in 1954 to control agricultural insect and mite pests on a variety of field, fruit, and vegetable crops. Technical-grade endosulfan is composed of two stereochemical is omers,atendosulfan and 3-endosulfan, in concentrations of approxi-mately 70% and 30%, respectively. Use data from 1987 to 1997 indicate an average domestic use of approximately 1.38 million pounds of active ingredient per year [U.S. EnvironmentalP rotectionA gency (U.S. EPA) 2002]. It has been found in at least 162 of the 1,569 current National Priorities List sites by the U.S. EPA (HazDat 2000). In India, it is widely used against a variety of agricultural pests. During 1999-2000, about 81,000 met-ric tons of endosulfan was manufactured in India, and in terms of tonnage its production was next only to mancozeb (103,000 metric tons) and monocrotophos (95,000 metric tons) (Anonymous 2001). Oral LD50 (lethal dose sufficient to kill 50% of population) endosulfan in rats is 80 mg/kg,a nd it has been classifieda s a moderately hazardous (class II) pesticide [World Health Organization( WHO) 2002]. Neurotoxicity is the major end point of concern in acute endosulfan exposure in human beings and experimental animals. No data are available for subacute or chronic exposure to endosul-fan in human subjects; however, the subacute and chronic toxicity studies of endosulfan in animals suggest that the liver, kidneys, immune system, and testes are the main tar-get organs [Agency for Toxic Substances and Disease Registry (ATSDR) 2000]. In recent years, there has been growing concern about toxicity of a number of chemi-cals, including pesticides, on the male repro-ductive system (Murray et al. 2001; Sharpe 2001). Reported effects of endosulfan on the male reproductive system in experimental animals have been variable, depending on species, age at exposure, dose, duration of exposure, and study end points. Routine gross and histopathologice xaminationo f the repro-ductive organs of male mice that consumed doses of 7.3 mg/kg/day for 13 weeks (Hoechst. Unpublished data) or 2.5-5.0 mg/kg/day for 2 years [Hack et al. 1995; Hoechst. Unpublished data; National Cancer Institute (NCI) 1978] revealed no toxic effects.

Later on, more detailed studies in adult rats exposed to 2.5, 5, and 10 mg/kg/day endosulfan for 5 days per week for 10 weeks showed reduced intratesticularsp ermatidc ounts, sperm abnormalities, and changes in the marker enzymes of testicular activities, such as lactate dehydrogenase,s orbitol dehydrogenase, 7-glutamyl transpeptidase, and glucose-6-phosphate dehydrogenase, providing further evidence of effectso n spermatogenesis(K hana nd Sinha 1996; Sinha et al. 1995). Exposureo f younger animals (3 weeks old) showed marked depletion of spermatid count as well as decreased daily sperm production at a dose of 2.5 mg/kg/day (Sinhae t al. 1997), which was earliers een only at 5 mg/kg/day in adult rats by the same investi-gators (Sinha et al. 1995). More recent studies have shown that exposure of pregnant rats to endosulfan at 1 mg/kg/day from day 12 through parturition leads to decreased sper-matogenesis in offspring (Sinha et al. 2001). Dalsentere t al. (1999) reporteds imilaro bserva-tions at 3 mg/kg/day but not at 1.5 mg/kg/day, and they attributed this to strain variation (Dalsenter et al. 2003). Thus, experimental studies suggest that endosulfan can affect the male reproductive system and also that these effectsa rel ikelyt o be greateri f exposureo ccurs duringt he developmental phase. Environmental exposuret o a single chemi-cal over a long period of time is very rare. We came across a situation where endosulfan was the only pesticide that had been aerially sprayed two to three times a year for more than 20 years on cashew nut plantations situ-ated on hilltops in some villages of northern Kerala,I ndia (Figure1 ). The populationl iving in the valley had a significant chance of expo-sure to this pesticide during aerial spray and subsequently through other contaminated environmental media. This population, there-fore, provided a unique opportunity to study the long-term health effects of endosulfan. In this article, we report the effects of endosulfan on male reproductive development.

Saiyed 2004 Endosulfani s a volatilea nd persistentc yclodiene pesticide that can migrate over a long distance throughv ariouse nvironmental medias uch as air, water and sediment. Once endosulfan is applied to crops, it can either persist in soil as a sorbed phase or be removed through several physical, chemicala nd biologicalp rocesses.R ecents tudies suggest that secondary emissions of residual endosulfan continue to recycle in the global sys-tem while they slowly migrated and are redeposited via wet deposition in the Northern Hemisphere. The occurrence of endosulfan in remote regions like the Great Lakes, the Arctic and the mountainous areas is well documented. Endosulfan can also enter the air as adsorbed phaseo nto suspendedp articulatem atter,b ut this process does not appear to be a major contributor to long ranget ransportli ke volatilization

ALAGH , 1988 residues in India in samples of soils, wheat grain samples, vegetables, milk and butter, also studies of pesticide residues in human fat, have been reported'upon in' India and in different states within the country. The results are revealing and indicate the nature of the problems being faced. The procedures and methods used by various workers, however, vary and so does the planning and periods of study. Without sound basic data representativeo f the total Indian situation, meaningful preventive measures, public education and legal action cannot be taken. Availablei nformationa nd data on pesticides residues in food, fibre, soil, water, air, humans, etc, does however suggest a close watch on the avoidable harmful side effects of pesticides and the need to draw flexible strategies and counter measures for public safety and environmental protection. PAN AFRICA, 2009 Endosulfan is neurotoxin causing convulsions and death. It is an endocrine disruptor, a reproductive toxicant, and there is increasing evidence that it is genotoxic. Epidemiological studies have provided evidence of birth defects, intellectual and behavioural impairment, and disrupted sexual development. Studies in West Africa have reported numerous poisonings and deaths amongst cotton farmers using endosulfan. The prevailing conditions of use, including inability to use suitable protective equipment, mean that endosulfan cannot be used safely in these countries. Environmental contamination and wildlife poisonings have also been reported. Endosulfan was registered only for use on cotton but there was evidence of its use also on vegetables. There is also concern about obsolete stockpiles of endosulfan. Endosulfan is an organochlorine insecticide used against aphids, thrips, beetles, larvae that feed on leaf tissue, mites, borers, grey worms, cotton caterpillar, white flies and leafhoppers. Endosulfan has been banned or severely restricted in 60 countries1 because of its high toxicity to humans and animals and its persistence in the environment. Endosulfan is used as a broad-spectrum, non-systemic poison to control a wide variety of insects and mites. It is a chlorinated hydrocarbon belonging to thE cyclodiene sub-group of the organochlorine family of pesticides, and is composed of stereoisomers alpha and beta ( and ), in the proportions / = 70/30. The chemical name is 6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9methano-2,3,4- benzodioxathiepin-3-oxide) and the molecular formula is C9H6Cl603S. It was developed in the mid-1950s. Endosulfan is sold under a variety of trade names, including Caiman, Callisulfan, Cotofan, Endocoton, Mistral, Phaser, Plexus, Rocky, Thiodan, and Thiofanex. Toxicity of endosulfan

Endosulfan is particularly neurotoxic for both insects and mammals, including humans. It was classified by the United States Environmental Protection Agency (US EPA) as Category Ib: highly toxic, based on an LD50 of 30 mg/kg for rats (US EPA,\ 2002), while the World Health organization (WHO) put it in Class II moderately hazardous, based on an LD50 of 80 mg/kg for rats (WHO, 2005). It is an antagonist of the chain of gamma-aminobutyric acid (GABA) receptors in the brain, reducing the uptake of chloride ions by neurons, which results in uncontrolled excitation (UNEP/FAO, 2007). It also inhibits calcium and magnesium uptake, and the enzyme ATPase. Both enzymes are involved in the transfer of nerve impulses. Among the most characteristic symptoms of poisoning by endosulfan are hyperactivity, tremors, convulsions, lack of coordination, dizziness, difficulty breathing, nausea and vomiting, diarrhoea, and in severe cases, impaired consciousness (ATSDR, 2000). Doses as low as 35 mg/kg have caused human death (IPCS, 2000). Chronic exposure to endosulfan can cause rashes and skin irritation among agricultural workers (US EPA, 2002). A number of studies demonstrate the high toxicity of endosulfan and formulations of endosulfan to aquatic organisms, including invertebrates (US EPA, 2002). It has also been established that endosulfan affects reproduction and the assessments of many\ agencies and scientific studies designate it as an endocrine disruptor. The effects observed include harm to the development of amphibians, reduced cortisol secretion in fish, disruption of the development of the genital tract and hormones in birds, testicular atrophy and reduced sperm production in mammals exposed to endosulfan (UNEP/POPS/POPRC.3/5, 2007). Environmental fate of endosulfan Persistence Based on laboratory studies, field studies, modelling, field monitoring and published papers, the US EPA concluded that endosulfan is a highly persistent chemical that can remain in the environment for a very long period, especially in acidic environment\ (US EPA, 2002). Endosulfan is oxidized in plants and soils to form mainly endosulfan sulfate and endosulfan diol (Goebel et al, 1982). The formation of endosulfan sulfate is due mainly to the action of micro-organisms, while endosulfan diol is the main product of hydrolysis (UNEP/POPS/POPRC.3/5, 2007). Microbiological mineralization is generally slow. The US EPA (2007) identified the aerobic soil half life (DT50) for the total endosulfan ( + isomers + endosulfan sulphate) as 1336 days; and the US State of California provides estimates of up to 2162 days for combined residues (CDPR, 2008). Dissipation under field conditions also varies largely; the European Union assessment reported, for the temperate regions, field DT50s ranging from 7.4 to 92 days for the + isomers. The field DT50 for total endosulfan is likely to

be considerably higher given that the EU laboratory value for the sulphate is 3 times higher than those of the + isomers (UNEP/POPS/POPRC.3/INF/9, 2007). According to the criteria for persistence set by the Stockholm Convention, of a DT50 greater than 183 days, endosulfan is persistent in soils, especially when taken as the sum of the isomers and the sulphate. The and isomers and endosulfan sulfate, are of low solubility in water, but are persistent, with halflives varying from 35 to 187 days under anaerobic conditions (ATSDR, 2000) the Stockholm Convention criteria for persistence in water is > 2 months. Vapour pressure values of the and isomers, the calculated Henrys Law constant (H), and the available monitoring data, all confirm that endosulfan is semi-volatile, with the volatility and the partitioning or interchange between air and water ensuring\ that it can be transported in the atmosphere over long distances. Endosulfan is mobile in the environment due to its volatility. Significant amounts of the pesticide volatilize from soil or the surface of leaves, especially immediately after application field studies have shown an 89% lose over 48 hours from cotton foliage at 400C (UNEP/POPS/POPRC.3/INF/9, 2007). The high water/air partition coefficients favour subsequent deposit of volatilised endosulfan on lakes. Endosulfan has been detected in samples of air, water, snow and biota in remote places like the Arctic, as a result of long-range atmospheric transport (PMRA, 2007). The persistence of endosulfan in the environment causes concern about the postapplication exposure of workers returning to the treated sites to perform agricultural tasks that result in contact with the foliage (such as pruning, thinning, harvesting or\ detection of harmful organisms). A short to intermediary period of post-application exposure is possible (1 day to 6 months). The post-application risk is managed by determining the safety deadline for specific tasks. Pesticide residues dissipate or degrade over time, and the safety deadline corresponds with the required time for the return to the treated places to be associated with acceptable exposure levels. According to the assessment made by the Pest Management Regulatory Agency (PMRA), the safety deadlines for endosulfan are generally long, and compliance may be unrealistic for producers, even with the minimum application rate (PMRA, 2007). Bioconcentration Endosulfan has a clear potential for bioaccumulation in both aquatic and terrestrial ecosystems. The octanol-water partitioning coefficients (log Kow) are respectively 4.74, 4.79 and 3.77 for the and isomers and endosulfan sulphate, indicating a high potential for bioaccumulation in aquatic biota. There is

also lot of data on the bioconcentration of endosulfan in various species of fish and freshwater invertebrates. The estimated bioconcentration factors (BCF) vary widely, and they range from 1.97 to 11,583 for aquatic organisms (although the US EPA, 2000 re-evaluated the last figure to 5,670). A trophic biomagnification factor >1 was calculated for the Southern Beaufort Sea and Amundsen Gulf food webs including marine mammals (Mackay & Arnold, 2005). Endosulfan has even higher octanol-air partitioning coefficients (Log Koa = 10.29, 10.29, 5.8) meaning that bioaccumulation is greater in terrestrial animals than aquatic life (Kelly & Gobas, 2003). It has also been found to bioaccumulate in plant foliage (Landers et al, 2008). Endosulfans intrinsic chemical, physical and toxicological properties, has resulted in its widespread contamination of the environment, including in remote environments through long-distance atmospheric transport. This, together with its high toxicity, has brought about a need for global measures to prevent further damage

CERILLO, 2005. Organochlorine pesticides are a wide group of chemicals, many of which persist in the environment. They are of interest because of reports, after their widespread use, of their ubiquitous persistence in different environmental media, of their ability to bioaccumulate and biomagnify in food chains, and of their capacity for long-range atmospheric transport (Longnecker et al., 1997). Although industrialized nations have restricted or banned many organochlorine pesticides, they continue to be manufactured in these nations for export to other countries. Moreover, some of these chemicals (e.g., endosulfans) are still used in industrialized countries, on the assumption that they pose little threat to the environment, wildlife, or human health. Endosulfan is a dienic compound containing six chlorine atoms. The commercial product contains the isomer I in the range of 6467% and the isomer II in the range of 2932%. It was introduced into the market in the mid-1950s. It is indicated both in non-food crops, such as cotton and tobacco, timber, and ornamental cultures, and in food crops, such as vegetables, fruits, corn, cereals, oilseeds, potatoes, tea, coffee, cacao, and soy bean (FAO Pesticide Management, 1993). In the\ past it has also been used to control termites and tsetse fly. Within the European Union (EU) there is only one declared producer, which manufactures endosulfan at a single production site in Germany. The vast majority\ of this volume is exported for use in tropical and subtropical countries, such as Latin America and Southeast Asia (Endosulfan Preliminary Dossier, 2003), but it is also sold in European countries. According to available information, Spain is the main consumer of endosulfan within the EU, accounting for almost half of the total volume, followed by Italy (about 20%), Greece, and France (about 15% each).

Its use decreased in some countries, for example, in France, between 1996 and 1997 (Endosulfan Preliminary Dossier,2003). Endosulfan is degraded in the environment and metabolized in living beings. Several transformation products have been identified in the environment, notably endosulfan sulfate (Endosulfan Preliminary Dossier, 2003). Endosulfan metabolites are distributed\ as a function of their lipophilicity. Endosulfan I and endosulfan II persist in the environment for 800 and 60 days, respectively, before their complete disappearance, and there is a continuous degradation of the II isomer to the I isomer (Doong et al., 1999). In mammals, commercial endosulfan is transformed into more hydrosoluble metabolites, mostly endosulfan sulfate, followed by ether and diol metabolites. All of these metabolites are bioaccumulated in adipose tissue, depending on their lipophilicity. Neurotoxicity is of major concern with acute endosulfan exposure of human beings and animals. Acute and chronic toxicity studies of endosulfan in animals suggest that the liver, kidneys, immune system, and testes are the main target organs (ATSDR, 2000). In terms of toxicity, the National Institute for Occupational Safety and Health (USA) recommends that endosulfan be recognized as a Group 1 pesticide. Pesticides in Group 1 pose a significant riskof adverse acute health effects at low concentrations, are carcinogenic, teratogenic, and/or neurotoxic, or cause reproductive effects (NIOSH, 1992). The consequences of the interference of endosulfan with the endocrine system were first reported in rodents, in which endosulfan treatment was associated with testicular atrophy and testosterone impairment (Maier- Bode, 1968; Gupta and Chandra, 1977; Gupta and Gupta, 1979). Recent literature has indicated that endosulfan causes some endocrine-disruptive effects in both terrestrial and aquatic species. The observed effects of endosulfan exposure include impaired development in amphibians; reduced cortisol secretion in fish; impaired development of the genital tract in birds; and altered hormone levels, testicular atrophy, and reduced sperm production in mammals (Endosulfan Preliminary Dossier, 2003). For example, endosulfan is reported to interfere with reproduction in newts at concentrations as low as 5 mg/g (Parket al., 2001). In vitro tests assigned estrogen-mimicking effects to a- and b-endosulfan and endosulfan sulfate (Soto et al., 1994). More recent reports have confirmed this observation (Vonier et al., 1996; Jin et al., 1997; Andersen et al., 1999), and some endosulfan metabolites (endosulfan ether and endosulfan diol) were also identified as estrogen mimics (Rivas et al., 2001). The United States Environmental Protection Agency (US EPA, 2000) declared that endosulfans potential action as an endocrine disruptor cannot be ruled out

because endosulfan-treated organisms exhibit toxic effects historically associated with endocrinedisrupting chemicals. The largest area of intensive greenhouse agriculture in Europe is near the Mediterranean coast of Southern Spain, where it has been practiced since the 1960s (Olea et al., 1996; Olea, 1997). This type of farming requires the use of large amounts of pesticides (Olea et al., 1999). The present study investigated the presence of endosulfan in women of reproductive age and children living in Southern Spain, analyzing samples of fatty tissue, placenta, umbilical cord serum, and human milk, in order to explore the distribution of metabolites in fatty and non-fatty tissues and fluids. Endosulfan is among the most frequent contaminants found in food, soil, or water in Europe (Endosulfan Preliminary Dossier, 2003). For example, in Spain, endosulfan was the most frequent pesticide detected in surface waters in Almeria (Ferna ndez Alba et al., 1998; Penuela and Barcelo , 1998; Ferna ndez Gutierrez et al., 1998) and Valencia (Hernandez et al., 1996). Endosulfan has also been found in air among other organochlorines in natural parks and industrial areas (Nern et al., 1996). Humans can be exposed to pesticides through their occupational activity (Olea et al., 1996), environment, or food (Herrera and Brotons, 1998; Jandacekand Tso, 2001). Thus, Delgado et al. (1994) studied dermal and respiratory exposure to endosulfan, and Martinez Vidal et al. (1998) and Arrebola et al. (1999) reported on the urinary excretion of endosulfan by pest-control operators and greenhouse workers, respectively. Whereas Arrebola et al. found only the commercial products in urine samples, Martinez Vidal et al. found endosulfans and the main metabolites (endosulfan ether, endosulfan lactone, and endosulfan sulfate) (Martinez Vidal et al., 1998). Studies of dissipation curves of endosulfan I, endosulfan II, and endosulfan sulfate in greenhouse air (Martinez Vidal et al., 1996) and of (Nern et al., 1996) demonstrated the ubiquity of this pesticide in this agricultural setting and its absorption by plastics and materials, which favors human occupational exposure. Among the general human population, contaminated food seems to be a common exposure route (La zaro et al., 1999; Endosulfan Preliminary Dossier, 2003). These data confirm other world-wide reports (Gunderson, 1995; Aguilera del Real et al., 1997; Antonius et al., 1998; Mathew et al., 1998; Doong et al., 1999; Araujo et al., 1999; Vanbarneveld, 1999; European Commission, 2001) that describe endosulfan as a common pesticide residue in fruits and vegetables

kang et al, 2001 . endosulfan has estrogenic properties and neurotoxicity.

EPA, 2002 Endosulfan is a broad spectrum contact insecticide and acaricide registered for use on a wide variety of vegetables, fruits, cereal grains, and cotton, as well as ornamental shrubs, trees, vines, and ornamentals for use in commercial agricultural settings. Endosulfan is formulated as a liquid emulsifiable concentrate and a wettable powder. there is direct evidence (measured residues) that endosulfan bioaccumulates in terrestrial systems and indirect evidence (modeling) that endosulfan has a significant potential to biomagnify in certain terrestrial food webs. Once endosulfan is applied to crops, it can either persist in soil or dissipate from the site of application through several physical, chemical, and biological processes. Recent studies suggest that residues of endosulfan volatilize and continue to recycle in the global system through a process of migration and dry/wet deposition in the northern Hemisphere. All home and residential uses ndosulfan in the form of fogger, insecticidal smoke, impregnated material, dust, E pressurized liquid, and pressurized spray. Food: Citrus (except non-bearing and nursery stock), artichoke, safflower, sugar beet, watercress, alfalfa, clover/forage (except grown for seed), corn (field/forage), endive, evening primrose, garden beets, garlic, and rapeseed (canola). Non-food: Indoor household uses, wood protectant, unseasoned forest products, ULV application, Douglas Fir, Juniper, Locust, Maple, and Willow (forestry use), forestry plantings. Commercially Grown Greenhouse/Out-of-Doors Ornamental Plants (Except for Commercially Grown Outdoor Trees and Shrubs) including, but not limited to, Aster,

Carnation, Chrysanthemum, Evening Primrose, Iris, Lilies, Marigold, Poinsettia, Snapdragon, Tulips, Croft Lily, German Lily, Hydrangea, Periwinkle, Rhododendron, Rose, Rhododendron, Canescens, Flowering Peach/Nectarine, Leatherleaf Fern, Holly Fern. Occupational- and Non-Occupational-Use Products Products containing endosulfan are intended for occupational use. Residential uses will not be included in this assessment, because of the above mentioned deletion. Occupational uses include applications to agricultural food and non-food crops, ornamental and/or shade trees, fruit and nut crops, ornamental herbaceous trees, and shrubs. Type of Pesticide/Targeted Pests Endosulfan [6, 7, 8, 9, 10-hexachloro-1, 5, 5a, 6, 9, 9a, hexahydro-6, 9-methano-2, 4, 3benzodioxathiepin-3-oxide] is a broad spectrum insecticide/acaricide. Examples of the type of pests that endosulfan is used to control include, but are not limited to, the following: Field Crops: Meadow spittlebug, Army cutworm, Aphids, Bean leaf skeletonizer, Cowpea curculio, Cucumber beetle, Flea beetle, Green stink bug, Leafhoppers, Mexican bean beetles, Cabbage looper, Cabbage worm, Cabbage aphid, Cucumber beetles, Whitefly, Cutworms, Diamondback moth, Corn earworm, Boll weevil, Bollworm, Lygus bugs, Thrips, Melonworm, Pickleworm, Rindworm, Squash beetle, Squash bug, Blister beetle, Potato beetle, Rose chafer, Pepper maggot, Cinch bug, Crown mite, June bug, Harlequin bug, Grape phylloxera, and Grape leafhopper. Orchard Crops: Aphids (including Apple aphids, Black cherry aphid, Black peach aphid, Green peach aphid, Rosy apple aphids, Rusty plum aphids, Wooly apple aphids), Apple rust mites, Green fruitworm, Tarnished plant bug, Tentiform leafminers, Whitefly leaf hoppers, Peachtree borer, Peach twig borer, Plum rust mite, Bud moth, Bud mites, Twig mites, Filbert aphid, Filbert leafroller, Filbert bud mite, Black pecan aphic, Pecan nut casebearer, and Spittlebug.

Ornamental Trees and Shrubs: Leather leaf fern borer, Aphids, Cyclamen mite, Rose chafer, Whitefly, Dogwood borer, Lilac borer, Colley spruce gall adelgid, Douglas fir needle midge, Walnut aphid, and Stink bug. SINHA 1997. Endosulfan, a chlorinated cyclodiene compound is used worldwide as insecticide & acaricide for the control of various pests in agriculture. The effect of endosulfan on laboratory animals reported so far is restricted to immunological, neurological and genotoxic studies (Toxicological Profile 1993). Few reports on the effect of endosulfan on reproductive functions are also available but are restricted to adult animals only (Dikshith et.al. 1984; Singh and Pandey, 1990; Sinha et.al., 1995). ENDOSULFAN (HSG 17, 1988) Production and Uses Present world production of technical grade endosulfan is estimated to be of the order of 10 000 tonnes per year. Endosulfan is used in a formulation as a non-systemic contact and stomach insecticide, mainly in agriculture, in the control of the tsetse fly, and in the control of home garden pests. Endosulfan controls a wide range of sucking and chewing insect pests, notably of the orders of Lepidoptera, Coleoptera, Heteroptera, Homoptera, Thysanoptera, Diptera, and some species belonging to the order of Acarina. It is especially used on non-food crops, such as cotton and tobacco, and on food crops, such as vegetables, fruits, corn, cereals, oilseeds, potatoes, tea, and coffee. It is also used

on numerous other crops. Endosulfan toxicity WHO (1986) classified endosulfan in the category of technical products that are moderately hazardous, based on an oral LD50 in the rat of 80 mg/kg body weight. Endosulfan can be absorbed following ingestion, inhalation, or skin contact. It is readily metabolized and eliminated and does not accumulate in the body. Acute intoxication may result in neurological manifestations, such as irritability, restlessness, muscular twitchings, and convulsions thatmay end in death. Endosulfan was essentially negative in short-term tests for genetic activity. It did not show any carcinogenic activity in mice or rats, but studies were limited by inadequate reporting or survival. Human exposure to endosulfan Food is the main source of exposure of the general population to endosulfan. Endosulfan residues in food (the sum of its alpha- and beta-isomers and endosulfan sulfate) have been found to be generally well below FAO/WHO maximum residue limits. In occupationally-exposed persons, both skin contact and inhalation can be important routes of absorption, when adequate safety precautions are not taken. a Adequate proprietary data on reproductive toxicity, teratogenicity, and neurotoxicity have become available since this evaluation was made in 1984; new carcinogenicity studies on the rat and the mouse are in progress. 2.3 Evaluation of Health Risks for Man The main hazard associated with endosulfan is acute intoxication

through overexposure. Such situations may be due to intentional or accidental overexposure or to gross negligence in occupational situations. In all other exposure situations, especially as far as the general population is concerned, the toxicity profile and the present exposure pattern do not indicate any appreciable hazard. 2.4 Fate in the Environment Both endosulfan isomers are fairly resistant to photodegradation, but the metabolites endosulfan sulfate and endosulfan diol are susceptible to photolysis. The half-life of endosulfan in water is estimated to be 4 days, but anaerobic conditions and/or a low pH will lengthen the half-life. In water, it is mainly degraded to endosulfan diol. In soil, the alpha-isomer disappears more rapidly than the beta-isomer. Endosulfan sulfate, the major degradation product in soil, is relatively persistent. These compounds are not prone to leaching. Biodegradation in soil and water is dependent on climatic conditions and on the types of microorganism present. 2.5 Evaluation of Effects on the Environment Endosulfan does not appear to be a problem with regard to persistence in biota. It is not readily bioaccumulated. In aquatic organisms, loss soon balances uptake and a fairly low plateau level of residues is achieved. Fish are extremely sensitive to endosulfan and the killing of fish has been reported as a result of the discharge of endosulfan into rivers. Agricultural run-off has not caused such a problem. However,

application of endosulfan to wetlands at recommended rates may well result in the killing of fish. Large-scale field experience with endosulfan has not resulted in any long-term adverse effects on the environment. Because there is little or no biomagnification, endosulfan applied at recommended rates is not hazardous for terrestrial animals. Toxicity for bees is low to moderate. Toxicity for birds is high in a laboratory setting, but no poisonings have been reported under field conditions.

Endosulfan is a neurotoxic organochlorine insecticide of the cyclodiene family of pesticides. It is an endocrine disruptor, and it is highly acutely toxic. Doses as low as 35 mg/kg have been documented to cause death in humans, and many cases of sub-lethal poisoning have resulted in permanent brain damage.

DENSLOW Organochlorine Pesticides

OCPs, a large group of structurally diverse compounds, are some of the most analyzed and studied environmental pollutants. OCPs can be found in great concentrations in the environment. For instance, in muck farms and contaminated lakes in Central Florida, Marburger et al.1) found concentrations in the flooded soils as high as 385_241m g/kg of dieldrin, 7173_1037m g/kg of DDTs (DDT_DDE_DDD) and 39,444_11,284m g/kg of toxaphene. Furthermore, the toxic effects of OCPs, their high chemical stability and their lipophilicity allows them to accumulate in animal tissue and makes them one of the most widely distributed types of pollu-Even though most OCPs have been banned for decades, they are still highly persistent in the environment. They were widely used from the 1950s to the 1970s, and as one might expect, they persist in the soils and also in the sediments at the bottom of lakes, even in remote areas, bioaccumulating and biomagnifying in wildlife.27) The presence of OCPs in remote areas is related to their physicochemical properties, which result in their long-range transport. The air masses that arrive to remote areas have already traveled long distances. Consequently, the pollutants inside them are very variable and it is almost impossible to determine their source, as the air masses may have passed by many different sources and the pollutants may have suffered many degradation processes. In the atmosphere, pollutants are either in the gaseous phase or attached to particles. HCHs (hexachlorocyclohexanes) and

DDTs together with some other low molecular weight compounds tend to be in the gaseous phase, rather than in the particle phase, in relatively clean environments.8) Besides, the concentration of OCPs in remote areas is enhanced by the global distillation effect that involves their migration from temperate to cold areas, where they can become trapped. This effect occurs both on the planetary scale9) and at regional levels, where high elevations serve as cold traps.10,11) There are many different types of chemical structures within the OCPs: compounds from the cyclodiene group such as aldrin and dieldrin; halogenated ethane derivatives such as DDT, DDE and methoxychlor; or cycloparaffins such as hexachlorocyclohexane. Many studies have reported the ability of OCPs to act as endocrine disruptors, leading to impaired development and reproduction in wildlife.1218) Toxicity Mechanisms of OCPs OCPs are known to affect multiple biochemical pathways leading to general toxicity in fish, wildlife and humans. Many of these pathways have been elucidated, but there is still a lack of information on the mechanisms of toxicity, suggesting that novel approaches to understand these pathways are necessary. The toxic effects of OCPs can be manifested into broad areas such as immunotoxicity, neurotoxicity, dioxin-like toxicity and endocrine-related toxicity. Included in these broad areas are hepatic, developmental, and reproductive toxicity. Although the majority of the studies have been done on mammals,

some effects have also been reported in fish. Table 1 summarizes the toxic effects of the OCPs reported in this review. 1. Immunotoxic effects Several research groups have shown that OCPs can affect the immune response. Harford et al.19) reported that endosulfan resulted in the modulation of phagocytic responses in crimsonspotted rainbowfish (Melanotaenia fluviatilis), golden perch (Macquaria ambigua) and Murray cod (Maccullochella peelii), three native Australian freshwater fish. Milston et al.20) found that a short-term exposure of Chinook salmon (Oncoryhnchus tshawytscha) to o,p_-DDE during early-life stages caused long-term humoral immunosuppression. Lahvis et al.21) correlated reduced immune response in bottlenose dolphins (Tursiops truncatus) with blood concentrations of p,p_-DDT, o,p_-DDE and p,p_-DDE. The biochemical pathways that these mechanisms utilize are diverse and inter-related. A genomic approach could help to show both the commonalities and the differences in gene expression on patterns that contribute to these effects. 2. Neurotoxic effects OCPs can also act as neurotoxic compounds. Epidemiological studies suggest a relationship between dieldrin concentrations in the brain and the neurodegenerative disorder Parkinsons disease (PD).22) PD provokes a progressive and selective dopaminergic neuronal degeneration in the substancia nigra pars compacta (SNc), the brain region that controls motor activity.

23) The loss of dopaminergic neurons results in depletion of striatal dopamine, resulting in irreversible motor dysfunction. 24,25) Oxidative stress, mitochondrial dysfunction, protein aggregation, and apoptosis are key mediators of nigral dopaminergic neuronal cell death in PD (reviewed by Kanthasamy et al.26)). Dieldrin induces apoptosis, alters dopamine levels, mitochondrial dysfunction, and protein aggregation, by activating a series of cell death signaling molecules.2729) Other studies show that the OCPs g -hexachlorocyclohexane, a -endosulfan, and dieldrin can have neurotoxic effects by interacting with the GABAA receptor.3033) It is possible that some of these disorders share common biochemical pathways with the immunotoxic effects, which could only be demonstrated by a systems biology approach with microarrays.

THESIS.Persistent Organic Polutants (POPs) are a set of chemicals that are toxic, persist in the environment for long periods of time, and biomagnify as they move up through the food chain . POPs have been linked to adverse effects on human health and animals, such as cancer, damage to the nervous, reproductive disorders, and disruption of the immune system. Because they circulate globally via the atmosphere, oceans, and other pathways. POPs released in one part of the world can travel to regions far from their source of origin (Sandra et al. 2006). With mounting evidence, indicating the long-range transport potential of these substances to regions where they have never been used or produced and the consequent threats they pose to the environment, the

international community has called for urgent global actions to reduce and eliminate their release into the environment (Burger et al., 2001). Organochlorines (OCs), represent an important group of POPs which have caused worldwide concern as toxic environmental contaminants (Law et al. 2003, Covacia et al., 2005) and (Wurl and Obbard, 2005). The lipophilic nature, hydrophobicity and low chemical and biological degradation rates of organochlorine pesticides have led to their accumulation in biological tissues and the subsequent magnification of concentrations in organisms, progressing through to the food chain (Tanabe, 2002 and Helberg et al. 2005). Specifically, one of the key 3 environmental concerns, regarding some POPs, is their occurrence in Polar Regions, at surprisingly high levels. Organochlorine pesticides (OCPs) are still widely distributed in the environment due to their persistency, semi-volatile nature resulting in longdistance transportation (Zhang H.B et al. 2006). Accumulation of OCPs also in the lipid content of animals is a common phenomenon due to their hydrophobic properties (Sijm and linde, 1995). Pesticide history and Classification A pesticide is a substance or a mixture of substances used for preventing, controlling, or lessening the damage caused by a pest (Parads, G. et al., 1995). A pesticide may be a chemical substance, biological agent (such as a virus or bacteria), antimicrobial, disinfectant or device used against any pest. ( USEPA, 2007 ). Pesticides are used to control organisms which are considered harmful. (Purdue.edu, 2007). Pesticides can save farmers money by

preventing crop losses to insects and other pests; in the US, farmers get an estimated four-fold return on money they spend on pesticides (Kollogg RL, et. al, 2000). One study found that not using pesticides reduced crop yeilds by about 10%. (Kuniuki S, 2001). 5 The first recorded use of pesticide to protect crops was 4,500 years ago (Miller, GT. 2002). In 1993 Paul Muler discovered that DDT was a very effective insecticide. In 1940s, manufacturers began to produce large amounts of synthetic pesticides and their use became widespread (Daily, H, et al., 1998). Pesticide use has increased 50-fold since 1950 and 2.5 million tons (2.3 million metric tons) of industrial pesticides are now used each year (Miller, G. T. 2002). In the 1960s, it was discovered that DDT was preventing many fisheating birds from reproducing, which was a serious threat to biodiversity. Rachel Carson wrote the best- selling book Silent Spring about biological magnification. DDT is now banned in at least 86 countries, but it is still used in some developing nations to prevent malaria and other tropical diseases by killing disease- carrying insects (Lobe, J. 2006). Endosulfan Endosulfan is a neurotoxic organochlorine insecticide of the cyclodiene family of pesticides. It is highly toxic and an endocrine disruptor, and it is banned in the European Union, Philippine, and several 22 countries. It is made by Bayer Crop Science, Makhteshim-Agan, and Drexel Company among other, and sold under the trade names Thionex, Thiodan, Phaser, and Benzoepin.

Because of its high toxicity and high potential for bioaccumulation and environmental contamination, a global ban on the use and manufacture of endosulfan is being considered under the stockholm convention (Environmental Health and Alliance, 2007). 1.10.1 Structure: Fig. (1.6): Structure of Endosulfan IUPAC name: 6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro6,9- methano -2,4,3- benzodioxathiepine - 3 -oxide.Molecular formula: C9H6Cl6O3S. 1.10.2 Properties Molar mass: 406.95. Density: 1.745. Melting point: 70-100 C. Solubility in water: 0.33 mg/L.

Endosulfan Endosulfan, an organochlorine, was developed in the early 1950s as an nonsystemic insecticide, acaricide and miticide (Berrill et al. 1998; Landis & Yu 2004; Rand 1995). Primarily, endosulfan, also known as Thiodan, is used in the control of sucking, chewing and boring insects and mites, including: aphids, leafhoppers, Colorado potato beetle, cabbage worms and other pests (Berrill et al. 1998; Health Canada 2004; Wan et al. 2005). Endosulfan is acutely neurotoxic to both insects and mammals, including humans; less is known regarding the toxicity of endosulfan to amphibians, as endosulfan residues can persist in organic matter within water bodies throughout the year, resulting in a continuous exposure of tadpoles and embryos to low levels (Boone & Bridges 2003; Bridges 2000; Bridges & Semlitsch 2000; Broomhall and Shine 2003; Harris et al. 2000;

Relyea 2004; Wan et al. 2005). Endosulfan exists in two forms: alpha and beta isomers (see Appendix B). These isomers are metabolized by microbial activity to more persistent forms, endosulfan-sulphate and endosulfan-diol (Berrill et al. 1998; Broomhall & Shine 2003). Interestingly, a combination of the two isomers are the most potent to the widest range of organisms (Berrill et al. 1998; Broomhall & Shine 2003; Wan et al. 2005). Although endosulfan has low solubility in water, direct aerial application over water bodies has proven to be fatal to fish, and toxic to other aquatic organisms (Berrill et al. 1998; Health Canada 2004). Endosulfan is a central nervous system stimulant, causing such symptoms as: excitability, melanophore aggregation, developmental abnormalities, depressed feeding and growth (Berrill et al. 1998; Besbelli & Szajewski 2000; Broomhall 2004; Harris et al. 2000; Park et al. 2001; Rohr et al. 2003). Endosulfan is known to disrupt neurotransmitter transmission in cholinergic neurotransmitter systems (systems which use acetylcholine as a neurotransmitter), altering the concentrations of melatonin, acetylcholine, serotonin, dopamine and norepinephrine (Goulet & Hontela 2003; Harris et al. 2000; Park et al. 2001; Sparling et al. 2001). There are a number of hypothesized biochemical pathways which are targeted by endosulfan which would lead to melanophore aggregation, and at present the affected pathway(s) of the afflicted P. regilla tadpoles of this experiment is not known. Endosulfan is known to disrupt neurotransmitter transmission in cholinergic neurotransmitter systems (systems which use acetylcholine as a neurotransmitter), altering the concentrations of melatonin, acetylcholine, serotonin, dopamine and norepinephrine (Goulet & Hontela 2003; Harris et al. 2000; Park et al. 2001; Sparling et al. 2001). There are a number of hypothesized biochemical pathways which are targeted by endosulfan, which would lead to melanophore aggregation. Catecholamines and melatonin have shown to increase in animals exposed to endosulfan due to endosulfan

stimulating the sympathetic nervous system (Park et al. 2001; Rohr et al. 2003; Scholz & Hopkins 2006). These neurotransmitters act as antagonists to MSH, and favor a decrease in melanophoric cAMP levels, and thus facilitate melanophore aggregation (Aspengren et al. 2003; Hadley & Goldman 1969; Novales & Davis 1969; White et al. 1987).

2006, Mathava Kumar, Ligy Philip Endosulfan(6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro6,9-methano-2,3,4-benzo(e)dioxathiepin-3-oxide) is a chlorinated pesticide (C9H6Cl6O3S) of the cyclodiene group. It is being used extensively throughout the world for the control of numerous insects in a variety of food and non-food crops. Its technical preparation consists of two stereoisomers, a and b-endosulfan (Fig. 1) in an approximate ratio of 70:30. It is extremely toxic to fish and aquatic invertebrates, and is a priority pollutant for international environmental agencies (Awasthi et al., 1999). The solubility of endosulfan in water is low, but it persists in soil and water environment for 36 months or more (Rao and Murty, 1980; Kathpal et al., 1997; Awasthi et al., 2000). Endosulfan contamination is frequently found in the environment even at considerable distances from the point of its original applications (Sethunathan et al., 2002; Siddique et al., 2003). It has also been detected in the atmosphere, soils, sediments, estuaries, surface and rainwaters, and food stuffs (United States Department of Health and Human

Services, 1990; Sujatha et al., 1999; Berrakat et al., 2002; Doong et al., 2002; Bhattacharya et al., 2003; Cerejeira et al., 2003; Golfinopoulos et al., 2003). As endosulfan is found in groundwaters, it is apparent that there is significant mobility of these chemicals through the soil (Masse et al., 1994; Johnson et al., 1995; Miller et al., 1995; Ro et al., 1997; Spark and Swift, 2002).

German Federal Environment Agency Umweltbundesamt, Berlin September 2004 Endosulfan is a widely used agricultural insecticide. It was introduced into the market in the mid 1950s. Plant production products containing endosulfan are still registered in a number of countries worldwide. Mammalian Toxicity In laboratory animals, endosulfan produces neurotoxicity effects, which are believed to result from over-stimulation of the central nervous system. It can also cause haematological effects and nephrotoxicity. The -isomer was generally found more toxic than the -isomer [106]. Investigation of chronic human toxicity exert endosulfan to be neither a carcinogen nor a reproductive toxin nor a teratogen in mammals. There are several results in vitro and in vivo showing no mutagenic effects, hence mutagenic effects in humans are unlikely.

Global scale Worldwide production of endosulfan was estimated at 10,000 metric tonnes in

1984 [155]. More recent data on production volumes could not be made available. However, current global production is likely to be significantly higher as use remains widespread. RELEASES TO THE ENVIRONMENT Endosulfan is released to the environment almost exclusively as a result of its use as an insecticide. There are no known natural sources of the compound. On a local scale environmental releases to the air or waste water may also occur from manufacturing of formulation operations. Its deliberate wide dispersive application on large areas, along with its physico-chemical properties makes it prone to spread over large distances as a consequence of post-application evaporation, Benefits of Endosulfan for the World Agriculture / Horticulture " Endosulfan has been registered and commercialized for more than 45 years in more than 60 countries around the world including those with high standard regulatory processes.. Used in many crops like cotton, soybeans, fruits and vegetables, endosulfan is an insecticide and acaricide which exhibits excellent efficacy against a number of difficult-to-control pests which can cause substantial loss of crops. Although the product is on the market for such a long time endosulfan is still an important tool for pest control in those crops and can not be replaced for the time being. Specially the unique mode of action and the respect of many beneficials make endosulfan a key tool in Integrated Pest Management and Resistance Management Programs. The National Registration Authorities from Australia published the following perspective in their registration review of Endosulfan (1998) concerning a potential ban of endosulfan: A simple ban of endosulfan could lead to other problems. This is because endosulfan has relatively low toxicity to many species

of beneficial insects, mites and spiders (that is, ones which prey upon or parasitise damaging insect pests). Other chemicals, necessarily substituted for Umweltbundesamt: Preliminary Risk Profile of Endosulfan 18.10.2004 38 endosulfan, would kill beneficial insects leading to population explosions of damaging pests which in turn would require more frequent sprays of harsher chemicals than if endosulfan had been used in the first place. In addition, because endosulfan is from a different chemical class than almost all other available insecticides, its use is very important for slowing the development of insecticide resistance to the other chemicals. Loss of endosulfan would, therefore, also lead to more insecticide use due to increasing resistance among insect pests. The net result is greater overall danger to agricultural workers and to the environment. As endosulfan can not be replaced simply by other existing registered products it takes time to find suitable solutions, probably mixtures of two active ingredients. Besides the efficacy studies, residue studies, toxicological studies and country registration needs to be performed. Although new insecticides have been introduced over the last decades, endosulfan is still appreciated by growers and recommended by extension services for the following reasons : Broad spectrum insecticidal/ acaricidal efficacy on difficult to control target pests. Tool for resistance management due to unique mode of action. Tool for IPM due to high selectivity on pollinators and respect of many beneficials. Excellent crop tolerance Cost effectiveness.

Broad spectrum insecticidal/ acaricidal activity Contrary to many new insecticides the biological efficacy of endosulfan allows to control a large spectrum of different pests : Lepidoptera : (e.g. Helicoverpa spp., Spodoptera spp.,Earias, Mamestra , Pieris .) Coleoptera : (e.g. Hypothenemus, Leptinotarsa., Anthonomus,, Meligethes ) Heteroptera : (e.g. Nezara , Piezodorus, Myridae) Homoptera : (e.g. Aphis spp, Bemisia spp) Mites (Acarina) : (e.g. Eriophydae) All these pests are damaging particularly cotton, soybeans, fruits, vegetables and plantation crops, which remain key markets for endosulfan. None of the modern insecticides cover the comprehensive pest spectrum like endosulfan. For the replacement of endosulfan it might be necessary to use several active ingredients in alternation or mixtures, as long as resistance management (see below) is not an issue. Further restrictions of its use or even loosing this "unique mode of action" would definitely negatively impact the other product's benefits. Furthermore endosulfan is the only tool available for certain target pests, especially in view of the minor use crop situation (increasingly important since many organophosphates are being cancelled for those uses; e.g. mites in blueberries, cyclamen mites in strawberries, pineapple fruit mites, green stink bugs in macadamia nuts, cow pea curculio control, lygus bug in alfalfa seed, only product that works on certain mites in ornamentals). Resistance management Umweltbundesamt: Preliminary Risk Profile of Endosulfan

18.10.2004 39 Despite the intensive use of endosulfan for more than 45 years all around the world only a few cases of temporary insect resistance have been reported. There are no reports about any significant product failures due to resistance problems. Endosulfan has a unique mode of action different to organophosphates, carbamates, synthetic pyrethroids, neonicotinoids, oxadiazine carboxylate, spinosad and all other classes of insecticides currently available or in development on the market. Furthermore there is no cross resistance in between endosulfan and synthetic pyrethroids, organophosphate, neonicotinoids or other chemical group used in crop protection. The reason for the very limited cases of resistance is connected with the multiple sites of action of endosulfan within the GABA receptor system as well as specific interaction with the detoxication system of pests . For these reasons Endosulfan is an important tool in Insecticide Resistance Management programs preventing or overcoming resistance against other chemical classes of insecticides. Endosulfan is the ideal product for difficult to control species like e.g. Helicoverpa armigera and white fly on cotton and vegetables in which respectively insect resistance against other insecticides has occurred (in Europe endosulfan is used mainly in southern countries like Spain, Greece, Italy Portugal and France, in areas with established pest problems where resistance to insecticides is becoming an increasingly important issue). Theoretically most of the pest can be controlled by other product groups as well like organophosphates and pyrethroids, but the loss of endosulfan would increase the use of these products with the consequence of increasing resistance. Furthermore it can be expected that due to restricted use of organophosphates in

future endosulfan will become even more important within the resistance strategies. Resistance management examples India: Endosulfan is recommended for use in a number of crops in India including cotton, rice, pulses, plantation crops, fruit crops and vegetables for control of various pests. It is highly recommended in IPM programs and also in Helicoverpa resistance management programs especially in cotton crop. Endosulfan is one of the very economic solutions for managing various pests in a number of crops. It is still one of the widely used molecules by farmers in India. It is recommended in the package of practices of almost all Agriculture Universities in India for control of various pests in different crops especially in Integrated Pest Management Programs due to its selectivity and lower toxicity to various beneficial insects. Selectivity on pollinators and beneficials Endosulfan provides high selectivity in favor of beneficial insects and pollinating insects. This allows predators and parasites of important pests to play an economic role in pest control and honey bees and bumble-bees to continue to be a vital part in agriculture/ horticulture through their activities as pollinators, e.g. Umweltbundesamt: Preliminary Risk Profile of Endosulfan 18.10.2004 41 in France Endosulfan is authorized for use during the flowering period.

IPEN Pesticide Working Group Project-2004 Endosulfan is a pesticide belonging to the organochlorine group of pesticides, under the Cyclodiene subgroup. Introduced in the 1950's, it

emerged as a leading chemical used against a broad spectrum of insects and mites in agriculture and allied sectors. It acts as contact and stomach poison and has a slight fumigant action1. It is used in vegetables, fruits, paddy, cotton, cashew, tea, coffee, tobacco and timber crops132. It is also used as a wood preservative and to control tse-tse flies and termites5. It is not recommended for household use. Intentional misuse of endosulfan for killing fish2,57and snails71 has also been reported. Endosulfan was also reported as used deliberately as a method of removing unwanted fish from lakes before restoring.132 Endosulfan was introduced at a time when environmental awareness and knowledge about the environmental fate and toxicology of such chemicals were low and not mandatory as per national laws. But now it is being detected as an important cause of pesticide poisoning in many countries. Q. How does endosulfan affect human beings ? Worldwide use of endosulfan increased with the ban/restriction in use of the more persistent organochlorine pesticides like DDT and endrin. Endosulfan is acutely toxic and has been implicated 5 in many cases of poisoning and fatalities. It has been

identified with a range of chronic effects, including cancer and impacts on hormonal systems, exhibiting similarities with its predecessors in the organochlorine class. Acute effects Endosulfan is highly toxic and can be fatal if inhaled, swallowed or absorbed through the skin. Acute toxicological data is adequately available. Acute oral toxicity is higher than dermal toxicity30. Absorption rate and toxicity is found to increase in the presence of solvents like alcohol and aromatic solvents12,24,34,61. A number of acute poisoning cases have been reported. Ingestion or breathing high levels of endosulfan may lead to convulsion and death. Endosulfan directly affects the central nervous system24,28 and recurrent epileptic seizures are also reported94. It is absorbed through skin and eye irritation may also result. Symptoms of poisoning include hyper activity, excitement, dyspnea (breathing difficulty), apnea (stoppage of breathing), salivation, loss of consciousness, diarrhea, anemia, nausea, vomiting, insomnia, blurred vision, cyanosis (bluish discoloration of skin due to want of oxygen), foaming at the mouth, tremor, dry mouth, lack of appetite, irritability, head ache, decreased respiration,

loss of memory, haematuria, albuminuria, confusion, dizziness, imbalance and lack of coordination4,5,7,30. Persons suffering from asthmatic and convulsive disorders are at high risk. Persons on protein deficient diet also possess high risk24,30. . Autopsy examination of an intentional ingestion (suicide) case has revealed damage to liver, lung and brain8,8a. Chronic effects There is experimental evidence of adverse effects of endosulfan on the male reproductive system, delaying sexual maturity and interfering with the sex-hormone synthesis118. Endosulfan is a proven endocrine disruptor6,9. It has potential to induce hypo thyroidism66. Long term health effects are not properly studied, experimented or documented world wide. Endosulfan exhibits estrogenic properties9,43, comparable to that of DDT9. It competes for estradiol for binding to estrogen receptors, thereby inhibiting hormonal function 107. The estrogenic potential of endosulfan increases in the presence of other estrogenic organochlorines 110 .It induces proliferation of human breast estrogen sensitive MCF7 cells49, (invitro) thereby increasing breast cancer risk117. It harms the reproductive system by affecting semen

quality, sperm count, spermatogonial cells, sperm morphology and other defects in male sex hormones11.Endosulfan [is having] have the capacity to alter the genetic material particularly chromosomes in mammalian cultures119. It is found to inhibit testicular androgen biosynthesis in lab animal experiments30 and exhibits significant risk in renal and testicular damage. It may have adverse effects on central nervous system by inhibiting brain acetyl cholinesterase16, causing uncontrolled discharge of acetyl choline. Endosulfan ingestion is known to affect the kidneys and liver50. It inhibits leucocyte and macrophage migration (this is the inhibition of the natural immune system by disrupting anti-body protection) causing adverse effects on humoral and cell-mediated immune system30. It is also a potential tumor promoter67. Many studies related to its acute and chronic toxicity in laboratory animals are available. Endosulfan is highly toxic to rats and mice13, 30.Some studies suggests its teratogenic28 and carcinogenic properties21 on rats and mice. It directly affects the central nervous system, causes liver and kidney (chronic glumerulonephrosis) damage6 in rats and mice. It also impairs the reproductory system of rats39. Behavioural and neurological changes have

also been noticed30. Thyroid follicular damage in mouse has been reported30,66. Endosulfan is known to damage the endocrine system, nervous system, circulatory, reproductory, respiratory and excretory systems and developing foetus.6,7,14,15,16,21,30,37 The National Institute of Occupational Health (India) have linked the higher prevalence of neurobehavioral disorders, congenital malformations in female children and abnormalities related to male reproductive systems to the continuous exposure to endosulfan spray. The study was conducted among children in one of the villages in Kasaragod District (in the South Indian State of Kerala) where endosulfan was aerially sprayed64. Endosulfan is implicated in the occurrence of adverse health effects particularly in rural communities in South East Asia, Southern Pacific and Sub-Saharan Africa.69,71 Q. Can endosulfan cause cancer ? Endosulfan is found to damage human red blood cells (RBC) at concentrations of 1ppb-1ppm10. Both alpha-endosulfan and beta- endosulfan are genotoxic to HepG2 cells8. Endosulfan is hepatotoxic 6, mutagenic, clastogenic and induce effects on cell cycle kinetics13. Endosulfan [is known] has been shown to cause chromosomal aberrations in hamster and mouse and sex linked mutations in Drosophila

13,30,58. Endosulfan has caused mutations in bacterial and yeast cell. It is also known to cause mutations in mammals13 A re-analysis data from a 1978 NCI (National Cancer Institute, US) study in Osborne-Mendel rats has revealed that endosulfan induced malignant neoplasms at all sites in male and female rats and endocrine organs in male rats21. Both sexes 6 developed lymphosarcomas and female rats had neoplasms of the reproductive system21. Endosulfan is also carcinogenic for the liver of female mice21. No accurate data related to the carcinogenicity of endosulfan in human is available but from field level reports, endosulfan can be highly suspected for having carcinogenic properties in human beings, especially in cases of chronic exposure13. In some reports it is referred to as having possible carcinogenic effects, effects in human immune and reproductive system68. Studies have also shown that it induces proliferation of human breast estrogen sensitive MCF7 cells in vitro9,49 which may lead to greater breast cancer risk. Studies also indicate the contribution of endosulfan in the combined effect of environmental estrogens in inducing breast cancer 117.

Q. What happens to endosulfan in the environment ? The fate of endosulfan released in the environment is different for the two isomers and also depends on the medium it gets deposited. Beta- endosulfan is more persistent than its alpha- isomer. Endosulfan sulphate is the main degradation product of both isomers, which is equally toxic37,66 and is itself more persistent in the environment than its parent compounds5,30. Endosulfan can be broken down by photolysis, hydrolysis and bio degradation. Endosulfan diol, endosulfan lactone, endosulfan ether etc. are some of the other byproducts5. Although the isomers are fairly resistant to photolysis, the break down products are susceptible132. On plant surfaces endosulfan rapidly degrades to metabolites30. Endosulfan is fairly immobile in soil and is highly persistent13,33. Major products in soil are endosulfan diol, endosulfan sulfate and endosulfan lactone. Endosulfan sulfate production increases with an increase in temperature30. Endosulfan will persist longer under more acidic conditions5,24. It persists longer under submerged conditions86. The half life of endosulfan varies from 60 days (alpha- endosulfan) to 800 days (beta-endosulfan)13. It enters air by volatilization from

plant and water surfaces13,33. Contamination by drift and particle transport also occur33. Ultra low volume (ULV)- application can drift several kilometers from point of application30. It has been detected in remote areas including the Arctic132 in air, snow-water and lake waters, rainfall and snow samples in Californian mountains and remote European mountain lakes 5,30,33,68,75,132. Beta-endosulfan is more stable in air. Endosulfan has been shown to be released from wood preservatives into room atmosphere over an one year period of observation5. In water endosulfan has a half life of 35 to 150 days13. It does not easily dissolve in water and may accumulate in bodies of fish and other aquatic organisms30,69. The break down products in water are endosulfan-diol and endosulfan sulfate. Endosulfan does not reach down much to ground water30, but has been proven to run off after spraying. But it has been detected from ground water at deep soil layers in concentration ranging from 0.009- 0.053 micro gram per litre up to 20 days after last spraying13. USEPA recommends that the levels of endosulfan in rivers, lakes and streams should not be more than 74 ppb28. But this limit is 15 times more than the concentration required causing reproductive damage in red spotted newt54. Several studies of bioaccumulation have been

conducted around the world and residues were found in aquatic organisms. USEPA considers endosulfan as having a high bioaccumulation potential in fish16, but not much evidence of bioaccumulation in higher trophic levels is available. Q. Is there any evidence of endosulfan contamination in the environment, food and human beings ? Reports of endosulfan residue in food, soil, air, body tissues etc are available from all parts of the globe. Residues were detected from air, water (surface and ground water)2,59 and soil in India2, water and sediments in Ghana45, marine water and sediments in India2,87, shallow ground water in Pakistan124, river water in China127, lagoons in Spain, surface and ground waters in Portugal92, ground and well waters in the Philippines133,134, coastal, estuarine and river sediments in Israel (high concentrations in Lake Kinneret)70, water in Benin, Malawi, Nigeria69 and from drinking, ground, surface and marine waters in South Africa97 (alarming levels in river water 6844843 ng/L)69, soil in Benin, Nigeria, Sudan and Zambia, sediments in Benin and Nigeria, vegetation in Madagascar, Zambia and Ghana69,97, Paddy fields in Mediterranean131, water from remote mountain

lakes in Europe (the Alps, Caledonian and the Pyrenees)75 and river and sea water in South East Asia71 etc. were found to be polluted with endosulfan. In Malaysia river sediments were found to have high levels (434micro grams/litre) of endosulfan 71. Reports from Central America shows air and wetlands93 in Costa Rica, shallow lakes, coastal waters, estuaries, well, surface and marine waters and sediments in Honduras, Mexico, Argentina and Jamaica are contaminated with endosulfan residues73,96,113,130.Endosulfan is one of the most frequently reported PTS in surface and ground water in Central America and the Carribean 73. Toxic levels of endosulfan are reported from the coastal lagoons of Mexico105 .In Guatemala, it is found more frequently and at higher levels in river water, well water and spring water73. It has been detected in municipal water system in Colombia73. Endosulfan residues were found in high levels from the gulf of 7 Mexico and from many parts of the USA104. Residues were also found in the Greenland biota 115 and biota including mammals of Arctic region132. High concentrations of alpha and beta endosulfan isomers and endosulfan sulphate have been detected in tree bark samples through out the

world, particularly in India and the Pacific Rim30. Endosulfan has been detected from food samples20 around the world- in Australian beef at 0.36 mg/kg32 (2 times the Australian limit and 4 times the international limit), in cows milk from tobacco farming areas in USA and food samples in USA and Canada5. Residues were detected in tomatoes from Brazil20, untreated leek in Argentina95, Spanish pepper samples from Finland, fish from India2, Kenya and Nigeria69, food and vegetables in Croatia70 and vegetables in Cyprus70. Residues were also found from sunflower seeds of untreated areas in Spain112 High levels of residues were found in red pepper and egg plants in Catania(Italy) 70. High residues have also been found in diary food, meat, fish, chicken, and vegetables in Eastern and Western South America72, cows milk in Brazil79, vegetable diet in Kuwait, vegetables, vegetable oil and seeds from India and animal samples from slaughter houses in India2. Endosulfan residues were detected from cows milk in Colombia far exceeding the WHO and FAO reference levels73. Fish from Kenya and Nigeria were heavily contaminated with endosulfan69. The European Union has banned import of fish from Tanzania, Uganda and Kenya due to high levels of endosulfan residues80. Residues were also detected

from animals samples in Benin, Nigeria, Cote d' Ivoire, Madagascar, South Africa and Kenya69. Presence of endosulfan in Lake trout in North America suggests a wide dispersal from areas of use to isolated lakes74. Endosulfan has also been detected from human tissues. It has been detected from cord blood samples obtained at the time of delivery46, human sera42,55 ,adipose tissue87 and human milk samples obtained from healthy lactating women in Spain44. Human breast milk from Egypt30,41, Colombia and Nicaragua73 and from cotton pickers in Pakistan2 were found to be contaminated. High levels of endosulfan were detected in human breast milk in Sub Saharan Africa69 and also from India128 . Residues were also detected in fat samples from children living nearby farms in Spain22. Blood, human milk and urine samples in Croatia were also found to be contaminated. Alarmingly high levels of endosulfan residues were observed in human blood and milk in a study in Kasaragod in Kerala, India26,26a,27,31,64. Q. How does endosulfan affect wildlife and domestic animals? There are many reports regarding the toxicity of endosulfan on wildlife. National Wildlife Federation

(USA) states that endosulfan is extremely toxic to wildlife and acutely toxic to bees13. It is acutely toxic to birds - mallard ducks24, quails and pheasants30. The alpha isomer is more neuro-toxic and its acute toxicity against mammals is more than three times that of the beta-isomer109. It is genotoxic and is a known endocrine disruptor in terrestrial and aquatic species132 Endosulfan is highly toxic to aquatic organisms even at recommended levels of application13,71,88,132. It is particularly toxic to fishes5,18,24,132 massive fish kills are reported from many places13. It also causes endocrine problems, reduction of protein in tissues and other health effects. The high toxicity to fish species is evidenced through studies on Gambusia affinis18, Rainbow trout, channel catfish, bluegill sunfish, minnow24,30,36. It affects metabolism in freshwater fishes by inhibiting transcription at some points126. It is known to impair the pheromonal systems leading to disrupted male choice and lowered mating success in female red spotted newts 54. It is known to affect the germ cell population of zebrafish embryos47. It exhibits antiestrogenic effects in fresh water catfish, reproductive problems in female teleostfish and opercoidfish30. It also bioconcentrates in aquatic organisms33. Reports

from South East Asia and Southern Pacific proves that endosulfan has detrimental effects on aquatic biota71. Reports prove its high toxicity to frogs, toads, annelids, snails, aquatic insects (damsel flies, midges, beetles etc), crustaceans (crab, shrimps, prawns etc), fishes and molluscs78, 114, 125. Endosulfan affects the hatching rate and larval survival of common Indian Toad82. It also affects the larval survival, growth rate, respiration and caused limb deformities in the streamside salamander103. Reports from Argentina and South Africa suggests that it affects the aquatic insect and macro-invertebrate populations in streams and rivers122,123. Studies show that it has contributed to the adverse effects on the wetland ecosystem in the Republic of Azherbaijan99. Besides being toxic to crab larvae29, it has been reported to disrupt the moulting of crabs and aquatic invertebrates76,76a. It is known to function as an endocrine disruptor in American alligators65. It has been detected from the nonviable eggs of Morelet's crocodile19 . It is also known to be affecting the egglaying of grass shrimps52 . It has been detected as one of the predominant organochlorine pesticides in the silverside fish in Argentina53. It has been related to the drastic population decline of anuran amphibians in Western USA in over the last 10 to 15

years48. Residues have been obtained from the liver of Eastern Box turtle40. It is found to accumulate 600 times the water concentration in mussels24. Endosulfan was found toxic to earthworms, causing a significant reduction in the growth rate and total protein content129. It is toxic to non target organisms16,120, like predators of several pests51. Endosulfan is highly toxic to soil micro arthropods17, 8 micro organisms, zooplanktons90, phytoplanktons, soil algae, actinomycetes, bacterial colonies etc.13,30. It has shown to cause chromosomal aberrations in Drosophila fly132. It is also toxic to mammals like rabbits13,30 and rats21,108. The disappearance of cats, frogs, bees, fresh water fishes etc. were reported form Kasaragod District in South India, where endosulfan was aerially sprayed27. Endosulfan is also highly poisonous to cats (LD50-2mg/kg) and dogs (LD50-76.7mg/kg). Reversible blindness and lack of muscle coordination has been reported in sheep and cattle grazing on endosulfan contaminated grass 24. Farmers from endosulfan sprayed area report of acute effects to cattle at time of spray and after, and leading to fatalities27. Acute poisoning of cattle and cattle deaths, due to the direct use of endosulfan on cattle

as an ecto-parasitic-control, are reported from Turkey100. Similar poisoning case is reported from USA also101. Chronic exposure to endosulfan leads to deleterious effects on metabolism and immune system of broiler chicken111. Endosulfan exhibits phytotoxic properties 7. Toxic effects on plants like root growth inhibition, stunting, burning of tips and margins of leaves and affected root permeability have been reported13. It is a prominent contaminant in vascular plants and lichen even in remote areas like the Arctic68,115. It is toxic to fresh water green algae90 and also to bluegreen algae91. It also affects diatom abundance, chrysophytes, cryptophytes and dinoflagellates121. Q. Are there reports of endosulfan poisoning from around the world ? Cases of endosulfan poisoning have been reported from many parts of the world. Accidental and intentional exposure leading to human fatalities and environmental tragedies has occurred. The following are some of the major cases of poisoning. Human poisonings In Sudan in1988, endosulfan barrels washed in irrigation canals caused fish kills and three people died after drinking water from the canal13. In the Philippines, endosulfan accounted for the largest

number of deaths due to pesticide poisoning reported in 199113. In Sulawesi, Indonesia, 32 cases of poisoning due to endosulfan have been reported from 1990 to 199356. In Columbia, 155 cases of poisoning (in 1994) and 60 cases (1993) of poisoning due to endosulfan were reported56. In Northern India, 18 cases of endosulfan poisoning have been reported in 1995-97 by accidental over-exposure during spray23. Endosulfan caused a rise in death numbers due to poisoning in Srilanka from 1994 to 199898. The misuse of endosulfan to kill snails has resulted in the largest number of poisoning cases with fatalities in Philippines in 1996 75. Poisoning due to consumption of endosulfan-contaminated food is also reported from Turkey (2003)102. Many cases of poisoningdeath in Guatemala, Costa Rica and other Central American countries have also been reported 73. In Feb 2002, two South African boys living near Ntabamhlophe, Kwa-Zulu Natal died following exposure to endosulfan. A police officer and 3 journalists were hospitalized after visiting the place several hours later80. In 2000, a case of 44 individuals who consumed food accidentally contaminated by endosulfan was reported in rural India. One individual died in the incident44. In 2004, 36 persons of all age groups in a rural area of

Jabalpur, India were poisoned after consuming wheat-grains or flour contaminated with endosulfan106. The worst of all the cases so far reported are from three nations--- Cuba, Benin and India. Pesticide Poisoning in Cuba : Endosulfan was responsible for the death of 15 people in the western province of Matanzas, Cuba in February 1999. A total of 63 people became ill after consuming food contaminated with endosulfan according to Cuban authorities83. The Benin Tragedy:- In Borgou province in Benin, endosulfan poisoning caused many deaths during 1999 - 2000 cotton season. Official records state that at least 37 deaths occurred and 36 were taken seriously ill. In the same region in1999 a boy died after eating corn contaminated with endosulfan25. The Kasaragod Tragedy, South India:People in 15 villages in Kasaragod in the South Indian State of Kerala were subjected to continuous exposure to endosulfan which was aerially sprayed three times every year for 24 years. Congenital Birth defects, reproductive health problems, Cancers, loss of immunity, neurological and mental diseases were reported among the villagers. Following a public

outcry a number of health based scientific studies confirmed that the health problems were directly linked to the exposure to endosulfan27,60,64. Poisoning of water bodies and fish kills A wide spread fish kill was observed in 1969, when 30 kilograms of endosulfan was discharged into the Rhine river in Federal Republic of Germany5,30. In 1975, an accidental spill of endosulfan caused a major fish kill in North Brook a tributary of the Dunk River in Eastern Prince County of Prince Edward Island. The Brook trout population was reduced from 2227-4147 to 45-246 30. In 1995, run-off from cotton fields contaminated with endosulfan resulted in the death of more than 24,000 fish along a 25-kilometer stretch of a river in Alabama13. Investigations showed that the pesticide had been sprayed according to label instructions.

Endosulfan is a synthetic chlorinated cyclodiene that is an environmental endocrine disruptor (1). It was introduced into the earth's environment in 1956 as a general use insecticide, and it was primarily used to protect food crops such as tea, fruits, vegetables, and grains as well as wood from a wide variety of insects and mites through contact poison.

Commercially used endosulfan is generally composed of its two isomers-ca-endosulfan and f3-endosulfan. Endosulfan is toxic not only to insects, but also to fish, animals, and humans (2,3). Autopsy examinations have revealed its damage to liver, lung, and brain (3). However, the data regarding its genotoxicity (4), especially that of its two isomers, are limited. Perhaps because its carcinogenicity and genotoxicity have not been confirmed, endosulfan is still widely used and continues to pollute the human environment not only in developing count. Endosulfan is an insecticide with estrogenic activity that is toxic to many fish and mammals. Some reports suggested that it could accumulate in aquatic animals (S!) and cause human fatalities (3). The genotoxicity of its two isomers, however, has not been confirmed.

KHAN AND SINHA 1996,Introduction Broad spectrum pesticides such as endosulfan (an acaroinsecticide), phosphamidon (a systemic insecticide) and mancozeb (a contact fungicide) are widely used to protect crops and grains from a variety of pests (Gruzdyev et al,

1983). These three pesticides have been reported to be somatic mutagens and clastogens because of their ability to induce chromosomal aberrations (Georgian et al, 1983; Sobti et al, 1983; Khan and Sinha, 1992, 1993), chromosome breakages detected as micronuclei (Usha Rani et at, 1980; Adhikari and Grover, 1989; Khan and Sinha, 1994a) and sister chromatid exchanges (Sobti et al., 1982; Jablonicka et al, 1989) in the haemopoietic cells of rodents and humans. Even in the primary spermatocytes of mice these pesticides induced various types of structural alterations in chromosomes, pairing impairments among homologues and division-disruptive changes (Khan and Sinha, 1994b)

NISHIMOTO et al. The widespread environmental pollution caused by the chemical substances such as pesticides is a serious problem for creatures including human (McClure et al., 2001; McKinlay et al., 2008; Boobis et al., 2008). Various chemical substances entering animal bodies are carried to the organs responsible for detoxification, such as liver and kidney, and excreted. On the other hand, some hydrophobic Interdisciplinary Studies on Environmental Chemistry Environmental Research in Asia, Eds., Y. Obayashi, T. Isobe, A. Subramanian, S. Suzuki and S. Tanabe, pp. 211217. by TERRAPUB, 2009. 212 S. NISHIMOTO et al. substances among these chemicals are accumulated in our body (Somogyi and Beck, 1993). In this study, we focused on the pesticidal chemicals used for the efficient cultivation of farm products. Many pesticides whose production and/or use are

prohibited in Japan are still in use in other countries and those farm products produced by using pesticides and/or dusted with such chemicals are consumed and also exported. Therefore, human cannot avoid exposure to these pesticides that may occur through residues on cereals, vegetables and fruits harvested in other countries. In some previous reports, influence of these pesticides on reproductive system has been studied (Welshons et al., 1999; Bretveld et al., 2007). However, the influence on immune system has not been well understood. We chose two pesticides for our investigation; methoxychlor possessing similar structure to DDT (Coats, 1983), and endosulfan (also referred as benzoepin) possessing similar characteristic property as endrin (Gegiou, 1974). S.C JOSHI 2005 The ever increasing problem of population since last few decades has put tremendous pressure on land. To feed the galloping population, there is a shift in cultivation of recalcitrant varieties to high yielding varieties and this is followed by a sizable variety of invading pests (Giridhar and Indira, 1997). To subvert these pests, variety of pesticides came to picture, one of these are fungicides. These were hailed as miracles of modern technology. Given todays extensive use of pesticides it is almost impossible for any one to avoid daily exposure to low level of several different pesticide residues. Pesticide incidents have more than doubled in the last 10 years. Each year 3 million people are poisoned by pesticides with 2,20,000 deaths (WHO, 1997). The most direct and most hazardous exposures occur among those who manufacture and formulate them, or apply them, or live or work near the areas where they are used heavily (Skjei & donald, 1983), which may have cumulative effects that lead to reproductive disorders (Lafuente et al.,

2000). Introduction of new more toxic and rapidly disseminating pesticides into environment has necessitated accurate identification of their potential hazards to human health. Many pesticides are extremely toxic to mammals and other non-target organisms (Ankley & Jensen, 2001). Studies are underway to address concern of potential persistent immunotoxic, reproductive, and neurotoxic effects of chronic and acute exposure to several pesticides. It is reported that pesticides may induce pathological changes in the testes and liver of rats (Dikshith and Datta, 1972; Joshi et al., 2003). Reproductive hormone profile among pesticide factory workers also have been reviewed (Padungtod et al., 1998). Occupational exposure to pesticides has increased parental risk of infertility whereas less is known about residential use of pesticides and the risk they pose to reproduction and development (Greenlee et al., 2004).

Sheo Mohan Prasada;2011. Introduction Heavy application of the chlorinated insecticide endosulfan (1), due to the restrictions in the use of other organochlorine insecticides such as endrin and DDT, has led to serious environmental contamination resulting in greater loss of crop productivity and inhibited growth of many beneficial micro-organisms (Dubois et al. 1996; Prasad, Kumar, and Zeeshan 2005).

PAROMITACHAKRABORTY Endosulfans. India is one of the major producers of endosulfan, with an average annual consumption of 3600 t

from 1995 to 2000 (38). The overall concentration range observed in the present study by AAS was 2404650 (1040 ( 610) pg m-3. The levels of the R-endosulfan isomer is comparable to the urban site of Guangzhou in China (28) and Tapachula, Chiapas in Mexico (32). Endosulfan sulfate (a breakdown product) contributed an average of 61% of the total endosulfan concentration, reflecting breakdown from past usage. Technical grade endosulfan comprises of two stereo isomers, R- and _-endosulfan in a ratio of 7:3. The overall average R/_ endosulfan in the present study is 4. Higher level ofR-endosulfan suggests ongoing use of technical endosulfan in India. In the PAS samples of Chennai elevated R/_-endosulfan ratios (average 2) possibly resulted from the extensive application of endosulfan in cotton and paddy fields in the surrounding state of Tamil Nadu (7). Kolkata has a higher range of endosulfans especially in the urban sites with the _-isomer being dominant. Kolkata is the capital city of West Bengal where technical endosulfan had been used for a longer period for an important winter season pulse crop, Bengal gram (39). _-Endosulfan has been found to bemorepersistent in Kolkata than R-isomer attributable to higher conversion of the latter to endosulfan sulfate in soil and plant (40). The highest levels of endosulfan sulfate were detected inMumbai (SI Figure 1d). Cotton cultivation is widespread in central and western India. Endosulfan is the single largest selling

insecticide in Central India, with an estimated 85% of it used on cotton (41). Hence the probable reason for the high range of R/_-endosulfan ratios from Mumbai (0.3-3, mean 1) and Goa (1-6,mean3) and the highest level of endosulfan sulfate may be due to the past application as well as ongoing use of endosulfan in the surrounding cotton growing fields.

HEALTH IMPACTS AND ECO-TOXICITY OF ENDOSULFAN INTRODUCTION Endosulfan, an organochlorine pesticide, is a broad spectrum contact insecticide widely used in pest control. It is used in a wide range of crops including cereals, coffee, cotton, fruit, oil seeds, potato, tea and vegetables. There is a global concern over the acute toxicity of endosulfan. Technically endosulfan is a mixture of two isomers alpha-endosulfan and beta-endosulfan a mixed proportion of 70% and 30% respectively. The endosulfan residues of toxicological concern are alphaendosulfan, beta-endosulfan and endosulfan sulfate. The sulphate is regarded as being equally toxic and of increased persistence in comparison with the parent isomers (USEPA-2010). The world Health Organization (WHO) classifies endosulfan in Category 2 (moderately hazardous)1. United States Environmental Protection Agency (USEPA) classifies endosulfan as Category 1b - highly hazardous2The Industrial Toxicological Research Centre (ITRC) in India the nodal centre for the Regional Based Assessment of Persistent Toxic Substances (PTS) for the Indian Ocean region by the United Nations Environment Programme-Global Environment Facility (UNEP-GEF) classifies endosulfan as Extremely Hazardous.3 The Intergovernmental Forum on Chemical Safety (IFCS) identified endosulfan as an acutely toxic pesticide that poses significant health problems for developing countries and economies in transition.4 According to the International Programme on Chemical Safety, INCHEM (1998) the Acute oral LD50(Lethal Dose) for rats is 80 mg/kg and the inhalation LC50(Lethal Concentration) (1 hour) for rats > 21 mg/L in air. However according to the

USEPA the LD50 for oral exposure is 30mg/kg bw and LC50 is less than and equal to 0.5mg/L. USEPA(2002) JMPR (Inchem)(1998) Acute Reference dose (acute Rfd) 0.015 mg/kg/day 0.02mg/kg/day Average Daily Intake (ADI) 0.006mg/kg/day (Referred to as Chronic Reference Dose) 0.006 mg/kg/day LD50 30mg/Kg bw 80 mg/kg/day Toxicity studies of Endosulfan have been conducted in animals. Animal toxicity studies are carried out to identify the target organs of toxicity and possible spectrum of effects. The effects of any chemical are determined by the dose, duration and the time of exposure. There is a close similarity between the spectrum of health effects observed in the human population exposed to endosulfan and those described in animal experiments. It has been demonstrated that much lower doses of toxicants may result in adverse health effects manifesting as functional or organic disorders in later life if the exposure takes place during the early developmental phase.5 The health and ecological hazards caused by exposure to endosulfan has been a global concern. Endosulfan persists in the environment and bioaccumulates in animals and plants, leading to instances of food contamination and eventually dietary exposure in humans6. Endosulfan's predominant toxicological effect is over stimulation of the centre nervous system, with little or no 1The WHO Recommended Classification of Pesticides by Hazard and Guidelines to Classification 2000 2002. Geneva:World Health Organization, International Programme on Chemical Safety/Inter-Organisation Programme for Sound Management of Chemicals. 2US EPA (16/11/07) Endosulfan Updated Risk Assessments, Notice of Availability, and Solicitation of Usage Information. Federal Register Environmental Documents. USEPA=

3Anon (1989). Toxicity Data Hand Book. Vol:III, Pesticide-A. Industrial Toxicology Research Centre( Council of Scientific and Industrial Research) Lucknow, India. 4IFCS. 2003. Acutely Toxic Pesticides: Initial Input on Extent of Problem and Guidance for Risk Management. Forum Standing Committee Working Group. Forum IV Fourth Session of the Intergovernmental Forum on Chemical Safety, Thailand, Nov 1-7. 5 Final Report ofThe Investigation of Unusual Illnesses Allegedly Produced by Endosulfan Exposure in Padre Village of Kasargod District (N.Kerala); NIOH Study 6 Vctor Briz et al.; Differential estrogenic effects of the persistent organochlorine pesticides dieldrin, endosulfan and lindane in primary neuronal cultures; ToxSci Advance Access published January 27, 2011 peripheral component. Endosulfan generally has been shown to have higher acute oral and inhalation toxicity than dermal toxicity.7 Absorption of endosulfan through the gastrointestinal tract is extremely efficient around 90% is absorbed. Similarly, absorption through the skin can be high; as much as 50%.8 EXPOSURE TO ENDOSULFAN DIETARY EXPOSURE Ingesting food that has been sprayed with endosulfan Drinking water from contaminated ground or surface Stores. OCCUPATIONAL EXPOSURE Skin exposure or inhalation during pesticide mixing, loading and/or applying a pesticide or reentering treated sites. ACCIDENTAL EXPOSURE Skin exposure or inhalation due to proximity to endosulfan use.9

VULNERABLE GROUPS Some populations are particularly sensitive to endosulfans neurotoxic effects; these include unborn children, infants and the elderly. Certain medical conditions also make people particularly sensitive to adverse affects. The Agency for Toxic Substances and Disease Registry (ATSDR) identifies people with liver or kidney disease; pre-existing anaemia or haematological disorders; neurological problems especially seizure disorders; people with HIV/AIDs and people with protein-deficient diets such as the malnourished poor, chronic alcoholics and dieters as vulnerable groups10 ACUTE TOXICITY OF ENDOSULFAN Acute endosulfan poisoning can cause convulsions, psychiatric disturbances, epilepsy, paralysis, brain oedema, impaired memory and death.11 Endosulfan is highly toxic and can be fatal if inhaled, swallowed or absorbed through the skin. Acute oral toxicity is higher than dermal toxicity.12 Symptoms of poisoning include hyper activity, excitement, dyspnea (breathing difficulty), apnea (stoppage of breathing), salivation, loss of consciousness, diarrhea, anemia, nausea, vomiting, insomnia, blurred vision, cyanosis (bluish discoloration of skin due to want of oxygen), foaming at the mouth, tremor, dry mouth, lack of appetite, irritability, head ache, decreased respiration, loss of memory, haematuria, albuminuria, confusion, dizziness, imbalance and lack of coordination.13 7US EPA (2010). Endosulfan. The Health Effects Divions Human Health Risk Assessment. EPA DP Barcode: D372569. June 2010. 134 pages. Docket No.: EPA-HQ-OPP-2002-0262-0178; http://www.regulations.gov 8GFEA-U. 2007. Endosulfan. Draft Dossier prepared in support of a proposal of endosulfan to be considered as a candidate for inclusion in the CLRTAP protocol on persistent organic pollutants. German Federal Environment Agency Berlin. 9 US EPA (November 2002) Endosulfan RED Facts. Pesticides: Re-registration. Available onlinehttp:// www.epa.gov/pesticides/reregistration/REDs/factsheets/endosulfan_fs.htm, accessed on 12/02/09 10ATSDR (2000) Toxicological Profile for Endosulfan. Agency of Toxic Substances and Disease Registry, Atlanta, USA.

http://www.atsdr.cdc.gov/toxprofiles/tp41.html 11Information for the consideration of Endosulfan, Provision of information to the Stockholm Convention Secretariat for use by the POPs Review Committee (POPRC), Pesticide Action Network (PAN) International, 30 June 2008. 12Susan Sang and Sania Petrovic (1999) Endosulfan- A Review of its Toxicity and its Effects on the Endocrine System WWF (World Wild Life Fund Canada) 13 1.Anon (1989). Toxicity Data Hand Book. Vol:III, Pesticide-A. Industrial Toxicology Research Centre( Council of Scientific and Industrial Research) Lucknow, India. 2.Anon (1984).Environment Health Criteria 40- Endosulfan. IPCS (International Programme on Chemical Safety) WHO Geneva. 3.Anon (Issue 2 May, 2000) Hazardous Substances Data Book (HSDB)- US National Library of Media Canadian Centre for Occupational Health and Safety. 4. Susan Sang and Sania Petrovic (1999) Endosulfan- A Review of its Toxicity and its Effects on the Endocrine System WWF (World Wild Life Fund Canada) CHRONIC TOXICITY Exposure through certain conditions of use (e.g. lack of protective equipment), and bystander exposure have been linked to congenital physical disorders, mental retardations and deaths in farm workers and villagers in developing countries.14 The sub acute and chronic toxicity studies of endosulfan in animals suggest that the liver, kidneys, immune system, and testes are the main target organs. A number of neurotoxicity studies were available, primarily in the rat. An acute neurotoxicity study in rats (gavages) showed a greater sensitivity of females compared to males 15 Long term exposure is linked to immunosuppression, neurological disorders, congenital birth

defects, chromosomal abnormalities, mental retardation, impaired learning and memory loss. 16 Long-term oral and dermal exposure in rats has been found to result in rapidly progressive Glomerulonephritis. This is a renal disease that affects the small blood vessels in the kidneys. Longterm oral and dermal exposure in male rats has been observed to cause aneurysms (blood-vessel dilations which if ruptured can even lead to death).17 Consumption of endosulfan at low dose for longer duration can ultimately lead to differential alterations of Monoamines (an important class of neurotransmitters) in various regions of the rat central nervous system.18 There is some indication that endosulfan can have adverse effects on the immune system at low levels of exposure.19 There is mounting evidence that organochlorine compounds can act as hormones. These compounds, including DDT, PCBs, and endosulfan, may also be part of the cause for the disease in the quality of semen, an increase in testicular and prostate cancer, an increase in defects in male sex organs, and increases incidence of breast cancer which has been observed in the last fifty years. 20 NEUROTOXICITY In laboratory animals, endosulfan produces neurotoxicity effects, which are believed to result from over-stimulation of the central nervous system. Possible mechanisms of neurotoxicity include (a) alteration of neurotransmitter levels in brain areas by affecting synthesis, degradation, and/or rates of release and reuptake, and/or (b) interference with the binding of neurotransmitters to their receptors.21Those exposed over prolonged periods have been found to experience cognitive and emotional deterioration, severe impairment of memory and inability to perform most daily tasks. Repeated exposure to a tolerated dose of endosulfan resulted in a deficit of behavioral responses responses involving both learning and memory. A serotonergic (activated by or capable of liberating serotonin, especially in transmitting nerve impulses) mechanism appeared to be involved significantly in endosulfan-induced learning impairment and negligibly in its memory disrupting action.22 Some have also experienced gross impairment of visual-motor coordination. Exposure has also been linked to conditions such as cerebral palsy, epilepsy and it may increase the risk of

Parkinsons disease.23 14Endosulfan: Draft Risk Profile; Stockholm Convention on Persistent Organic Pollutants (POPs) Review Committee (POPRC);July 2009 15Memorandum: Revised findings on the health effects of the active ingredient: endosulfan; Office of Environmental Health Hazard Assessment ; June 2008 16Information for the consideration of Endosulfan, Provision of information to the Stockholm Convention Secretariat for use by the POPs Review Committee (POPRC), Pesticide Action Network (PAN) International, 30 June 2008. 17Galatone. V, Environment Canada (09/01/09) Endosulfan: Canadas submission of information specified in Annex E of the Stockholm Convention pursuant to Article 8 of the Convention. Available online-

http://www.chm.pops.int/,accessed on 05/01/09) 18 M.K. Lakshamana, T.R. Raju; Endosulfan induces small but significant changes in the levels of noradrenaline, dopamine and serotonin in the developing rat brain and deficits in the operant learning performance; Toxicology 91 19(ATSDR). 1993. Toxicological profile for endosulfan. Atlanta, GA: U.S. Department of. Health and Human Services, Public Health Service. 20Hileman, B. 1994. Environmental estrogens linked to reproductive abnormalities, cancer. C&EN (31 January): 19-23; Soto, A.M. 1993. Testimony before the Subcommittee on Health and the Environment, U.S. House of Representatives, 21 October 1993. 21Endosulfan: Draft Risk Profile; Stockholm Convention on Persistent Organic Pollutants(POPs) Review Committee (POPRC);July 2009 22The neurobehavioral toxicity of endosulfan in rats: a serotonergic involvement in learning impairment; V.Paul, E.

Balasubramaniam and M. Kazi; European journal of pharmacology; EJPTOX40086 (1994) 23Jia. Z & Misra. H (2007) Developmental exposure to pesticides zineb and/or endosulfan renders the nigrostriatal dopamine system more susceptible to these environmental chemicals later in life. Neurotoxicology. Vol. 28 (4): p727-35 IMMUNOTOXICITY The immune system is adversely affected by endosulfan because exposure decreases the white blood cell count. These cells are vital for functions such as fighting infections, allergies and for tumour suppression.24 Endosulfan inhibits leucocytes and macrophage migration (this is the inhibition of the natural immune system by disrupting anti-body protection) causing adverse effects on humoral and cell-mediated immune system.25 REPRODUCTIVE SYSTEM TOXICITY There has been a major concern regarding the effect of endosulfan on the reproductive system. It harms the reproductive system by affecting semen quality, sperm count, spermatogonial cells, sperm morphology and other defects in male sex hormones26. The study confirmed that endosulfan displayed dose-dependent deformities, behavioural abnormalities and mortality. Detailed studies in adult rats exposed to endosulfan for 5 days per week for 10 weeks showed reduced intratesticular spermatid counts, sperm abnormalities, and changes in the marker enzymes of testicular activities, providing further evidence of effects on spermatogenesis27 There is experimental evidence of adverse effects of endosulfan on the male reproductive system, delaying sexual maturity and interfering with the sex-hormone synthesis.28 Although the lack of a comparison population in this study makes it difficult to know whether there was an association between endosulfan and infertility, the high prevalence of exposure among women of reproductive age is of serious concern. Pre- and post-natal exposures to endosulfan have been confirmed by measures of residues in human breast milk, placenta, cord blood, and adipose tissue. 29 ENDOCRINE DISRUPTION

Contradictory opinions on the potential for endocrine disruption have been presented. The latest health effects analysis of endosulfan released by the Stockholm Convention's Persistent Organic Pollutant Review Committee (POPRC) published that in weight-of-the-evidence evaluation by Plunkett (2008) considering all of the available data (published in vitro and in vivo data, published human data, and unpublished toxicological data submitted as part of the pesticide registration process), it was demonstrated that the potency of endosulfan in the available in vitro studies was very low, with potencies in the range of 105 to 106 times less than the naturally occurring hormones and even natural phytoestrogens that are present in the human diet. However there are several studies that prove that endosulfan is an endocrine disruptant. The commission of European union has recently published a priority list of endocrine disrupting substances, placing endosulfan in Group II substance, i.e. a High Production Volume Chemical with 24 1. Breast Cancer Network (NZ) (07/11/08) Application HRC07003: An application by ERMA for the reassessment o endosulfan and formulations containing endosulfan under section 63 of the Act. Available online h tt p://www.breastcancernetwork.org.nz/docs/Endosulfan_Submission.doc 2. Galatone. V, Environment Canada (09/01/09) Endosulfan: Canadas submission of information specified in Annex E of the Stockholm Convention pursuant to Article 8 of the Convention. Available online- h tt p://www.chm.pops.int/ , accessed on 05/01/09) 25Susan Sang and Sania Petrovic (1999) Endosulfan- A Review of its Toxicity and its Effects on the Endocrine System WWF (World Wild Life Fund Canada) 26Pandey N, Gundevia F, Prem A S, Ray P K. (1990) Studies on the Genotoxicity of Endosulfan, an organochlorine insecticide in mammalian germ cells; Mutant Res; Vol. 242; ISS 1, P 1-7.

27Ameliorating effect of vitamin C on murine sperm toxicity induced by three pesticides (endosulfan, phosphamidon and mancozeb). Khan PK, Sinha SP. (1996) Mutagenesis 11(1): 33-36. 28Effect of Endosulfan on Male Reproductive Development (NIOH Study) Habibullah Saiyed et al, volume 111 | number 16 | December 2003 Environmental Health Perspectives 29 1. Food and Drug Administration. Total Diet Study: Summary of Residues Found Ordered by Pesticide Market Baskets b 91-3 97-1, June 1999 2. Campoy C, Jimenez M, Olea-Serrano MF, Moreno-Frias M, Canabate F, Olea N, Bayes R, MolinaFont JA. Analysis of organochlorine pesticides in human milk: preliminary results. Early Hum Dev. 2001 Nov;65 Suppl:S18390. 3. Shen H et al; Concentrations of persistent organochlorine compounds in human milk and placenta are higher in Denmark than in Finland. Hum Reprod 23(1):201-10. a potential for endocrine disruption.30It has potential to induce hypo thyroidism31. It competes for estradiol for binding to estrogen receptors, thereby inhibiting hormonal function. The estrogenic potential of endosulfan increases in the presence of other estrogenic organochlorines.32Endosulfan has been shown to have hormone disruption activity on diverse animals ranging from newts to zebrafish. There are disturbing reports suggesting endocrine effect of pesticides including endosulfan on humans. Studies have described impaired thyroid function in pesticide formulators exposed to endosulfan and other pesticides. Estrogenic effects of organochlorine pesticides on human uterine leiomyoma cells in vitro have been demonstrated.33 Endosulfan has even shown endocrine-disrupting activity in two different neuronal populations, Cebellar Granular Cells (CGC) and Cortical Neurons (CN) through their direct interaction with neuronal Estrogen Receptors (ER).34

CONGENITAL PHYSICAL DEFORMITIES A relationship has been observed between maternal exposure and foetal malformations in the skull, ribs and spine of rats.35 Physical malformartions observed in humans include cleft palates, harelips, club feet, limb malformations, eye deformities and extra fingers and toes. 36 CARCINOGENICITY & GENOTOXICITY Role of endosulfan in causing carcinogenicity cannot be conclusively established. However there is considerable evidence that endosulfan can be genotoxic. It has been found that exposure to sublethal doses of endosulfan and its metabolites induce DNA damage and mutation37. Genotoxicity has been displayed in HepG2 cells (Human liver carcinoma cells)38 and hepatocyte-derived transformants39. Endosulfan has caused mutations in bacterial and yeast cell. It is also known to cause mutations in mammals.40 Silva & Beauvais (2010), concluded that endosulfan is considered to be genotoxic on the basis of evidence of genotoxicity in tests for gene mutation, chromosomal aberration and DNA damage in open literature studies, despite other tests being negative.41 A re-analysis data from a 1978 National Cancer Institute, US (NCI) study in Osborne-Mendel rats has revealed that endosulfan induced malignant neoplasms at all sites in male and female rats and 30Knauf. W., E.F. Schulze New finding on the toxicity of Endosulfan and its Metabolites to Acquatic Organisms. Mede. Fac. Landbouwwet Riijkuniv. Gent 38(3), 717-732, (1973) 31Anon (Nov 12 2001) World Wildlife Fund Comments on reregistration of Endosulfan submitted to Public Information and Record Integrity Branch, Information Resources and Services Division (7502c) Office of Pesticide Programme, Environmental Protection Agency, Washington. 32Grumfeld HT, Bonefeld-Jorgensen EC (2004) Effects of invitro estrogenis pesticides on human oestrogen receptor alpha and beta mRNA levels. Toxicol.Lett 2004 Aug 1;151(3);467-80

33Report of the committee to study and analyse the effects of aerial spray of Endosulfan in the cashew plantations of PCK Ltd. in Kasaragod disrict 34V. Briz et al; Differential estrogenic effects of the persistent organochlorine pesticides dieldrin, endosulfan and lindane in primary neuronal cultures; ToxSci Advance Access published January 27, 2011 35 Singh. N, Sharma. A, Dwivedi. P, Patil. R & Kumar. M (2007) Citrinin and endosulfan induced teratogenic effects in Wistar rats. J Appl Toxicol. Vol. 27 (2): p143-151 36 Rupa. D, Reddy. P, Reddi. O (1991) Reproductive performance in population exposed to pesticides in cotton fields in India. Environ Res. Vol. 55(2): p123-8 37Bajpayee M, Pandey AK, Zaidi S, Musarrat J, Parmar D, Mathur N, Serth PK, Dhawan A. 2006. DNA damage and mutagenicity induced by endosulfan and its metabolites. Environ Mol Mutagen 47(9):682-92. 38Li D, Liu J, Li J. 2010. Genotoxic evaluation of the insecticide endosulfan based on the induced GADD153-GFP reporter gene expression. Environ Monit Assess. 2010 Jul 14. [Epub ahead of print]. 39Hashizume T, Yoshitomi S, Asahi S, Uematsu R, Matsumura S, Chatani F, Oda H. 2010. Advantages of human hepatocyte-derived transformants expressing a series of human cytochrome P450 isoforms for genotoxicity examination. Tox Sci, online May 27, doi:10.1093/toxsci/kfq154. 40Romeo F. Quijano, MD (Oct/Dec 2000). Risk Assessment in a third world reality: An Endosulfan case History. International Journal of Occupational and Environment Health. Vol. 6, No. 4. 41Silva MH, Beauvais SL. 2010. Human health risk assessment of endosulfan. I: Toxicology and hazard identification. Regulatory

Toxicology and Pharmacology 56 417. endocrine organs in male rats. Both sexes developed lymphosarcomas (a diffused malignant cancer of lymphatic cells of the immune system) and female rats had neoplasms(tumor) of the reproductive system. Endosulfan is also carcinogenic for the liver of female mice.42 No accurate data related to the carcinogenicity of endosulfan in human is available but from field level reports, endosulfan can be highly suspected for having carcinogenic properties in human beings, especially in cases of chronic exposure. In some reports it is referred to as having possible carcinogenic effects, effects in human immune and reproductive system.43 Studies have also shown that it induces proliferation of human breast estrogen sensitive MCF7 cells in vitro which may lead to greater breast cancer risk.44 Studies also indicate the contribution of endosulfan in the combined effect of environmental estrogens in inducing breast cancer.45 TERATOGENICITY In the studies presented to the Stockholm Convention, no teratogenic effects were identified in animal studies. 46 However some studies suggest its teratogenic and carcinogenic properties on rats and mice. 47Therefore, in absence of adequate data, it would be difficult to conclude endosulfans teratogenic effects. BIOACCUMULATION Endosulfan stores easily within the fatty tissues of living organisms, and it accumulates in concentration whilst exposure continues that is, the organism absorbs endosulfan at a greater rate than it can be excreted. Studies have shown that both aquatic and terrestrial species can accumulate concentrations of endosulfan to a significant extent48, but the susceptibility to bioaccumulation varies greatly between species for example, oysters and bivalves appear to accumulate very little endosulfan, whilst some fish species accumulate endosulfan much more readily49. Terrestrial species show a greater relative potential for accumulation than aquatic species, and monitoring data has shown that concentrations of endosulfan have increased over time in beluga whale blubber samples from the Canadian Arctic50, the tissue of freshwater tetra in Brazil51 and even in plants. Two year

old conifer needles in Western national parks of the USA were found to have three times the concentration of endosulfan that one year old needles had. This characteristic, teamed with endosulfans high toxicity, means there is significant potential for damage.52 42Reuber MD , (Aug 1981) The role of toxicity in the carcinogenicity of Endosulfan. Sci. Total Environ.;20(1); 23-47. 43Anon (Dec 2002) Regional Based Assessment of Persistent Toxic Substances- Arctic Regional Report Chemicals- United Nations Environmental Programme- Global Environment Facility 44 1. Soto AM; Chung K L, Sonnen Schein C(1994).The Pesticides Endosulfan, Toxaphene and Dieldrin have estrogenic effect on human estrogen sensitive cells. Environmental Health Perspectives Vol 102,Iss 4; P 380-3. 2. Preziosi P (1998) Naturo and Anthropogenic Environmental Estrogens The Scientific Basis for risk Assessment. Endocrinedisruptors as environmental signalers- An Introduction to Pure and Applied Chemistry; Vol 70,No. 9 ;P 1617-1631. 45 Ibarluzea Jon J, Fernandez MF, Santa Marina L, Olea Serrano MF, Rivas AM, Aurrekoetxea JJ, Enposito J, Lorenzo M, Torne P, Villalobos M,Pedraza V, Sasco AJ,Olea N (2004). Breast cancer risk and the combined effect of environmental estrogens- Cancer causes control.2004 Aug; 15 (6):591-600 46Addition information on endosulfan, Stockholm Convention, POPRC 4,

UNEP/POPS/POPRC.4/INF/14 47Anon (Feb 2001), Endosulfan Fact sheet (ToxFAQs) Agency for Toxic Substances and Disease Registry (ATSDR), US Dept of Health and Human Services, Public Health Services, Division of Toxicology, Atlanta Georgia 48PAN North America (13/06/00) Endosulfan Deaths in Benin. Pesticide Action Network Updates Service (PANUPS) 49EU Endosulfan proposal. (27/08/08) UNEP/POPS/POPRC.4/14. UNEP. Available online

http://chm.pops.int/Convention/ US/Default.aspx , accessed on 12/02/09

POPsReviewCommittee/Chemicalsunderreview/tabid/43/language/en-

50Stern. G & Ikonomou. M (2003) Temporal trends of organochlorine contaminants in SE Baffin (Pangnirtung) beluga, 1982-2002. Synopsis of Research conducted under the 2001-2003 Northern Contaminants Program. Ottawa ON, Indian and Northern Affairs Canada: p358-361 51Jonsson. C & Toledo. M (1993) Bioaccumulation and elimination of endosulfan in the fish yellow tetra (Hyphessobrycon bifasciatus). Vol. 50 (4): p572-577 52POPRC4/5: Endosulfan http://chm.pops.int/Portals/0/docs/from_old_website/documents/meetings/poprc/chem_review/Endosulfa n/Endosulfan_AnnexD_e.pd f ECOLOGICAL IMPACT Technical grade endosulfan is a mixture of two biologically-active isomers, the alpha and beta isomers, which differ in physico-chemical and fate properties. The beta isomer is generally more persistent and the alpha isomer is more volatile. The major transformation products found in the fate studies are endosulfan diol (hydrolysis) and endosulfan sulfate (soil metabolism).53 Ecological risks are also of concern regarding Endosulfan. The environmental risk assessment suggests that exposure to endosulfan could result in both acute and chronic risks of concern for terrestrial and aquatic organisms. Exposure to endosulfan has resulted in both reproductive and development effects in non-target animals, particularly birds, fish and mammals54. Indiscriminate use of endosulfan plays a significant role in obstructing the catabolic activity of the plants.55. USEPA recommends that the levels of endosulfan in rivers, lakes and streams should not be more than 74 ppb56. But this limit is 15 times more than the concentration required causing

reproductive damage in red spotted newt (a common Salamander).57 The effects of endosulfan on non-target species can be swift and devastating. Through surface run off, evaporation, or seepage into ground water stores, a variety of wildlife species as well as humans can be at risk from its harmful effects.Farmers in Benin have observed birds and frogs dying after eating insects sprayed with endosulfan. 58Endosulfan is considered to be very toxic to nearly all kinds of organisms. It is highly to moderately toxic to birds and extremely toxic to aquatic organisms (notably fish but also amphibians, shrimp and prawns, aquatic snails and plants and coral reef organisms). 59 Endosulfan has a relatively high potential to bioaccumulate in fish . Endosulfan is classified as highly toxic to birds and mammals on an acute exposure basis and moderately toxic to birds on a subacute dietary basis. Chronic toxicity data on birds and mammals revealed that reproduction and growth were the most sensitive endpoints.60 In laboratory studies it has also shown high toxicity in rats, and it appears that female rats are 45 times more sensitive than male rats.61 POTENTIAL FOR LONG-RANGE ENVIRONMENTAL TRANSPORT: (i)Evidence of long-range environmental transport of endosulfan and endosulfan sulfate is confirmed by Arctic monitoring data; (ii)Levels of 0.9 and 3.02 ng/g of endosulfan in the blubber of elephant seals in the Antarctic provide evidence of potential concern for endosulfan found in areas distant from its sources of release but the toxicological significance is not known. Other data, however, also show lower levels in other areas of the globe; (iii)Overall persistence (Pov) for the endosulfan family is in the region of 10 days for tropical air and soil. The Arctic contamination potential after 10 years of continuous releases was between 53US EPA (November 2002) Endosulfan RED Facts. Pesticides: Re-registration. Available onlinehttp:// www.epa.gov/pesticides/reregistration/REDs/factsheets/endosulfan_fs.htm, accessed on 12/02/09 54 USEPA; Endosulfan-RED facts; November 2002; EPA-738-F-02-012

55A. R. Chopade, A.Y. Nalawade, N.S. Naikwade; Effects of pesticides on Chlorphyll content in leaves of medicinal plants ; Poll res.26 (3):491-494(2007) 56 Anon (Feb 2001),Endosulfan Fact sheet (ToxFAQs) Agency for Toxic Substances and Disease Registry (ATSDR), US Dept of Health and Human Services, Public Health Services, Division of Toxicology, Atlanta Georgia 57Park D, Hempleman S C, Propper C R ( July 2001) Endosulfan exposure disrupts Pheromonal system in the red spotted Newt- A Mechanism for subtle effects of environmental chemicals. Environmental Health Perspectives 109(7); 669-673. 58 Ton. P et al. 2000. Endosulfan deaths and poisonings in Benin. Pesticides News 47. 59UNEP & GEF (2002) United Nations Environment Programme Regionally Based Assessment of Persistent Toxic Substances: SubSaharan Africa Regional Report. UNEP Chemicals. Geneva, Switzerland. 60US EPA (November 2002) Endosulfan RED Facts. Pesticides: Re-registration. Available onlinehttp:// www.epa.gov/pesticides/reregistration/REDs/factsheets/endosulfan_fs.htm, accessed on 12/02/09 61UNEP & GEF (2002) United Nations Environment Programme Regionally Based Assessment of Persistent Toxic Substances: SubSaharan Africa Regional Report. UNEP Chemicals. Geneva, Switzerland. 0.1 and 1.0%62. 62 Stockholm Convention on Persistent Organic Pollutants POPs Review Committee (POPRC); UNEPPOPS-POPRC.4-POPRC-4-5; Evaluation of endosulfan against the criteria of Annex D

SoH nia Ribeiro,2001 *Endosulfan (6, 7, 8, 9, 10, 10-hexachloro-1, 5, 5a, 6, 9, 9ahexahydro-

6, 9-methano-2, 4, 3-benzodioxathiepin-3-oxide) is an organochlorine insecticide. This toxicant is a central nervous system poison (WHO, 1988; Naqvi and Vaishnavi, 1993). Acute intoxication may result in neurological manifestations, such as irritability, restlessness, muscular twitching, and convulsions that may end in death. Due to its low persistence in the environment, it is commonly used in Portugal in several cultures (including rice and corn) to control a wide range of insect pests. ENDOSULFAN= GUJARAT 2011 It acts as a poison to a wide variety of insects and mites on contact. Although it may also be used as a wood preservative, it is used primarily on a wide variety of food crops including tea, coffee, fruits, and vegetables, as well as on rice, cereals, maize, sorghum, or other grains. Endosulfan is a broad spectrum, generic insecticide used in a variety of crops to control over 60 different pests. The unique chemistry of Endosulfan has ensured that the farmer is able to protect his farm ecosystem while dealing with pest infestation. Farmers used Endosulfan extensively in cross pollinated crops where successful Honey bee pollination plays an important role. Endosulfan is the only in-use generic pesticide known to be soft on pollinators such as Honey bees and beneficial insects. India is the second largest producer of fruits and vegetables in the world. It is also one of the largest producer and exporter of honey. Hence, there is a great need of a bee-soft pesticide for the Indian farmers. It is extensively used in agriculture to control insect pests. It has been recommended for controlling insects pest in various crops namely paddy, grams, sugarcane, cotton, groundnuts, mustard, brinjal, cabbage, cauliflower, wheat, maize, tea, mango, cashew etc. Formulations of endosulfan include emsulsifiable concentrate, wettable powder, ultra-low volume (ULV) liquid, and smoke tablets. It is compatible with many other pesticides and may be found in formulations with dimethoate, malathion, methomyl, monocrotophos, pirimicarb, triazophos, fenoprop, parathion, petroleum oils, and oxine-copper. It is not compatible with alkaline materials. Technical endosulfan is made up of a mixture of two molecular forms (isomers) of endosulfan, - (alpha) and (beta) isomers. Registration status, approved usage Endosulfan is registered for import, manufacture for use in the country and export. Endosulfan is a wide spectrum insecticide, which acts mainly as a contact and stomach poison and controls many important chewing and sucking insects pests in various

crops. Three formulations of Endosulfan are registered under the Insecticides Act, 1968 for use against various crop pests. Three formulations i.e., 35% EC, 4% DP and 2% DP of Endosulfan are registered under the insecticides Act for controlling insect pests in various crops viz., rice, wheat, jowar, pulses, sugarcane, cotton, jute, maize, vegetables, tobacco, cardamom, tea, coffee, cashew, mango, cocoa, citrus, groundnut, mustard, safflower, til etc,. 8.2 Toxicity data and Toxicokinetics of Endosulfan. The pesticides are backbones of agriculture and food production. The pesticide chemicals are known poisons for insects and pests when applied in recommended concentrations. It is expected that beneficial creatures like honeybees, earthworm, and frogs are not affected or less affected. It is also expected to posses selective action on pests in recommended concentration an minimum or no harm to the animals, birds and human populations. Endosulfan has been classified by The US EPA as Category I: "Highly Acutely Toxic" compound based on LD50 value of 30 mg/kg for female rats. The World Health Organization classifies it as Class II "Moderately Hazardous" based on a rat LD50 of 80 mg/kg. The pesticide Endosulfan is used in agriculture for more than 50 years showing broad spectrum active without development of any resistance. It is also sparing useful honeybees. In recent years some episode of endosulfan related (supposed to be) toxicity as noticed in village Padre in Kerala State as reported by local medical practitioners. Endosulfan in bulk is manufactured mostly in India and used in Indian agripractice and exported to some countries. Kerala is hardly using 1% Endosulfan and 99% is used in other parts of India like Maharashtra, Karnataka, Punjab, Andhra Pradesh, Tamil Nadu, Gujarat, etc. It is very interesting to note that there are no reports of pesticide (Endosulfan) toxicity in the other states, farmers, sprayers and factory workers. Endosulfan is the third largest selling insecticide worldwide. It accounts for a global market in excess of 40 million liters valued at over US$ 300 million (` 1350 crores). Indian companies account for over 70% of this market which has come at the cost of the European manufacturers. The replacement value of Endosulfan by patented alternative is estimated to be in excess of US$ 1 billion (` 4500 crores). As a result, Endosulfan is today in the eye of the storm in the battle of patented versus generic pesticides. Endosulfan is widely used in India on cross pollinated crops and it is soft on pollination honey bees and natural predators The states marked in green have been using Endosulfan in the range of 1000 KL to 1200 for over 30 years. There have been no reports of any effects on human or environment in these regions. The states marked in black are Kerala and Sikkim. Use of Endosulfan in Kerala is suspended and Sikkim has no significant use of pesticides due to its climate.

NIOH FINAL REPORT 2002 Neurobehavioral Problems: Nervous system is the target organ for endosulfan toxicity. Higher prevalence of learning disability and neurobehavioral disorders were found in study population. Paul et al. (1994) found significant increases in serotonin concentration in the cerebrum and midbrain of rats after 90 days of treatment with 2 mg/kg/day endosulfan, and in this study, spontaneous motor activity was significantly increased in the treated animals. Furthermore, they also found a correlation between the increase in serotonin and inhibition of a learning paradigm. Lakshmana and Raju (1994) also reported changes in the concentrations of dopamine, noradrenaline, and serotonin in various brain areas of endosulfan-treated rats. In this case, treated rats took 29 % more time to learn a behavioral task; however, it was not determined which neurotransmitter(s) change may have been responsible for the behavioral change.

PAN 2008 Endosulfan residues are also commonly found in human placental tissue, umbilical cord blood and breast milk, and endosulfan is transferred to the foetus and newly-born infant (Cerrillo et al 2005; Fukata et al 2005; Damgaard et al 2006; Torres et al 2006; Shen et al 2007, 2008; Pathak et al 2008). Residues of endosulfan in breast milk in Bhopal, India in 2003 were 8.6 times the average daily intake levels recommended by the World Health Organisation (Sanghi et al 2003). At least some of the residues in humans are believed to have resulted from the consumption of food containing residues of endosulfan (Campoy et al 2001; Sanghi et al 2003; Carreno et al 2007). Residues of endosulfan have been found in biota and in environmental media at locations far distant from where it has been released. It has been found in biota and ice in both the Arctic (Vorkamp et al 2004; Kelly et al 2007; Stern et al 2005) and

Antarctic (Miranda-Filho et al 2007). It has been found in grasses on Mt Qomolangma (Everest) region of the Tibetan Plateau (Wang et al 2007a) and in spruce needles of the Central Himalayan region (Wang et al 2006b); in lichen in the Canadian Rockies (Daly et al 2007a), and in lichen and conifers in the western national parks of the USA (Landers et al 2008). (ii) Endosulfan has been consistently measured in air all over the world, including in remote locations in the Arctic, high mountain areas in Asia (Himalayas), Europe, and North America, as well as tropical mountains in Costa Rica. Levels of endosulfan in the air are frequently amongst the highest of the pollutants measured. The Global Air Passive Sampling study resulted in a geometric mean value of 62 pg/m3 for total endosulfan, well above the next most abundant, PCPs with a mean of 17 pg/m3. In the polar regions endosulfan levels (2.0 pg/m3) were second only to PBDEs (3.7 pg/m3), and well above DDT (0.5 pg/m3), dieldrin (0.14 pg/m3) and the other POPs (Pozo et al 2006). It has also been consistently measured in precipitation: in snow in the Canadian Arctic (Tuduri et al 2006) and US national parks (Hageman et al 2006; Mast et al 2007), as well as in ice in the Italian Alps (Herbert et al 2004) and Antarctica (Deger et al 2003); and in rain in Asia (Kumari et al 2007), Africa (GEF SSA 2002), Europe (Carrera et al 2002; Quaghebeur et al 2004; Scheyer et al 2007), North America (Kuang et al 2003; Carlson et al 2004; Sun et al 2006; Tuduri et al 2006; Brun et al 2008), and Latin America (Laabs et al 2002). Residues of endosulfan in the Caribbean are believed to have resulted from deposition in dust carried from the African Sahara/Sahel region (Garrison et al 2006). Levels of endosulfan have continued to increase in the Arctic, in beluga (Braune et al 2005) and in air (NCP 2003) at the same time as the levels of most POPs have 3

declined. Similar increases have been observed in the freshwater fish char; residues were 2.2 times higher in 2002 than they were in 1992 (Evans et al 2005). (iii) Endosulfan is semi-volatile. It evaporates from the surface of soil and plants after application. Laboratory studies indicate 25-30% dissipates from the soil surface over 24hrs, and 64% from plant leaves (GFEA-U 2007). Field studies in Australia have found 70% of endosulfan is lost from cotton fields through volatilisation (Kennedy et al 2001; Sutherland et al 2004). The atmospheric half-life of endosulfan under experimental conditions is 27 days. Experimental measures have found a half-life of endosulfan of > 2.7 days and for beta endosulfan of > 15 days. Measurements at Alert in the Canadian Arctic have shown an atmospheric half-life there of 38 years for alpha endosulfan (Hung et al 2002, 2005). Conclusion: There is sufficient evidence that Endosulfan meets the criterion on potential for long-range environmental transport. Adverse effects Annex D screening criteria (i) Evidence of adverse effects to human health or to the environment that justifies consideration of the chemical within the scope of this Convention; or (ii) Toxicity or ecotoxicity data that indicate the potential for damage to human health or to the environment. Evidence (i) Endosulfan is one of the main causes of poisoning in humans in many countries (Kishi et al 2002; Oktay et al 2003; Roberts et al 2004; Wesseling et al 2005). Many deaths have resulted from occupational and accidental non-occupational exposure, as well as self-poisoning, in a number of countries in Africa, Asia and Latin America (PANNA 1999; EJF 2002; El Hindi et al 2006; GEF CAC 2002; Venkateswarlu et al 2000; Glin et al 2006; Mingxin 2007). Acute effects have also been reported in New Zealand and USA (ERMANZ 2007b; Associated Press 2007).

Chronic effects reported in humans include birth defects, congenital reproductive disorders, long-term brain damage, recurrent convulsions, epilepsy, autism, delayed sexual maturity, endometriosis, menstrual disorders, early menarche, male breast enlargement, various cancers, congenital intellectual disability, cerebral palsy, psychiatric disturbances, and vision impairment and loss (Aleksandrowicz 1979; Pradhan et al 1997; Quijano 2002; NIOH 2003; Saiyed et al 2003; Roberts et al 2007; Venugopal 2008). Many deaths in animalsincluding fish, wildlife, pets, and livestockhave also been reported, as well as congenital deformities, miscarriages, infertility, stunting of growth, and dwindling populations (PANNA 1996; Ton et al 2000; GEF CAC 2002; GEF SSA 2002; Quijano 2002; NIOH 2003; Schulz 2004; Glin et al 2006; ERMANZ 2007b). (ii) Toxicological data indicate that endosulfan is very toxic to mammals by skin contact, inhalation or ingestion (GFEA-U 2007). It causes a range of acute neurological 4 effects, including convulsions and death (ATSDR 2000). It damages the liver and kidneys and is toxic to the adrenal gland and pancreas (ATSDR 2000). It causes oxidative stress (Omurtag et al 2008). It is toxic to and suppresses the immune system (ATSDR 2000; Kannan et al 2000; Pistl et al 2003; Garg et al 2004; Lafuente et al 2006; Narita et al 2007). It depresses testosterone levels and may cause reproductive toxicity in humans (ATSDR 2000). It interferes with the steady state levels of oestrogen causing proliferation of MCF-7 human breast cancer cells, and its effects on the endocrine system indicate that endosulfan is likely to cause the onset and/or development of mammary tumours (Soto et al 1994, 1995; Bradlow et al 1995; Toniolo et al 1995; Berrino et al 1996; Dorgan et al 1996; Andersen et al 2002; Cossette et al 2002; Rousseau et al 2002; Grunfeld & Bonefeld-Jorgensen 2004;

Kojima et al 2004; Bonefeld-Jorgensen et al 2005; Wozniak et al 2005; Je et al 2005; Laville et al 2006; Lemaire et al 2006; Wong & Matsumura 2006; Chatterjee et al 2008). It targets the prefrontal cortex of the brain (Cabaleiro et al 2008), and may be implicated in Parkinsons disease (Wang et al 2006a; Jia & Misra 2007a). It causes adverse behavioural effects (ATSDR 2000). Exposure in utero causes teratogenic effects (Singh et al 2006). Many studies have shown it to be genotoxic and mutagenic in human cells, rodents, hamsters, fruit fly, fish, tadpoles, oysters, bacteria, microalgae and plants (Yadav et al 1982; Sobti et al 1983; Pandey et al 1990; Lu et al 2000; ATSDR 2000; Jamil et al 2004; Lajmanovich et al 2005; Bajpayee et al 2006; Neuparth et al 2006; Pandey et al 2006; Antherieu et al 2007; Perez et al 2007; Sharma et al 2007a; Wessel et al 2007; Akcha et al 2008; Menone et al 2008). It is also a tumour promoter (Fransson-Steen et al 1992; Dubois et al 1996; Warngard et al 1996; ATSDR 2000). Toxicity is increased with protein-deficient diets, which are a problem in some of the countries in which endosulfan is still used. Endosulfan is very toxic to aquatic organisms especially juveniles, and its use results in disruption of the aquatic food chain. Concentrations of endosulfan found in rivers greatly exceed the hazardous concentrations that adversely affect 5% of fresh water and marine organisms as identified in Bollmohr et al (2007). It is also toxic to amphibians, reptiles, snails, aquatic plants, coral reef organisms, birds, bees, earthworms, and beneficial insects and microorganisms, and is incompatible with IPM (Elzen 2001; Bostanian & Akalach 2004; Bastos et al 2006; Schneider et al 2006; Alizadeh et al 2007; Benam et al 2007). Conclusion: There is sufficient evidence that Endosulfan meets the criterion on adverse effects. Endosulfan is an insecticide still in widespread use in many countries, on crops like cotton, soy, coffee, tea and

vegetables, but also banned in 55 countries because of high toxicity to humans and nearly all other organisms, and its persistence in the environment. Human Exposure Exposure to endosulfan is high. Apart from occupational exposure which has resulted in many poisonings (see below), residues in food and drinking water are widespread globally at sufficiently high levels to constitute a threat to human health, and to result in consistent findings of human body burdens. Endosulfan contaminates breastmilk, adipose tissue, placental tissue and umbilical cord blood, meaning that the unborn child is exposed, and then re-exposed on birth through breast milkboth exposures taking place at critical periods of development where oestrogenic substances, such as endosulfan, can have a profound life-long impact. Acute toxicity The primary acute effect is on the nervous system, causing hyperexcitation and convulsions, and nervous system mediated effects on respiration and heart. Death results from low levels of exposure. Long-term toxicity Endosulfan is an endocrine disruptor. It is oestrogenic and antiandrogenic in human cells, and causes breast cancer cells to grow. It also interferes with male hormones, causing chronic depression of testosterone. It is toxic to and suppresses the immune system, as well as promoting allergic responses.

It is linked to long-term neurological effects such as epilepsy, and may cause Parkinsons disease. Birth defects have been seen in laboratory studies and in human populations exposed to endosulfan. Many studies show endosulfan to be mutagenic and genotoxic, and there is evidence of cancer in both laboratory animals and exposed human populations. Environmental effects Endosulfan is extremely toxic to fish and has caused massive fish kills. It is also highly toxic to all other aquatic organisms and demonstrates a range of chronic effects, including genotoxicity, reproductive and developmental effects. Toxicity is increased by increased temperatures: more problems can be expected with global warming. It is also highly toxic to birds, bees, earthworms, beneficial insects and microorganisms. Environmental contamination is widespread and has been found in soil, ground and surface waters, marine sediments, air, rainfall, snow and ice pack, grasses and tree bark all over the world-from areas in which it is still in use to high remote mountain lakes and the Arctic and Antarctic regions. Levels of contamination in aquatic systems are frequently high enough to be toxic to aquatic organisms. Endosulfan is persistent in the environment and biomagnifies in terrestrial food chains.

Alternatives There are many effective alternatives to endosulfan as evidenced by its having been banned in so many countries. For example, there is a thriving organic cotton industry particularly in Africa, replacing one of the main uses of endosulfan worldwide. Chemical Profile Common name Endosulfan Common trade name Thiodan Other related chemicals Technical grade endosulfan is a mixture of two isomers, alpha endosulfan (64-67%) and beta endosulfan (2932%) (GFEA-U 2007). Chemical names and form 6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9methano-2,4,3-benzodioxathiepin-3-oxide 6,9-methano-2,4,3-benzodioxathiepin-6,7,8,9,10,10hexachloro-1,5,5o,6,9,9-hexahydro-3-oxide It is a cream-to-brown coloured solid that may appear crystalline or in flakes, with a distinct smell like turpentine (ATSDR 2000). Molecular formula and structure C9H6Cl6O3S alpha endosulfan (endosulfan I):

H H Cl Cl Cl Cl Cl Cl O OS O beta endosulfan (endosulfan II): H H Cl Cl Cl Cl Cl Cl O O S O Chemical group Organochlorine, cyclodiene subgroup

CAS numbers Technical endosulfan 115-29-7 Alpha endosulfan 959-98-8 Beta endosulfan 3321-65-9 Endosulfan sulphate 1031-07-8 Trade names Trade names include: Agrisulfn, Afidan, Aikido, Akodan, Alodan, Axis, Benzoepin, Beosit, BIO 5462, Bromyx, Caiiman, Chlorbicyclen, Chlorthiepin, Crisulfan, Cyclodan, Cytophos, Devisulfan, Endel, Endocel, Endocide, Endocoral, Endocoton, Endofan, Endoflo, Endomight Super, Endopol, Endosan, Endosol, Endosulphan, Endotaf, Endoxilan, Enrofn, Ensure, ENT 23979, Flavylan 350E, FMC 5462, Galgofon, Galgptal, Global E, Goldenleaf Tobacco Spray, Hexasulfan, Hildan, HOE 2671, Insectophene, Isolan, Kop-thiodan, Lucasulfan, Malix, Misulfan, NIA 5462, Niagara 5462, Novasulfan, Palmarol, Parrysulfan, Phaser, Rasayansulfan, Red Sun, Rocky, SD-4314, Sharsulfan, Sialan, Sonii, Sulfan, Technufan, Thifor, Thimul, Thiodan, Thiofanex, Thiofor, Thioflo, Thiomet, Thiomul, Thionate, Thionex, Thiosulfan, Thiosulfax, Thiokil, Thiotox, Tionel, Tionex, Tiovel, Tridane, Termizol p, Veldosulfan, Vulcn, Zebra Ciagro.1 It is also found in mixtures with other insecticides, such as with methomyl in Methofan (MAI 2008), with cypermethrin in Callisulfan and chlorothalonil in Mistral

(Glin et al 2006); with deltamethrin in Decisdn; and with dimethoate, isoxathion, malathion, monocrotophos, pirimicarb, triazophos, fenoprop, parathion, petroleum oils, and oxine-copper (El Hindi et al 2006; GFEA-U 2007). It is available as emulsifiable concentrate, wettable powder, ultra-low volume liquid, and smoke tablets (GFEA-U 2007). Inerts and contaminants Inerts may include unidentified alcohol solvent emulsifiers, petroleum distillate emulsifiers, suspension agents, talc, and wetting agents (ATSDR 2000). Since these components are unidentified, their contribution to the hazardous effects of formulated endosulfan products is unknown. The Environmental Risk Management Authority of New Zealand (ERMANZ 2007b) identified common impurities in technical grade endosulfan as endosulfan alcohol, endosulfan ether, hexchlorocyclopentadiene, and residual solvent. Endosulfan is also contaminated with pentachlorobenzene (PeCB). This chemical is a Persistent Organic Pollutant (POP) (still undergoing evaluation). It is persistent, highly acutely toxic to aquatic organisms and acutely toxic to humans, and is fetotoxic (POPRC 2007). Epichlorohydrin is reported to have been used as a

stabiliser in technical grade endosulfan and it is not known if it is still used. Epichlorohydrin is mutagenic and carcinogenic in animals, and possibly carcinogenic in humans. It causes sterility in male rats, and damages the central nervous system, respiratory tract, liver, blood, eyes, and skin (ATSDR 2000). At least one study has found a formulated product (Technufan) to be more toxic than the technical grade endosulfan, causing damage to proteins specific to pulmonary cells in humans (Skandrani et al 2006). Metabolites The principle metabolite is endosulfan sulphate. Other metabolites include endosulfan diol, endosulfan ether, endosulfan hydroxy carboxylic acid, endosulfan hydroxyether, and endosulfan lactone (GFEA-U 007). Endosulfan sulphate is usually included with the alpha and beta isomers of endosulfan as total endosulfan, or endosulfan (sum) in measurement of residues. The sulphate is regarded as being equally toxic and of increased persistence in comparison with the parent isomers (US EPA 2007a). Mode of Action in Insects Endosulfan is a non-systemic insecticide and acaricide with contact and stomach action (Kidd & James 1991). Uses Endosulfan is used to control a wide range of sucking and

chewing insects including aphids, thrips, beetles, foliar feeding caterpillars, mites, borers, cutworms, bollworms, bugs, whiteflies, leafhoppers, snails in rice paddies, earthworms in turf, and tsetse flies. Major crops to which it is applied include soy, cotton, rice, and tea. Other crops include vegetables, fruit, nuts, berries, grapes, cereals, pulses, corn, oilseeds, potatoes, coffee, mushrooms, olives, hops, sorghum, tobacco, and cacao. It is used on ornamentals and forest trees, and has been used in the past as an industrial and domestic wood preservative (ERMANZ 2007b; GFEA-U 2007; Kidd & James 1991). Endosulfan is reported to be one of the most commonly used pesticides in India in recent years, particularly on rice and cotton against thrips, stem borer, whorl maggot, case worm, boll worm and bud worm (Jayashree & Vasudevan 2007b). It is used to control ectoparasites on farm animals and pets in Ghana (Darko & Acquaah 2008). It is also used on sand-based sports fields in New Zealand to kill earthworms and prevent formation of worm casts (Tasman District Council 2008) and there are anecdotal reports that it is also used on the grass strips between aircraft runways again to kill earthworms, which might encourage the presence of birds. In May 2007 Codex Alimentarius cancelled maximum

residue limits (MRLs) for endosulfan in a number of crops including apple, broad bean, cabbage, carrot, cauliflower, common bean, cotton seed oil, garden peas, grapes, lettuce, maize, onion, orange, peach, pineapple, plums, rape seed, rice, spinach, sugar beet, sunflower seed, and wheat. These uses are no longer supported. However they adopted MRLs for avocado, broccoli, cacao beans, celery, cherries, coffee beans, cottonseed, cucumber, custard apple, eggplant, hazelnuts, litchi, macadamia nuts, mango, melons, papaya, persimmon, potato, soybean, squash, sweet potato, and tomato. Due to pressure from India and China MRLs for tea will continue for 4 years (Codex 2007 India is regarded as being the worlds largest producer and user (Li & Macdonald 2005), with more than 60 endosulfan manufacturers and formulators, mostly the latter (WebIndia 2007). Absorption and distribution In animals absorption through the gastrointestinal tract is very rapid and efficient, with >90% absorbed in rats. Absorption through the skin has been as high as 50% in rats (GFEA-U 2007). Endosulfan is readily metabolised in animals by microsomal enzymes, initially to endosulfan sulphate and endosulfan diol; and excreted in urine and faeces. It has been found distributed to adipose, liver, kidney, heart,

spleen, testes, epididymis, prostate, seminal vesicle, milk and muscle. There is a greater accumulation in adipose tissue of females than males, possibly because of more rapid excretion by males. One study in rats concluded that it is rapidly eliminated from the body after ingestion ceased, with an estimated half-life of approximately 7 days (ATSDR 2000). Mode of action in mammals Endosulfan has an affinity with the GABA (gammaaminobutyric acid) receptors in the brain and acts as a non-competitive GABA antagonist. Binding of GABA to its receptors induces the uptake of chloride ions by neurons and blockage of this uptake by endosulfan results in a state of uncontrolled excitation (UNEP/FAO 2007). Acute toxicity Tests are generally carried out using technical grade endosulfan, which consists of both alpha and beta endosulfan. Alpha endosulfan is about 3 times more toxic than beta (ATSDR 2000). US EPA (2002) toxicity categories: oral category I: highly toxic inhalation category II: moderately toxic dermal category III : slightly toxic World Health Organisation Recommended Classification by Acute Hazard (WHO 2005): Class II moderately hazardous.

UNEP/FAO (2007) describes endosulfan as being highly toxic through oral or dermal exposure. Lethal doses Lethal dose, LD50, is the dose that kills 50 % of test animals. LD50s for endosulfan are: Oral LD50 rat = 10- 23 mg/kg (female); 48-160 mg/kg (male) Dermal LD50 rat = 500 mg/kg (female); >4000 mg/kg (male) Inhalation LC50 rat = 0.0126 mg/l (female); 0.0345 mg/l (male). (GFEA-U 2007) Acute effects The primary effect of acute exposure is on the nervous system. Inhalation: ataxia, hyperexcitability, trembling, convulsions, reduced reflexes of the cornea, pupil and skin; irregular respiration, and lung lesions (ATSDR 2000). Oral: hyperactivity and hyperexcitation, seizures, tremors, convulsions, clonic spasms, brain oedema, salivation, dilated pupils, poor muscle tone, head swaying, vomiting, diarrhoea, rapid respiration, decreased respiration, noisy breathing, shortness of breath, lung congestion, emphysema, cyanosis, respiratory paralysis, haemorrhage of heart muscle, lung and kidney; death (ATSDR 2000). Dermal: shortness of breath, increased respiratory rate,

rapid heart rate, decreased heart rate, nausea, vomiting, abdominal pain. Endosulfan has also caused death of livestock (calves) and laboratory animals through a single dermal exposure (ATSDR 2000). Skin and eye irritation Skin: mild irritation in rats, including erythema, slight oedema and dry rough scaling skin (ATSDR 2000). Eyes: category I irritant, causing residual opacity (US EPA 2002) No and Lowest Observed Adverse Effects Levels The No Observed Adverse Effects Level (NOAEL) is the lowest dose of the chemical given to a test animal at which no effects are observed, and the Lowest Observed Adverse Effects Level (LOAEL) is the lowest dose of the chemical given to a test animal at which an effect is observed. NOAEL (acute oral, rat) = 1.5 mg/kg/day (US EPA 2002) NOEL (acute oral, rabbit) = 0.7 mg/kg/day (CAL EPA 2007) LOAEL (acute oral, rat) = 3 mg/kg/day, causing convulsions in female rats within 8 hours of dosing (US EPA 2002). However ATSDR (2000) reported the lowest LOAEL as 1.8mg/kg/day causing convulsions in pregnant rabbits after 10 days of force-feeding endosulfan.

Sub-chronic toxicity / intermediate The sub-chronic NOAELs and LOAELs are: NOAEL = 3.85 mg/kg/day (male rat), 2.3 mg/kg/day (mice) LOAEL = 23.41 mg/kg/day (male rat), 7.4 mg/kg/day (mice) (ATSDR 2000) LOAEL (short-term dermal) = 3.74 mg/kg/day (rat) (US EPA 2007b) NOAEL (short-term inhalation) = 0.2 mg/kg/day (rat) (US EPA 2002, 2007a) Systemic effects The primary systemic targets are the liver and kidney, but it also causes haematological and respiratory effects as a result of generalised effects on the central nervous system. Observed effects on rats include: inflammation of lungs; congestion and degeneration of kidney tubules renal necrosis, renal haemorrhage; congestion and necrosis of the liver; blood in small intestines, diarrhoea; decreased haemoglobin and red blood cell count in protein deficient rats; damage to the membrane of red blood cells, increasing permeability and impairing enzyme

activity, even at very low doses (1 ug/kg) that are 500-fold lower than the generally permissible level for residues in food of 500 ug/kg; calcification of heart and coronary and mesenteric arteries, aneurisms, cardiotoxicity through oedema and swelling of myocardial cells, heart and circulatory failure; reduced body weight gain, reduced appetite; and hyperglycaemia, more marked in older animals (Daniel et al 1986, ATSDR 2000; Choudhary et al 2003). Endosulfan is toxic to both the adrenal gland and the pancreas, causing degranulation of the beta cells of the islets of Langerhans in the later (ATSDR 2000). The resulting effects on blood glucose levels are complex: On the one hand it is toxic to adrenocortical cells in fish (at low doses that do not decrease cell viability) and impairs their production of cortisol (Bisson & Hontela 2002; Dorval et al 2003). Cortisol is antagonistic to insulin and promotes the breakdown of glycogen, which then increases blood sugar levelsso in this instance endosulfan acts to reduce blood sugar levels by reducing cortisol. On the other hand, in the pancreas of rats it is toxic to the B cells of the islets of Langerhans, which produce the insulin necessary to reduce blood sugar

levels, so in this case it acts to raise blood sugar levels (Kalender et al 2004). Endosulfan causes oxidative stress (Omurtag et al 2008), which is implicated in its neurotoxic effects (Jia & Mizra 2007a,c), damage to the adrenal gland (Dorval & Hontela 2003; Dorval et al 2003), and in cancer (Antherieu et al 2007). The brain is particularly sensitive to oxidative damage from sublethal levels of endosulfan, at least in fish (Ballesteros et al 2008). Chronic toxicity The chronic NOAEL and LOAEL are: NOAEL = 0.57 mg/kg/day (female dog), 0.67 mg/kg/day (male dog) LOAEL= 1.75 mg/kg (dog) Immune system The immune system is more sensitive, reacts more rapidly, and at lower doses, to the effects of pesticides than other organ systems, so effects on this system can indicate subclinical toxic states (Pistl et al 2003). A number of studies show that endosulfan is toxic to, and suppresses, the immune system, as well as promoting allergic responses. Endosulfan induces immunosuppressive effects decrease in humoral antibody and cell-mediated immune responseat low dose levels that do not induce any other sign of toxicity; and decreases macrophage function

(ATSDR 2000). The ATSDR (2000) concluded humans may be at risk of adverse immune effects following exposure to endosulfan. Effects include decreased serum IgG levels, decreased antibody titer to tetanus toxin, inhibition of leukocyte and macrophage migration, and increased albumin-to-globulin ratio (ATSDR 2000; Abadin et al 2006). It was also immunosuppressive and immunotoxic in sheep blood cells, causing significant suppression of phagocytes, decreased activation of lymphocytes, and decreased migration of leukocytes (Pistl et al 2003). In broiler chicks, endosulfan reduced total leucocytes, T-lymphocytes, macrophages, and caused atrophy and haemorrhage of the thymus gland (Garg et al 2004). Endosulfan is highly immunotoxic to fish, causing suppression of phagocytic function of the head kidney immune cells of Australian freshwater fish (Harford et al 2005). Exposure to endosulfan in utero and through postnatal lactation resulted in immunosuppressive effects on adult rats, with female rats being more susceptible than males (Lafuente et al 2006). Endosulfan promotes allergic disease in humans by enhancing mast cell degranulation: it caused rapid, doserelated release of hexosaminidase (a marker for the granules that contain preformed allergic mediators) from human mast cells, and enhanced IgE-mediated release

(Narita et al 2007). Endosulfan also induces the death of human Natural Killer T-cells, which are part of the immune system involved in tumour suppression (Kannan et al 2000), hence endosulfan assists the development of tumours. Endocrine disruption Endosulfan is an endocrine disruptor in mammalian species, and in fish, birds and amphibians, affecting both male and female reproductive hormones. In male rats endosulfan caused a dose-related decrease in testosterone, luteinizing hormone and follicular stimulating hormone, with depressed activity of steroidogenic enzymes and testicular cytochrome P450-dependent monoxygenases. The testosterone remained chronically depressed, but the other effects were reversible. ATSDR (2000) concluded that endosulfan may potentially cause reproductive toxicity in humans. Studies of the effects on female hormone levels in animals are inconsistent. However studies on human cells indicate that endosulfan is oestrogenic, interfering with the steady state levels of oestrogen receptors (Grunfeld & BonefeldJorgensen 2004); and causing proliferation of MCF-7 human oestrogen-sensitive breast cancer cells (Soto et al 1994, 1995; Bonefeld-Jorgensen et al 2005), and oestrogen-sensitive ovarian cells (Wong & Matsumara 2006). The simultaneous exposure of breast cancer cells to

endosulfan and growth factors increased the oestrogenic effect of endosulfan on cell growth (Cossette et al 2002). It activates the oestrogen receptor (ER) alpha but weakly antagonises ER beta. This increases its harmful action: because ER beta can oppose the cell proliferation effects of ER alpha, antagonising ER beta increases the risk of cell proliferation (Lemaire et al 2006). Other studies have demonstrated the oestrogenmimicking effect of endosulfan on enzymes in pituitary tumour cells (Bulayeva & Watson 2004) and in breast cancer cells (Li et al 2006a), which subsequently leads to cell proliferation, transformation, differentiation and migration. In Bulayeva & Watsons study, the oestrogenic effect of endosulfan showed a bimodal response: it occurred at very low subpicomolar levels and at higher levels, but not medium levels, a similar response pattern to that of the natural hormone oestradiol. As well as mimicking oestrogen, endosulfan is also antiandrogenic (Andersen et al 2002; Kojima et al 2004; Chatterjee et al 2008). It also induces the activation (Hunter et al 1999) and proliferation of progesterone receptorsanother oestrogen-mimicking effectin human breast cancer cells (Soto et al 1995), and it decreases the activity of progesterone (Jin et al 1997; Chatterjee et al 2008). Exposure to endosulfan at very low concentrations may

cause breast cancer by interfering with a number of hormonal mechanisms: It has recently been found to activate aromatase in cancer cells in the human placenta at low concentrations that do not cause cytotoxicity (Laville et al 2006); aromatase is the enzyme that catalyses conversion of androgens to oestrogen. It significantly increases the ratio of 16-hydroxyestrone (a tumour promoting oestrogen) to 2-hydroxyestrone (non-genotoxic), increasing breast cancer cell proliferation, development, and promotion (Bradlow et al 1995). It potentiates 17beta-estradiol (Andersen et al 2002), the primary natural oestrogen, elevated levels of which are strongly linked to breast cancer (Toniolo et al 1995; Berrino et al 1996; Dorgan et al 1996). It causes changes to intracellular oestrogenic signalling that increase the risk of breast cancer at very low concentrations (e.g. 10-10 M)by mimicking the effects of oestrogen and raising cellular calcium levels, which then cause rapid secretion of prolactin which in turn causes cell proliferation (Rousseau et al 2002; Wozniak et al 2005; Watson et al 2007). Endosulfan interferes with mammary gland development increasing the number of alveolar budsby increasing telomerase reverse transcriptase mRNA transcriptional

activity (Je et al 2005). Endosulfans ability to act as a xeno-oestrogen may also cause it to contribute to cervical cancer and endometriosis, which are regarded as being oestrogen dependent disorders (Lemaire et al 2006; Foster & Agarwal 2002). Nervous system Endosuflan targets the prefrontal cortex of the brain, which is involved in cognitive tasks, selective attention, shortterm working memory, response inhibition, behavioural flexibility, sexual and maternal behaviour, and depression (Cabaleiro et al 2008). Endosulfan changes the brain levels of the neurotransmitters dopamine, noradrenalin and serotonin (ATSDR 2000). In particular, endosulfan blocks the receptors for the GABA neurotransmitter in nerve cells (Vale et al 2003). GABA neurotransmission is important in gestational brain development (Roberts et al 2007). It also has age-related effects on other amino acids in the prefrontal cortex of the brain (Cabaleiro et al 2008). There is emerging evidence that exposure to endosulfan may increase the risk of Parkinsons disease. Mice exposed to endosulfan from postnatal days 5 to 19 exhibited only insignificant changes in dopamine, acetylcholinesterase and alpha-synuclein levels; however when re-exposed as adults they showed significantly depleted dopamine, increased levels of alpha-synuclein

and increased acetylcholinesterase activity in the brain (Jia & Misra 2007a). Endosulfan, at low concentrations (10nmol/l-10umol/l) also inhibits proteasome activity which degrades proteins like alpha-synuclein (Wang et al 2006a). The loss of dopamine and the accumulation of alpha-synuclein are both associated with Parkinsons disease. Low doses of endosulfan reduced the threshold for seizures produced by electrical stimulation in rats (ATSDR 2000). Endosulfan can cause behavioural effects such as aggression and increased time to learn tasks, even after exposure has ceased; and impaired learning and memory processes, extreme sensitivity to noise and light, and muscle spasms (ATSDR 2000). Reproductive & developmental effects LOEL (repro) = 6.2 mg/kg/day (rat) NOEL (devel) = 2.0 mg/kg/day (rat) LOEL (devel) = 6.0 mg/kg/day (rat) (ATSDR 2000) Endosulfan has caused a number of adverse effects on male reproductive parameters in rats, reducing fertility: degeneration of seminiferous tubule epithelium, reduced sperm count, altered spermatogenesis, increased abnormal sperm, testicular necrosis, and aspermatogenesis (Dalsenter et al 1999; ATSDR 2000;

Sinha et al 2001). Effects were reported to be greater if exposure occurred during the developmental phase (Saiyed et al 2003). Adverse effects on male offspring have occurred even at dose levels that were not toxic to the mother (Sinha et al 2001). ATSDR (2000) concluded that, for humans, exposure during the period of testicular maturation may result in disturbed spermatogenesis at sexual maturity (ATSDR 2000). Endosulfan has also been shown to reduce implantation in female mice, and to increase oestrus (Hiremath & Kaliwal 2002). Exposure in utero to endosulfan has caused embryotoxic effects in animal studies including increased resorptions and skeletal variations, decreased birth weight and length, and increased aggressive behaviour in newborn rats, but the findings are regarded by the ATSDR (2000) as inconclusive. More recently Singh et al (2006) reported similar teratogenic effects, as well as accumulation of cerebrospinal fluid in the brain, underdeveloped cerebrum, incomplete ossification of skull bones, and malformations of the liver, kidneys, ribs and renal pelvis. A study with human sperm in vitro showed that low concentrations of endosulfan (0.1nM) strongly inhibited the ability of sperm to fertilise ova (ATSDR 2000). Endosulfan disrupts the retinoid signalling pathway in

cells, and this is thought to explain the teratogenic effect of long-term exposure to low levels of the chemical, as has been experienced in India (congenital heart and skeletal abnormalities). Retinoids play an essential role in the proliferation, development and differentiation of cells and disruption can lead to malformation or abnormal development of the eye, brain, heart, and limbs (Lemaire et al 2005). Genotoxicity / mutagenicity Studies have yielded inconsistent results leading ATSDR (2000) to conclude it is not genotoxic in rats. However, endosulfan has caused mutagenic and genotoxic effects in human lymphocytes and liver hepatoblastoma cells; in rat and mouse spermatogonial cells; in rat, mouse and hamster bone marrow; in rat foetal liver cells; in fruitfly; in fish gill, kidney and erythrocyte cells; in tadpoles; in oysters; in Chinese hamster ovarian cells; in bacterial systems (Salmonella, E. coli and Saccharomyces); in microalgae; and in the root tip cells of the wetland macrophyte Bidens laevis L (Yadav et al 1982; Sobti et al 1983; Pandey et al 1990; Lu et al 2000; ATSDR 2000; Jamil et al 2004; Lajmanovich et al 2005; Bajpayee et al 2006; Neuparth et al 2006; Pandey et al 2006; Antherieu et al 2007; Sharma et al 2007a; Wessel et al 2007; Akcha et al 2008). The effects include formation of DNA adducts, DNA strand breaks, sister chromatid

exchange, micronucleus induction, chromosomal aberrations and gene mutations. Of the 27 studies reported by the ATSDR, 17 were positive for genotoxic effects. Sister chromatid exchange was increased at least 5-fold in rat embryos. ATSDR (2000) described endosulfan as an efficient mutagen in Drosophila (fruit fly), and concluded that the data showed endosulfan to be mutagenic and clastogenic, although some of this effect may be caused by the stabiliser epichlorohydrin used in some formulations. In a first attempt to test endosulfans genotoxicity in plants, Perez et al (2007) found it to be genotoxic to the wetland macrophyte Bidens laevis L (bur marigold, beggars tick) at environmentally relevant concentrations. It interacted with the mitotic spindle at concentrations of only 5 ug/l, well below the NOAELs reported for chronic toxicity (0.57, 0.67 mg/kg/day). In microalgae it caused DNA strand breaks at concentrations of only 1 ug/l (Akcha et al 2008). Both these findings of genotoxicity occurred at doses well below concentrations found in the aquatic environment (run-off in Southern USA has contained concentrations as high as 100 ug/l, in India up to 66.5 ug/l, and in Australia up to 45 ug/l (Menone et al 2008)). GFEA-U (2007) concluded that there is evidence of mutagenicity, clastogenicity and effects on cell cycle kinetics.

Cancer Evidence of the carcinogenicity of endosulfan is regarded as being inconclusive. Some studies have found an increase in the total number of malignant tumours and pulmonary adenomas, and increases in total number of carcinomas, hepatic carcinomas, and sarcomas in female rats, and lymphosarcomas in male rats (e.g. Reuber 1981). However the poor quality of the studies precludes firm conclusions according to ATSDR (2000). Alpha endosulfan is a tumour promoter causing a significant and dose-related increase in hepatocytes (liver cancer cells) (Fransson-Steen et al 1992; ATSDR 2000), and to rapidly inhibit gap junctional intercellular communication (GJIC) in liver cells (Dubois et al 1996; Warngard et al 1996). The tumour promoting effect is suggested to be through the inhibition of GJIC (ATSDR 2000). Endosulfan has not been classified by the International Agency for Research on Cancer (IARC) as a carcinogen, and was described by the International Programme on Chemical Safety (IPCS) (2000) as not carcinogenic, but it is increasingly being described as a potential carcinogen in humans (Antherieu et al 2007). The authors of this recent study found that it generates reactive oxygen species causing oxidative stress, and that this results in endosulfan having mutagenic effects and causing

increased DNA strand breaks. It was found to inhibit apoptosis, which could contribute to mutant cell survival and therefore have possible carcinogenic effects. Sohn et al (2004) demonstrated that endosulfan induces oxidative stress and inhibits cellular respiration via the generation of reactive oxygen species. Oxidative stress is also described in the section on Systemic Effects. Other effects Endosulfan has been shown to be a nonspecific inducer of drug metabolism by inducing microsomal enzyme activity (ATSDR 2000). Toxic interactions Alcohol may interfere with the metabolism of endosulfan delaying its elimination from the body and increasing its toxic effect (ATSDR 2000). There is also a suggestion that dimethoate and endosulfan act synergistically in human poisoning (ATSDR 2000). Endosulfan in combination with methyl parathion significantly increased adverse effects on rat behaviour compared with dosage of either of the pesticides alone, even at doses that did not cause overt toxicity, or kidney or liver pathology (Castillo et al 2002). Endosulfan and zineb in combination exhibited significantly higher toxicity to human cells than either by itself (Jia & Misra 2007b). Endosulfan increases the potency of the drug diazepam

(ATSDR 2000). Endosulfan increases the convulsant action of picrotoxin, a poisonous alkaloid in a fruit from Southeast Asia known as ligtang, aria, bayat, Indian berry or Levant nut (ATSDR 2000). Gender differences Female rats are 4-5 times more sensitive to the lethal effects of endosulfan than male rats. There is a lack of data to determine if this is also true of other species (ATSDR 2000). The difference is attributed to differences in metabolism of endosulfan. Sensitive populations A protein deficient diet caused a 20-fold increase in sensitivity to the lethal effects of endosulfan in rats. It also enhances the anaemia-inducing capacity of endosulfan (ATSDR 2000). Increased mortality also occurred in pregnant rats (ATSDR 2000). The foetus and newborns are more sensitive than adults at least for neurotoxic effects (ATSDR 2000). The ATSDR (2000) lists the following groups of people who may be particularly vulnerable to the effects of endosulfan: people with liver or kidney disease, pre-existing anaemia or haematologic disorders, or neurological problems especially seizure disorders such as

epilepsy; people with compromised immune systems such as AIDS/HIV patients, infants and the elderly; and people with protein-deficient diets such as the malnourished poor, chronic alcoholics, dieters, and the elderly. It is possible that diabetics may also be at increased risk from endosulfan, given its complex effects on blood glucose levels including the ability to significantly elevate them (Kalender et al 2004). Additionally endosulfan, in a mixture with hexachlorocylclohexane (HCH) and monocrotophos, was more toxic to diabetic rats and especially those that were also malnourished than to healthy rats (Benjamin et al 2006). Human Exposure Exposure guidelines EU: ADI (acceptable daily intake) = 6 ug/kg bw ARfD (acute reference dose) = 15 ug/kg bw (UNEP/FAO 2006b) US: Minimum Risk Level (acute, oral) = 5 ug/kg/day Minimum Risk Level (chronic, oral) = 2 ug/kg/day (ATSDR 2000) Occupational exposure In 2007, the US EPA concluded that occupational

assessment for endosulfan indicates short- and intermediate-term risks for mixers, loaders, and applicators for the majority of uses, even with maximum Personal Protective Equipment (PPE) and engineering controls (US EPA 2007a). 10 Protective clothing requirements in the USA are a coverall over long-sleeved shirt and long pants, chemical resistant footwear plus socks, chemical resistant gloves, chemical resistant head gear when exposed overhead and a respirator (US EPA 2002). Occupational exposure has resulted in residues of endosulfan and its metabolites in the blood of agricultural workers who sprayed endosulfan in greenhouses in Spain (Arrebola et al 2001), and in residues in the male offspring of women agricultural workers in Spain (Carreno et al 2007), as well as numerous fatalities and severe adverse health effects globally (see Symptoms and Consequences of Poisonings and Cases of Poisonings). Non-occupational exposure Non-occupational exposure is very common. In France endosulfan was found to be a ubiquitous air pollutant in residences in the Paris area, found in 79% of homes, and in some of them at levels higher than those found in greenhouses. At this stage (2006) endosulfan was still permitted for use in France but only on some fruit and

vegetables, and its presence in homes appeared to result from drift and contaminated plant matter. It was also found, on the hands of 20% of the general population sampled in Paris (Bouvier et al 2006). Reuse of containers Reuse of containers for storing food, water, milk, oil, etc, or as drinking vessels, has caused a number of poisonings. For example, in Benin an 8 year old died after using a discarded Callisulfan container to scoop drinking water from a canal (Glin et al 2006). Contaminated clothing Contaminated work clothing can be a problem; for example in Benin 4 children died after work clothes left on the roof of a house after spraying were exposed to overnight rain that leached out the endosulfan into the vessel used for drinking water (Glin et al 2006). Residues in food and drink Non-occupational exposure frequently occurs as a result of residues in food and drinking water. Such residues are commonplace where endosulfan is used, and are of such magnitude in some countries as to constitute a significant risk to health. Residues also contribute to the body burden of endosulfan. Campoy et al (2001) found a positive correlation between intake of contaminated vegetables and the presence of endosulfan-lactone in breast milk in Spain. Deaths have been reported from consuming

contaminated fish after East African fishermen used endosulfan for catching it (GFEA-U 2007); and two people are reported to have suffered neurological symptoms are consuming food contaminated with endosulfan in Turkey (Oktay et al 2003). For residues throughout this document, unless otherwise specified, the values given are for total endosulfan i.e. alpha + beta + the sulphate. Otherwise, AE = alpha endosulfan; BE = beta endosulfan, ES = endosulfan sulphate Endosulfan contamination of vegetables and fruit is widespread. It was one of the most commonly detected residues in the 5000 most widely consumed foods in the USA, and in fruit and vegetables in Europe (Campoy et al 2001). Between 2001 and 2006 it was found in oil, strawberries, peppers, celery and cucumber in Barcelona, Spain (Fontcuberta et al 2008); in red pepper and eggplant from Italy; and in vegetables and grapes from Cyprus (GEF M 2002). An analysis of organic waste material in Germany found high concentrations in lettuce and smaller amounts in tropical fruit peels and flowers (Taube et al 2002). In New Zealand 32% of tomatoes sampled contained endosulfan with levels up to 972 ug/kg (NZFSA 2007). It has also been found in capsicum, courgette, cucumber, pears, vegetable oil, salad dressing, peanuts and peanut butter. Eating only one tomato per day, without the other

foods, results in a daily intake of approximately 81 ug of total endosulfan, or 1.18 ug/kg of body weight for the average woman (68.7 kg), for one out of three women. Endosulfan intake is likely to be higher than this given the residues in other foods. Subclinical adverse effects could result from this level of intake considering that dose levels of only 1 ug/kg have caused damage to red blood cells, increasing permeability and impairing enzyme activity in rats (Daniel et al 1986), and 0.5 ug/l has caused DNA damage in fish (Sharma et al 2007a). In India, all samples of cauliflower, brinjal and okra taken at Ranchi, Jharkhand were found to contain endosulfan at levels as high as 2,470 ug/kg (Shahi et al 2005); and almost all samples of the fruits ber, grapes and guava (Kumari et al 2006). It is also found in rice (Jayashree & Vasudevan 2007b) and mangoes (Singh et al 2008a) in India. It has been found in sesame seeds, groundnuts, chilli powder in India; and in canned pineapple, chillies and onion in Sri Lanka (GEF IO 2002). 100% of 28 samples of a complete daily vegetarian diet, taken in Hisar City, were contaminated with endosulfan, with residues up to 354 ug/kg (Kumari & Kathpal 2008). It has been found in Ghana, in 36% of lettuces at up to 1,300 ug/kg, which is nearly 3 times the MRL (Amoah et al 2006). It was found in pekmez, the traditional grape molasses of Turkey (Erdogrul 2007b). It has been found

in vegetables in El Salvador and Colombia (GEF CAC 2002), Argentina, and in tomatoes in Brazil (Araujo et al 1999). Endosulfan has even been found in leeks that were not treated with the pesticide, efficiently removing it from the soil (Gonzalez et al 2003). In fact the authors suggested that leeks may be grown as a means of decontaminating the soil. Endosulfan has been reported to contaminate animal feed (Deka et al 2004), and to be excreted in goats milk when goats ingest it even up to 20 days after ingestion ceases (Nag et al 2007). It has been found in 27% of cows milk samples in Bundelkhand (Nag & Raikwar 2008) and Haryana in India (Sharma et al 2007b), Brazil (Ciscato et al 2002), Colombia (GEF CAC 2002), and Argentina (Maitre et al 1994); in butter with 11% of samples above 11 the MRL in Haryana (Kumari et al 2005); and in butter in Turkey (Nizamlioglu et al 2005). It has recently been found in fresh milk (0.12 ug/kg), yoghurt (0.34 ug/kg), and cheese (9.06 ug/kg) in Ghana (Darko & Acquaah 2008). Endosulfan has also been found in meatin Australian beef at levels up to 4 times the MRL (Campoy et al 2001) as a result of cattle consuming cotton waste (Agrow 1996; NRA 2000), and in New Zealand beef exported to Korea as a result of illegal use on cattle to control ticks (Collins

2005). It has also been found in lamb and pork in Spain (Garrido Frenich et al 2006), and in goat and chicken in India (Singh et al 2008b). Endosulfan has been found in 4.2% of samples of seafood products from Southern China (Guo et al 2007), in cockles, oysters and mussels from coastal lagoons in Ghana (Otchere 2005), mussels in India (GEF IO 2002), and cockles, mussels and fish in Malaysia. It has also been found in freshwater fish in a number of countries including Australia (Nowak 1990), India (Kole et al 2001; Singh & Singh 2007), USA (Hinck et al 2008), Zambia (Syakalima et al 2006), Benin and Kenya (Pazou et al 2006a, 2006b), Tanzania (Henry & Kishimba 2006), Nigeria (GEF SSA 2002), and Uganda (Kasozi et al 2006). It has been found in all trout sampled from the Great Lakes of USA and Canada (GEF NA 2002), in fish from remote lakes of USAs western National Parks (Ackerman et al 2008), and in estuarine fish in Argentina (Lanfranchi et al 2006). It has been found in farmed prawns in India (Amaranemi 2006), and in shrimps and oysters in Jamaica (GEF CAC 2002). Endosulfan has been found in honey in Turkey (Erdogrul 2007a), and in 23.4% of beeswax sampled from honeybee colonies in France (Chauzat & Faucon 2007). It has been found in every sample of wine corks analysed in the USA (Strandberg & Hites 2001), and was the most

frequent residue in olives and olive oil sampled in Spain (Guardia Rubio et al 2006). It was recently found in herbal drug materials in Korea (Oh 2007), and in infant formula in Spain (Mezcua et al 2007). Endosulfan has been found in 22% of samples of cottonseed in Punjab, India (Blossom & Singh 2004); and in cottonseed in Pakistan (Parveen 1993). It has been found in mustard oil and vegetable oil in India (GEF IO 2002). Endosulfan has also been found commonly in drinking water, including in the Philippines (Bouman et al 2002), Morocco (El Bakouri et al 2007), India (Shukla et al 2006; Kumari et al 2008; Jayashree & Vasudevan 2007a), Pakistan (Tariq et al 2004), USA (US EPA 2002), the Beijing Guanting reservoir in China (Xue et al 2005; Xue & Xu 2006), Colombia at concentrations of 116.6 ug/l (GEF CAC 2002), and in household water supplies in a soy growing area near Cordoba, Argentina (Rulli 2006). Levels of 1.85 ug/l were found, in 2003, in water samples from the Selangor River in Malaysia, which is used for drinking purposes (Leong et al 2007). In Sri Lanka endosulfan has been found throughout the tea ecosystem: in fresh tea leaves (1475 ug/kg), made tea (446 ug/kg), the soil (1058 ug/kg) and water bodies (27 ug/kg) (Bishnu et al 2008). Tobacco

As endosulfan is used on tobacco in some countries, and residues have been found in both tobacco and cigarettes (Papadopoulou-Mourkidou & Milothridou 1990), exposure is also likely to occur through the smoking or chewing of tobacco that has been sprayed with endosulfan. In utero Findings of endosulfan and its metabolites in both maternal and umbilical cord sera indicates that these chemicals pass though the placental barrier and the foetus is exposed in utero (Torres et al 2006), and this may have critical effects on physical development and cognitive functioning of the child (Shen et al 2007). Pathak et al (2008) found residues of endosulfan in 60% of pregnant women in their survey in Delhi, with levels of 6.9 ug/ml in maternal blood and 5.9 ug/ml in cord blood, with the mean values (3.7 and 2.27) indicating a 60% transfer from mothers to newborns. They expressed great concern about this high rate of transfer and the consequent effects on the growth and development of the infant. Carreno et al (2007) said the source of exposure in their study on residues in young men appeared to be consumption of contaminated food, mainly fruit and vegetables or contaminated water; however they also found an association between maternal employment in agriculture during pregnancy and serum levels of endosulfan sulphate in the males aged 18 to 23 years,

indicating maternal transfer. Through breast milk Endosulfan and its metabolites are commonly found in breast milk (see next section), hence the exposure to the newly born infant is equally common. In 2003 infants in Bhopal, India were found to be consuming, through breast milk, 8.6 times more endosulfan than the acceptable daily intake levels recommended by the World Health Organization. The mothers were all unemployed, and the residues was thought to have occurred as the result of eating contaminated fish and vegetables 78.9% of 422 vegetables tested in another survey were found to contain endosulfan residues (Sanghi et al 2003). Health Effects and Poisonings Absorption and accumulation Endosulfan is absorbed across the skin and absorption is increased with oils or lotions on the skin, and through cuts. It is also absorbed through the gastrointestinal tract and through inhalation. (ATSDR 2000) Most published studies on body burdens of organochlorine pesticides have not included endosulfan (Torres et al 12 2006), but where they have, it is almost without exception found, and it can be assumed that endosulfan is a common human contaminant. It has been found in breast milk, adipose tissue, placental tissue, and umbilical cord

blood (Cerrillo et al 2005). Most of the available studies on the body burden of endosulfan relate to Southern Spain, an area of intensive greenhouse agriculture: Carreno et al (2007) found endosulfan or its metabolites in 100% of the blood samples from 220 young males; Lopez-Espinosa et al (2008) found endosulfan or its metabolites in 16% of the young boys aged 0-15 years who were sampled; Botella et al (2004) found them in 78% of adipose and 96% of serum samples from 200 postmenopausal women; Torres et al (2006) found them in maternal adipose tissue and serum and in umbilical cord serum of 61% of 72 women giving birth by caesarean section (only 26% of the adipose samples); ATSDR (2000) reports that endosulfan was found in the adipose tissue of 30-40% of children hospitalised in an agricultural region in Spain, assumed to be from repeated dietary exposure; other studies from Spain have found endosulfan in female adipose tissue (Hernandez et al 2002; Cerrillo et al 2005, 2006; Ibarluzea et al 2004); in breast milk (Campoy et al 2001; Cerrillo et al 2005); in placenta and umbilical cord blood (Cerrillo et al 2005); and in

blood serum (Arrebola et al 2001; Martinez Vidal et al 2002). In Portugal, endosulfan sulphate was present in the highest mean level and greatest frequency of detection of all organochlorines measured, in blood serum of male and female students, with the mean level in females being even higher than in women in Spain (Lino & da Silveira 2006) with levels up to 1295.5 ug/l. Endosulfan was found in all samples of breast milk from 280 women, and in all placental samples from 130 women, in Denmark and Finland (Damgaard et al 2006; Shen et al 2007, 2008). Endosulfan and/or its metabolites have been found in breast milk in Egypt (Saleh et al 1996), Madagascar (at 12 ug/gm), South Africa (GEF SSA 2002), El Salvador (GEF CAC 2002); in cord blood samples from pregnant Hispanic farm workers in USA (Cooper et al 2001); in serum of men in India (Singh et al 2008b); and in more than 50% of the sera samples from 21 couples in Canada seeking fertilisation assistance (Younglai et al 2002). In the Asia Pacific region, it has been found in breast milk in Kazakhstan (Lutter et al 1998), India (Sanghi et al 2003: endosulfan levels were higher than any other pesticide), Indonesia (Burke et al 2003), in cotton pickers in Pakistan (GEF IO 2002); and in maternal serum (90%) and umbilical cord tissue (70%) of 32 pregnant women in

Japan (Fukata et al 2005). In Indonesia alpha endosulfan was found in 80% of the samples from rural areas, but in none of the samples from urban areas, suggesting exposure was through contact with the spray rather than as residues in food. A survey of cacao farmers in Nigeria found that 29% of them carried residues of endosulfan with a mean level of 50 ug/kg (upper level not given) (Sosan et al 2008)well above the 1 ug/kg level found to cause adverse effects in protein deficient rats. The only study (Ramesh & Ravi 2003) that could be located in which endosulfan was not found when sampled for was from Kasargod District in India where villagers had suffered severe health effects from prolonged aerial spraying of endosulfan of a cashew plantation. However, this study, which was commissioned by the plantation corporation, was later exposed when it was found the blood samples had not in fact been taken from the villagers (Yadav & Jeevan undated). A second study (Saiyed et al 2003) found that 78% of the male children sampled had significant levels of endosulfan in their serum (mean = 7.47 ug/l) and these were still significantly higher than a control group 10 months after spraying of endosulfan ceased. Residues have also been found in the liver, kidney and brain of humans (ATSDR 2000).

Symptoms and consequences of poisonings Acute effects The most prominent signs of acute poisoning by endosulfan are hyperactivity, tremors, decreased respiration, shortness of breath, salivation, and convulsions (ATSDR 2000). Effects reported as a consequence of occupational inhalation and dermal exposure to endosulfan are primarily neurotoxic: headaches, irritated eyes, malaise, nausea, vomiting, dizziness, confusion, agitation, disorientation, irritability, weakness, shortness of breath and irregular respiration, tachycardia, brachycardia, abdominal discomfort after meals; diarrhoea, impaired consciousness, writhing, muscle twitching, and convulsions (ATSDR 2000; UNEP/FAO 2006a). Effects experienced by 22 people spraying rice and cotton were worse for those who had cuts on their legs from the sharp rice leaves (ATSDR 2000). Effects of ingestion of endosulfan involve respiratory, cardiovascular, haematological and gastrointestinal systems, as well as the liver and kidney. Symptoms include nausea, gagging, vomiting, headache, dizziness, diarrhoea, agitation, writhing, convulsions, sustained epileptic state, loss of consciousness, cyanosis, shortness 13

of breath, foaming at the mouth, and noisy breathing (ATSDR 2000). Clinical signs include elevated haemoglobin and white cell count, decreased blood pressure, hyperglycaemia, metabolic acidosis, kidney failure, liver congestion and fatty degeneration, hypoxia, aspiration pneumonia, clots in pulmonary arteries and aorta, cardiogenic shock, and death (ATSDR 2000). A strong odour of sulphur is reported as characteristic of endosulfan poisoning (Ramaswamy et al 2008). Severe metabolic acidosis has been reported in a case of unintentional endosulfan poisoning in Turkey (Yavuz et al 2007). Autopsies have revealed oedema of the brain and lungs, haemorrhage of the medullary layer of the kidney, necrosis of the kidney tubules, acute lung emphysema, congested lungs, inflammation of the stomach and small intestine, and chromatosis of the neurons (ATSDR 2000). Death has been reported to have occurred from 3 hours to 10 days after ingestion, and has occurred from cardiorespiratory arrest, heart failure, pulmonary oedema, and cerebral hernia from massive cerebral oedema (ATSDR 2000). Foetal death occurred after a 5-month pregnant woman ingested endosulfan to provoke abortion. A relatively low concentration of endosulfan in the blood of the mother,

470 ug/kg, caused relatively quick death of the foetus (Sancewicz-Pach et al 1997). Long-term effects Chronic liver and kidney damage may be expected from both short-term high level and long-term low level exposures (ATSDR 2000), as well as a range of other effects. Neurological One case of occupational inhalation, in an Israeli chemical factory, led to long-term brain damage with psychiatric manifestations. The man suffered long-term cognitive and emotional deterioration, severe memory impairment, and inability to perform small tasks, with impaired visualmotor coordination. The acute phase included malaise, repeated convulsions and impaired consciousness, and after recovery from these the person became disoriented and agitated (Aleksandrowicz 1979). In a person who attempted suicide, recurrent convulsions over two weeks were followed by a slow recovery but his mental activity was still severely impaired a year later and he required medication to control seizures (ATSDR 2000). Acute poisoning symptoms in a teenage girl were followed by psychosis, cortical blindness and limb rigidity (Pradhan et al 1997). An agricultural pilot exposed to endosulfan showed

persistent nonspecific epileptic foci in the cerebral frontal lobes (ATSDR 2000). The ATSDR (2000) concluded that humans with a predisposition to seizure disorders through hereditary or environmental causes may be at increased risk to the adverse effects of endosulfan. A recent study in California has linked maternal nonoccupational exposure to endosulfan and dicofol to autism in children amongst women living within 500m of its application during the first trimester of pregnancy, suggesting exposure by inhalation of drift. The rate of autism was 6 times higher than expectedpossibly caused by endosulfans interference with GABA-mediated neurotransmission, which plays an important role in gestational brain development. The first 8 weeks of foetal life is the period during which the central nervous system development begins (Roberts et al 2007). Cancer The ATSDR (2000) concluded we do not know whether endosulfan has ever affected the ability of people to fight disease or has ever caused cancer in people. One epidemiological study on environmental oestrogens and breast cancer found an increased risk of breast cancer (but not statistically significant) amongst women with elevated adipose tissue levels of endosulfan (in combination with DDE, aldrin and lindane) in a hospitalbased

case-control study involving 198 cases and 260 controls (Ibarluzea et al 2004). Reproductive There have been several reports of adverse reproductive outcomes linked with endosulfan. Rupa et al (1991) found decreased male fertility, increased stillbirths, neonatal deaths and congenital birth defects (such as anencephaly, cleft palate, harelip, club foot, limb malformations, eye deformities and extra fingers or toes), amongst families of those exposed to endosulfan and other pesticides in Indias cotton fields. Endosulfan was one of 8 organochlorine pesticides measured in significantly higher concentrations in mothers who gave birth to boys with cryptorchidism than in women who gave birth to normal boys, in a study in Denmark and Finland (Damgaard et al 2006). Cryptorchidism has also been linked to endosulfan in southern Spain, where higher rates coincide with intensive use of the insecticide and high rates of residues in childrens adipose tissue (40%) (Saiyed et al 2003). Chronic effects on male reproductive development were found in villagers in Kasargod, south India exposed to endosulfan, including delayed sexual development (significantly reduced development of pubic hair, testes, and penis) and reduced synthesis of testosterone. These findings correlate with animal studies that show

endosulfan inhibits steroidogenesis and spermatogenesis (Den Hond & Schoeters 2006). Additionally, there were six cases of congenital malformations3 undescended testicles, 3 congenital hydrocele, 1 congenital inguinal herniacompared with one only (a congenital inguinal hernia) in a comparable less exposed group. Endosulfan was detected in the serum of 78% of the children in the study group and in 29% of the control group, but at a 14 median level 5.45 times greater than that of the less exposed control group (Saiyed et al 2003). Other effects noted in Kasargod and attributed to exposure to endosulfan included: congenital deformities of hands, feet and heart especially in females; other congenital deformities including a child born with her bladder outside her body; endometriosis, early menarche, frequent menstrual disorders, male breast enlargement; liver cancer, haematological cancers, brain tumours (neuroblastoma); congenital mental retardation, cerebral palsy, delayed mental and psychomotor development, learning disabilities, low IQ; psychiatric disturbances including committing suicide, epilepsy;

 frequent illness, skin diseases; ear nose and throat problems; and vision impairment and full loss of vision. (Quijano 2002; Yadav & Jeevan undated; NIOH 2003; Venugopal 2008). A survey by the Kasargod District Committee found a disability rate 73% higher than the norm for Kerala State, and the rate of locomotor disability and mental retardation taken together was 107 % higher than the norm (Yadav & Jeevan undated; NIOH 2003). Cases of Poisonings Numerous intentional and unintentional deaths have occurred from ingestion of endosulfan, and poisonings are reported for Benin, Colombia, Costa Rica, Cuba, Guatemala, India, Indonesia, Malaysia, Philippines, New Zealand, South Africa, Sri Lanka, Sudan, Turkey and USA. It is regarded as one of the main causes of poisoning in many countries (Kishi 2002) including in Asian countries (Roberts et al 2004), Turkey (Oktay et al 2003), Latin America (Wesseling et al 2005), and West Africa (Glin et al 2006). Intentional An analysis of poisonings from pesticides in the Warangal district of Andhra Pradesh, India during 1997 to 2002, revealed that endosulfan and monocrotophos caused the majority of the 1,817 deaths (8040 hospital admissions

for pesticides with a 22.6% fatality rate). 96% of these hospital admissions were intentional poisonings (Srinivas Rao et al 2005). Such deaths are still continuing with one of the most recently reported being that of a 26 year-old woman in Chandigarh (Ramaswamy et al 2008). In 2005, 17 males were admitted to just one hospital in Turkey, that of Ondokuz Mayis University in Samsun, after ingesting endosulfan (Karatas et al 2006). According to Roberts et al (2003), in Sri Lanka the replacement of banned organophosphates by endosulfan after 1995 led to a rise in deaths from endosulfan poisoning, from 1 in 1994 to 50 in 1998. At this point endosulfan was also banned and deaths from it fell to 3 over the following 3 years. Gunnell et al (2007) reported that, following the Sri Lankan restrictions on WHO Class I pesticides in 1995 and endosulfan in 1998, the rate of self-poisoning fell significantly with 19,769 fewer suicides in 19962005 as compared with 198695. No other trends appeared to be associated with this decline. In Malaysia, 15 endosulfan poisoning incidents were recorded for the years 1987 and 1988, but whether they were intentional or not is not reported (Arumugam 1992). Environmental fate Persistence In soil Endosulfan is persistent in the soil. The Stockholm

Convention on Persistent Organic Pollutants (POPs) defines persistence as having a half-life (DT50) in soil of > 183 days. Endosulfan is oxidised in plants and soil by microbial action to form endosulfan sulphate, and by hydrolysis to form endosulfan-diol. Aerobic conditions alpha endosulfan DT50 = 12-39 days (mean = 27.5) beta endosulfan DT50 = 108-264 days (mean 157) total endosulfan DT50 = 288-2,241 days, or 9 months to 6 years (GFEA-U 2007); 1,336 days (US EPA 2007c) Anaerobic conditions These are expected to considerably extend the half-lives in soil (GFEA-U 2007). Tropical soils DT50 total endosulfan in Brazil was 161-385 days, i.e. it is less persistent in tropical soils. In water The half-life in water varies from 35-187 days under anaerobic conditions (ATSDR 2000). The Stockholm Convention regards a chemical as being persistent in water if its half-life is > 2 months: endosulfan is persistent in water under some conditions. Increasing pH increases hydrolytic breakdown of endosulfan, which is the main degradation process in

seawater, thus endosulfan is more persistent in acidic conditions (GFEA-U 2007): At 25 oC, DT50 at pH 7 is 10-20 days, and at pH 9 it is 0.2 days (GFEA-U 2007). In seawater, which is alkaline, the half-life of alpha endosulfan was 22 days, and of beta endosulfan 8.3 days (GFEA-U 2007). 20 The half-life in a water/sediment microcosm in a Brazilian wetland was measured as up to 63.6 days (DT50) for beta endosulfan and 20.4 for alpha endosulfan (Laabs et al 2007). In plants The estimated half-life of endosulfan in cotton plant material in Australia is 65 days (Kennedy et al 2001). One study on rice in India found that foliar concentrations of alpha endosulfan initially of 48,400 ug/kg reduced to 13,300 ug/kg after 75 days, but were still present at 7,400 ug/kg after 225 days. The rice at harvest contained 2,200 ug/kg endosulfan sulphate (Jayashree & Vasudevan 2007b). Bioaccumulation, bioconcentration, and biomagnification Bioaccumulation is a general term for the accumulation of substances in an organism through respiration, food intake, absorption through the skin, etc, resulting in the

organism having a higher concentration of the substance than the concentration in the surrounding environment. Bioconcentration is bioaccumulation that occurs when uptake is from water only; the octanol-water partition coefficient (KOW) of the substance is used to determine its ability to bioaccumulate. Biomagnification is the bioaccumulation of a substance up the food chain, and it can result in higher concentrations of the substance than would be expected if water were the only exposure mechanism. The biomagnification factor (BMF) is the ratio of the concentration of the chemical in the organism to the concentration in its food, and biomagnification occurs if the ratio is >1. The Stockholm Convention (and the US) recognises a chemical as bioaccumulative if it has a bioaccumulation or bioconcentration factor (BCF) > 5,000 in an aquatic species, or a log Kow>5. The EU recognizes bioaccumulation when the BCF is > 2000 (Kelly et al 2007). Estimates of log Kow (GFEA-U 2007): alpha endosulfan = 4.65 beta endosulfan = 4.34 endosulfan sulphate = 3.77 The US EPA (2007d) put the range of log Kow for the 3 forms of endosulfan as 3.55-4.78. Alpha endosulfan is more bioaccumulative than the beta isomer or the sulphate.

The measured BCF in aquatic organisms covers a wide range: oysters and bivalves = < 100 whole fish, fresh and marine = 2,400 to 11,000 freshwater green algae = 2,682 Daphnia = 3,278 (DeLorenzo et al 2002; GFEA-U 2007) The highest BCF was for the fish yellow tetra, with a value of 10,994 for the alpha isomer, 9,908 for the beta isomer and 11,583 for both isomers together (Jonsson & Toledo 1993). These figures are well above the Stockholm Convention requirement of 5,000. The US EPA (2007d) estimated a BCF range of 1000 to 3000. DeLorenzo et al (2002) noted that the BCF of 3,278 for Daphnia is similar to those reported for the POPs pesticides DDT, dieldrin and chlordane of 2,500-12,000. They also noted that this BCF of 3,278 was for neonates and it is expected to be higher for adult Daphnia, which have a nearly five times higher lipid content. US EPA (2007d), however, concluded a range of 20-600 for aquatic invertebrates. The US EPA (2002) considered endosulfan to have a high potential to bioaccumulate in fish, and hence may affect animals higher in the food chain. However US EPA (2007d) reported a calculated BMF in aquatic organisms

as up to 1.38 for piscivorous fish and regarded it as not significant. Even if the threshold of 5,000 for a BCF for aquatic species is not met, the Stockholm Convention accepts other evidence of bioaccumulationin particuar evidence of high bioaccumulation in other species, or monitoring data in biota indicating that the bioaccumulation potential of the chemical is sufficient to justify its consideration within the scope of this Convention. Such evidence certainly exists. Vorkamp et al (2007) found endosulfan residues in every single species, both terrestrial and aquatic, that they tested in Greenland (see section on Contamination of biota, p27 for details). The authors reported that, whilst there was significant accumulation of endosulfan in tissue, and some evidence of biomagnification in fish (wolfish and caplin), there did not appear to be appreciable biomagnification higher up in the aquatic food chain as levels in fish eaters were generally not higher than in fish. However, Kelly & Gobas (2003) and Kelly et al (2007) have proposed that the biomagnification of endosulfan (particularly the beta isomer) is greater in the terrestrial food chain that the marine food chain, because it has a high log Koa (octanol-air partition coefficient), which is of greater importance than the Kow in air-breathing animals. A high Koa causes slow respiratory elimination.

 log Koa alpha endosulfan =10.29 log Koa beta endosulfan =10.29 log Koa endosulfan sulphate =5.18 Armitage & Gobas et al (2008) concluded that chemicals with a log Kow between 1.75 and 12, and a log Koa > 5.25 have the potential to bioaccumulate in air-breathing organisms, hence endosulfan clearly has the potential to biomagnify in terrestrial animals. Kelly et al (2007) summarised residues of endosulfan found in the Artic food web in Hudsons Bay 1999-2002 to illustrate bioaccumulation (units are ug/kg lipid equivalent, geometric means), especially of beta endosulfan in beluga, a small arctic whale that feeds mainly on fish: 21 alpha beta sulphate lichen - 0.03 sediment 0.16 0.33 0.16 cod (B. saida) - 2.91 salmon 0.41 0.85 0.18 beluga (m) - 12.56 0.86 beluga (f) - 4.87 0.58 ringed seals (f) 3.02 0.19 ringed seals (m) 0.30 2.26 0.32 eider ducks 2.32 - scoters - - 0.87 Kelly & Gobas (2003) established the following predicted

BMFs for beta endosulfan in male wolves at various ages, showing significantly higher biomagnification than found in the aquatic food chain (see preceding paragraph), even at a 1.5 years, and increasing considerably with age: 1.5 years 2.25 years 13.1 years 5.3 17.9 39.8 Kelly et al (2007) calculated BMFs for endosulfan ranging from 2.5 to 28 for different herbivorous and carnivorous terrestrial organisms. US EPA (2007d) calculated a BMF of 7 for male beluga and 3 for female beluga. These figures clearly show that endosulfan has the potential to significantly biomagnify in the food chain, and findings of widespread contamination in the Arctic region, with levels increasing over time, support this. Bioaccumulation is occurring even in the aquatic food chain despite some analyses not indicating this. Braune et al (2005) reported that over 20 years monitoring of POPs in the Canadian Arctic, there was a 3-fold increase in age-adjusted concentrations of endosulfan sulphate in beluga at the same time as most other POPs were declining. Similarly, Evans et al (2005) reported increasing residues of endosulfan in the freshwater fish charthey were 2.2 times higher in 2002 than 1992. Bioaccumulation is also occurring in non-polar regions. Menone et al (2000) found residues of endosulfan sulphate in the liver, gonads, fat and muscle of fish from a coastal

lagoon in Argentina, at levels ranging up to 0.018 ug/kg of tissue. They calculated a BMF of 4.02, higher than that for DDT. Lanfranchi et al (2006) found endosulfan biomagnified in striped weakfish also in Argentina. In Zimbabwe, endosulfan was found to accumulate in the tissues of wild rats feeding in to soya bean fields to which endosulfan was applied (Kuvarega & Taru 2007). Levels in liver were generally higher (0.36-5.8 ug/kg) than those in muscles (0.09-5.17) and fatty tissues (0.08-4.78 ug/kg). Measurements taken in Ghana indicate that bioconcentration may be occurring in freshwater fish there, according to Darko et al (2008): endosulfan levels in fish (up to 0.42 ug/kg) were, on average, 10 times those found in water samples, although the mean level in the sediments was about 14 times the levels measured in fish. Bioaccumulation has even been found in plants: a BCF of 30.9 was found for endosulfan sulphate in bulrushes from a lake in Argentina, which appear to have sequestered it from the sediment (Miglioranza et al 2004b). This BCF was significantly higher than those for other POPs such as DDT (1.1), chlordane (2.1), dieldrin (2.1), and heptachlor (3.8), with the exception of HCH (61.1). An analysis of airborne pollutants in the western national parks of the USA, carried out from 2002 to 2007, found evidence of the bioaccumulation of endosulfan in vegetation, the degree of accumulation increasing with

forest productivity and proximity to sources of endosulfan. The mean level of total endosulfan in two-year-old needles of White Fir (7573 ug/kg lipid) was 3 times higher than the levels in one-year-old needles (2448 ug/kg). The degree of bioaccumulation varies between species with firs accumulating substantially higher concentrations than pines. The coniferous forests of the western USA and Alaksa are estimated to be removing between 1,280 and 7,210 kgs of endosulfan from the air annually, amounting to about 2% of the USAs annual usage (Landers et al 2008). There was no estimation of how much other vegetation types might also be accumulating, but there were signficantly higher levels of endosulfan in some lichen species than in the conifers (nearly 6 times higher). Wang et al (2007a) found that the bioconcentration factor (BCF) of grass for POPs increased with increasing elevation, indicating that the cold condensation of POPs at high-elevation sites may increase the potential threat to vegetation and the food chain in the mountain ecosystem. Atmospheric Transport and Deposition The potential for atmospheric transport of endosulfan is indicated by: its ability to volatilise from soil and plants; its atmospheric half-life; and its ability to interchange between air and water, and

air and air-borne particlesknown as partitioning both of which affect uptake into the atmosphere, transport and subsequent deposition. That endosulfan is actually being transported by air from the fields and glasshouses where it is used and then redeposited, often at very remote locations, is evidenced by the residues found in air, fog, rain, snow and ice described below. Volatility Endosulfan is semi-volatile. It evaporates from the soil surface and even more so from plants, particularly during the first week after application. The rate of evaporation increases with temperature, humidity, and wind (Bedos et al 2002). It also evaporates from water: the volatilization half-life from surface waters is greater than 11 days and possibly greater than 1 year (ATSDR 2000). Laboratory studies (at 21-22 oC, and 50% relative air humidity): 25-30% dissipated from the soil surface to air over 24 hours; 64% dissipated from bean leaves; and alpha endosulfan is much more volatile than the beta 22 isomer, and the sulphate is much less volatile, with only 5% released to the air from plants within 24 hours (GFEA-U 2007).

Field studies: At maximum temperature of 40 oC, there was 82% loss over 24 hrs, and 89% over 48 hrs, from cotton foliage; and at maximum temperature of 29 oC, there was 49% loss over 24 hrs, and 69% over 48 hrs, from cotton foliage (GFEA-U 2007). In one study in Australia, approximately half of endosulfan applied to soil surfaces dissipatedthe alpha isomer in 35 days, and the beta isomer in 5-8 days. Even with heavy rainfall for the first few days after application to freshly tilled soil, 34.5% of alpha endosulfan evaporated from the soil within 20 days (GFEA-U 2007). Sutherland et al (2004) claimed 70% of endosulfan volatilises within 2 days. Kennedy et al (2001), based on studies on endosulfan application in cotton fields in Australia, concluded that 70% of endosulfan is lost from the cotton field through volatilisation, with only 8.5% remaining on the field one month after spraying; 2% is carried away in run-off, 1% remains in the soil, and the remainder is degraded by plants or microorganisms). In other words about 73% of endosulfan applied leaves the site of application and contaminates the wider environment. Atmospheric half-life The Stockholm Convention regards a chemical as having the potential for long-range atmospheric transport if its

half-life in air is > 2 days. Endosulfan has an atmospheric half-life of 27 days (+ 11 days), at 75 oC under experimental conditions. The much lower temperature in the trophosphere would result in a much longer half-life in the air there. Experimental measures have found a half-life for alpha endosulfan of >2.7 days and for beta endosulfan of >15 days. However measurements at Alert, Nunavut in the Canadian Arctic have shown an atmospheric half-life there of 38 years for alpha endosulfan (Hung et al 2002, 2005). Partitioning Interchange between air and water, which affects uptake into the atmosphere and redeposition in rain or snow, is described by Henrys Law constant (H)the higher the value, the higher the deposition. Estimates of H for endosulfan vary widely, from 0.71 to 12.9 Pa m3 mol-1 for the alpha isomer at 200C (GFEA-U 2007) Gas-particle partitioning for endosulfan is poorly understood. Values given by GFEA-U (2007) are 0.166 for alpha endosulfan and 0.228 for beta endosulfan adsorbed to particles at 00C, falling to 0.018 and 0.024 at 200C. Residues in air Endosulfan, along with PCPs, is regarded as the most abundant POPs pollutant of the global atmosphere (Pozo Unless otherwise specified, the values given are for total endosulfan

i.e. alpha + beta + the sulphate. AE = alpha endosulfan; BE = beta endosulfan, ES = endosulfan sulphate et al 2006). It has been found in air from one end of the world to the other: the Global Air Passive Sampling (GAPS) study carried out over just 4 months in 2004-2005 found residues of alpha endosulfan in the air at every one of 41 sites with the exception of Antarcticafrom Alert in the Canadian Arctic through Europe, Africa, Asia and Central America, to Chile and Australia in the south (see Table 1 for a sample of these)including in a number of urban areas (Pozo et al 2006). An earlier pilot study carried out over 2002-2004 found much higher levels in Toronto, Canada (0.104 ng/gm) and at Cape Grim, Tasmania in Australia (0.719 ng/gm), probably resulting from Australias own use of endosulfan. Residues at Whistler Mountain in Canada (0.321 ng/gm) were believed to have come from India (Harner et al 2006). Residues in air are generally described in terms of the distance over which they have travelledlong-range to remote locations such as the Arctic, and Antarctic; regional distribution including to local mountain ranges; and short range including localised spray drift and ambient or background air concentrations resulting from drift and/or volatilisation. In most atmospheric studies alpha endosulfan is present in greater amounts than the beta isomer (Tuduri et al

2006), but at an order of magnitude lower than elsewhere in the environment. Long range Endosulfan was first noticed in the Artic air in 1986-87, and was measured from early 1993 through to the end of 1997 at concentrations of 0.0042-0.0047 ng/m3 and has been routinely found there, widely distributed, ever since (GFEA-U 2007). Unlike the other POPs, concentrations have not decreased with timedue to ongoing use they Table 1: Residues found in GAPS study Country Location TE (ng/m3) Argentina Bahia Blanca 18.704 Canary Island Telde 1.760 Ghana Accra 0.952 Bolivia Potosi 0.510 S Africa DeAar 0.350 Canada Whistler 0.129 Turkey Ismir 0.126 Iceland Malin Head 0.107 USA Georgia 0.107 Kuwait Kuwait City 0.101 Bermuda Bermuda 0.077 Canada Alert 0.069 Philippines Manilla 0.066 Malaysia Dum Valley 0.066 China Chengdu 0.047

Australia Cape Grim 0.027 Source: Pozo et al 2006 23 are actually increasing (NCP 2003)and are exceeded only by HCH and HCB (Hung et al 2002). It has been found at many sites across the Canadian Arctic, in 90% of air samples at Amerma in the Russian Arctic (GFEA-U 2007), and recently in the European Arctic at Bjrnya (Bear island) (Kallenborn et al 2007). Modelling data has also shown that endosulfan released in Central Europe may spread over the Northern Atlantic as far as Greenland (GFEA-U 2007). Regional transport Endosulfan has been described as one of the most abundant and ubiquitous air contaminants in continental North America (GFEA-U 2007). Air sampling at 40 stations (31 in Canada, 5 in USA and 4 in Mexico, Belize and Costa Rica) found alpha endosulfan at 39 of the stations and beta at 30 (Shen et al 2005). In Canada endosulfan has been measured in the air in both agricultural areas and in so-called receptor sites demonstrating regional atmospheric transport, including in the Great Lakes Basin, the City of Toronto, the Fraser Valley in British Columbia, Prince Edward Island, Quebec, Ontario and Saskatchewan. 100% of air samples collected at 5 sites across Canada in the summer of 2003

contained endosulfan, at high levels of 5.71 ng/m3 (AE) (Yao et al 2006). Alpha endosulfan was present in 100% of air samples taken on the campus of the University of Maryland beside the Choptank River, Chesapeake Bay over a 7-month period Apr-Nov, with a maximum concentration of 0.68 ng/m3. The endosulfan was thought to come from sources outside the Choptank River watershed area (Kuang et al 2003). Endosulfan has also been measured in the atmosphere in a number of sites from northern Michigan to southern Louisiana (Hoh & Hites 2004). Regional transport of endosulfan has been identified in China and over the Yellow Sea. The beta isomer was found, at levels up to 0.025 ng/m3, in all 9 day and nighttime samples taken at the Ocean University of China, which is in an urban setting in Qingdao on the Yellow Sea coast (Lammel et al 2007). Very high concentrations of endosulfan have also been reported in Guangzhou and Hong Kong and are thought to originate in agricultural areas of East China where it is used in cotton fields, and to have been carried from there by the winter monsoon. Levels at a suburban site in Guangzhou were as high as 2.5 ng/m3 (AE) and in urban Hong Kong 0.84 ng/m3 (AE) (Li et al 2007). The air around Lake Taihu contained residues of alpha endosulfan up to 0.888 ng/m3 (AE) (Qiu et al 2004), with an estimated deposition rate of 25

ng/m2/day in summer (Qiu et al 2008) indicating that it is rapidly moving out of the air and into the Lake. Other studies have found lower levels of alpha endosulfan in urban Hong Kong and on top of the islands highest point (Louie & Sin 2003). In Chile alpha endosulfan was found in a concentration gradient that decreased from 0.099 ng/m3 (AE) in the north to 0.035 ng/m3 (AE) in the south (Pozo et al 2004). Mountains Endosulfan has been found in the air around a number of mountains. Temperate mountains in particular experience effective upslope air movement which brings with it residues originating from regional applications, with residue levels increasing with elevation (Daly et al 2007b). It has been found as a ubiquitous air contaminant in the mountains of the Canadian Rockies, the California Sierra Nevada (Daly et al 2007a), and the USAs western national parks (Landers et al 2008). It has also been found in air in the European Central Pyrenees (0.006 ng/m3 AE) and the High Tatras (0.042 ng/m3 AE) (van Drooge et al 2004), and in concentrations higher than any other POP in the air in the Mt Everest region (Li et al 2006b). More recently endosulfan has also been found in the air (and soil) in neotropical montane forests in Costa Rica, by

Daly et al (2007b), as a result of regional transport. They found endosulfan to be ubiquitous in the air across the country with very high concentrations at some lowland sites (e.g. 2.3 ng/m3), and lesser concentrations in the mountain air. However the reverse was true for residues in the soil with higher residue levels (e.g. 3,175 ng/kg) in the mountain soils. The authors explained a process whereby endosulfan used in lowland agriculture is carried by easterly winds to the downwind mountains; forced atmospheric uplift causes the formation of orographic clouds and subsequent rainfall. The drop in temperature with elevation greatly increases the scavenging by the rain of endosulfan from the air (only slightly scavenged at the higher temperatures in the lowlands), leading to greater deposition in rainfall and a build up of residues in the soil because of lower evaporation. The authors suggested that the high soil residues in the mountain forest may be a factor in the observed decline in amphibia in the mountains. Short-range drift and ambient air contamination Localised air contamination also occurs, as a result of short-range spray drift and/or volatilisation, exposing people and the environment to unacceptable levels of endosulfan in both rural and urban areas. Endosulfan was the most prevalent organochlorine pesticide in rural air in South Korea (Yeo et al 2003), and

in urban Seoul (Yeo et al 2004). It was also present in air samples taken in Izmir, Turkey (Sofuoglu et al 2004). In France, endosulfan was found in 79% of indoor air samples in the Paris area (Bouvier et al 2006), and in 100% of air samples taken in urban Strasbourg during July (Scheyer et al 2005). Endosulfan was found in 66 out of 75 rural air samples taken in California, on the roofs of community buildings such as schools in both rural and urban communities. The mean concentration in urban areas was 3.8 ng/m3, and in rural areas was 180 ng/m3 (Lee et al 2002). 24 It has been found in the air in Belize (GEF CAC 2002), and India (GEF IO 2002). Ambient air concentrations of endosulfan were found in both farming and non-farming areas of Prince Edward Island in Canada, where it is used in potato cultivation (White et al 2006). In air-monitoring studies facilitated by scientists from PAN North America, using their Drift Catcher equipment, endosulfan was found in every sample collected at a Florida elementary school surrounded by fields of cabbage. The maximum level of endosulfan found (626 ng/m3) was 1.8 times the 24- hour acute and subchronic 1-year child Reference Exposure Level (REL) of 340 ng/ m3 for endosulfan calculated from the US EPAs inhalation

NOAEL. All samples contained alpha endosulfan, and 88% contained beta endosulfan. 38% were above the 24-hour acute and subchronic 1-year-old child REL (PANNA 2007). Repeat sampling between 1 October and 6 December 2007, found detectable levels of endosulfan in 87% of samples. Nine samples (23%) were above the child REL of 340 ng/m3. The highest concentration of total endosulfan observed for a 24-hour period was 1376 ng/m3 (mainly composed of the alpha isomer) and this was 4 times the 24-hour acute 1-year-old REL and 2.8 times the 7-year-old REL (PANNA 2008). Atmospheric Deposition Snow Endosulfan, like other POPs has a marked tendency to deposit out of the air (cold condensation) in remote environments at high latitudes and at high elevations. This is caused by low temperatures and efficient scavenging from the atmosphere, by snow, of both particle-bound and gas-phase pesticides (Hageman et al 2006; Daly et al 2007b). Alpha endosulfan was first identified in snow in the Canadian Artic in 1986 (Ellesmere Island) and concentrations measured through to 1989 ranged up to 1.34 ng/m3 (Tuduri et al 2006; GFEA-U 2007). In 2002-03 endosulfan residues were amongst the most frequently detected in snowpack in seven national

parks in arctic, sub-arctic and alpine regions of the US. The highest concentration was 1500 ng/m3 at Sequoia National Park, the majority of which was regarded as resulting from regional transport. Residues in the Arctic region (peaking at 170 ng/m3) were regarded as the result of long-range transport (Hageman et al 2006). Alpha and beta endosulfan were again consistently detected in snowpack near Lake Superior in 1999/2000, at concentrations ranging up to 0.181 ng/l AE plus 0.035 ng/l BE (Burniston et al 2007). It was one of the most frequently detected pesticides in snow in the Rocky Mountain and Glacier National Parks in the Western US, found at levels up to 2.5 ng/l (AE) (Mast et al 2007). Alpha endosulfan was found in 100% of samples of snow taken from the Punta Indren glacier in the Italian Alps at concentrations ranging from 0.05 to 0.168 ng/l (Hebert et al 2004). It appears that very little monitoring has been carried out in Antarctica. However endosulfan sulphate has been found in an ice core sample from the Antarctic region at a concentration of 300 ng/l (Deger et al 2003). Rainfall Endosulfan has been found at high levels in rain in various parts of the world, especially in countries still using the pesticide.

In six years of monitoring of rainfall at Lakes Superior, Erie and Ontario, endosulfan was found in 95% of samples, with concentrations of the beta isomer always higher than the alpha isomer. For 20 years, between 1980 and 2000 endosulfan was monitored in rainfall in Prince Edward Island, Nova Scotia, New Brunswick and Newfoundland: the alpha isomer was found in 5.3% of samples and the beta isomer in 11.5% (Tuduri et al 2006; Brun et al 2008). Samples taken at seven US and Canadian sites around the Great Lakes between 1997 and 2003 showed residues of endosulfan peaked in summer corresponding to maximum usage in nearby agricultural areas (Sun et al 2006). Carlson et al (2004) reported mean concentrations of endosulfan as 1.18 ng/l in the rain at five sites round the Great Lakes (US). Between 1996 and 1998 endosulfan was found in rainfall samples collected at remote high mountain lakes in the Pyrenees, Alps and Caledonian mountains of Europe (Carrera et al 2002). Endosulfan has also been measured in rainfall in urban areas: between 2000 and 2003, endosulfan was measured in rain in the urban areas of Cuiaba, Brazil at 107 ng/l (Laabs et al 2002), and in Chicago and Jersey City, USA (Sun et al 2006). In 2000, the beta isomer was found in 28%, and alpha isomer in 13%, of rainfall samples, taken on the campus of the University of Maryland beside the

Choptank River, Chesapeake Bay over a 7-month period Apr-Nov, with a maximum concentration of 81 ng/l (beta) (Kuang et al 2003). Endosulfan was found in 85% of rainwater samples taken in urban Strasbourg and rural Erstein in France, in 200203, the highest concentration being 3667 ng/l (AE + BE) in Strasbourg; and is also reported for Flanders in Belgium (Scheyer et al 2007). Between 1997 and 2000 endosulfan and its metabolite was the pesticide most frequently detected in weekly rainfall sampling in Flanders, Belgium (Quaghebeur et al 2004). Endosulfan was found in 100% of samples of rainwater collected in Hisar, India in 2002, total endosulfan at levels up to 3,020 ng/l (Kumari et al 2007). Endosulfan was also been found in rainwater in Malawi (GEF SSA 2002). 25 Dust Deposition of endosulfan in dust is also of concern. Monitoring in the Caribbean, at the Virgin Islands and Trinidad, has identified endosulfan at all sites, blown there in dust from Mali in the African Sahara/Sahel region (Garrison et al 2006). Climate change and deposition Endosulfan is more easily deposited onto water than it is

onto ice, and it is expected that as the ice in the Arctic presumably also Antarcticdiminishes with increasing global temperatures, the deposition of endosulfan in polar waters will increase and hence become more available to plants and animals in the marine food-web (NCP 2003). Environmental Contamination Endosulfan residues can be found throughout the world, throughout the environment. In remote regions such as the Arctic, this results from long-range atmospheric transport; in other regions it is from local use. Residues in air, rainfall, and snow are covered in the preceeding section on transport and deposition. Soil Contamination Very high levels of contamination have been found in Ethiopia, with total endosulfan reaching levels of 56,000 ug/kg in the soil of a state farm where organochlorine insecticides have been used for about three decades. The Upper Awash Agro Industry Enterprises grows cotton, fruits, vegetables, allium flowers and cereals, and their annual endosulfan use is 29,000 litres, estimated to be about 10 tonnes active ingredient (Westbom et al 2008). In Australian cotton fields where endosulfan is applied 3 times throughout the cotton-growing season, residues from the previous seasons spraying were found in the soil at the beginning of the next season (at 10 to 80 ug/ kg), indicating a continual build up in the soil (Kennedy et

al 2001). Similarly, in Tamil Nadu, India, 100 to 1,000 ug/kg residual endosulfan sulphate was found at the beginning of the rice season, with this level increasing to 22,000 ug/kg during the season-still present at 5,500 ug/kg at 210 days after application (Jayashree & Vasudevan 2007b), again indicating accumulating residual levels. Although at much lower levels, endosulfan contamination of the soil in cotton fields of the Punjab has been shown to increase with time, by a factor of 5 over the course of 3 years: in 1995 the residue after application was 7.3 ug/kg reducing to 2.4 ug/kg after 22 days. But in 1998 in the same field the levels were 35.6 ug/kg reducing to 22 ug/kg after 22 days, despite the insecticide being applied at the same rate (Vig et al 2008). All samples of soil taken in cotton-wheat, paddy-wheat and sugarcane fields in Haryana, India, were contaminated with endosulfan at levels ranging up to 39 ug/kg (Kumari et al 2008). It has also been found in the soils of in the northern Indo-Gangetic plains (Singh et al 2007); Hong Kong (Zhang et al 2006), and the Taihu Lake Region of China (Wang et al 2007b); Ghana (Ntow 2001), Senegal and Kenya (Westbom et al 2008), Portugal (Gonalves & Alpendurada 2004), Czech Republic (Shegunova et al 2007), rice fields in Spain (Gamon et al 2003), Colombia

(at 350 ug/kg), Honduras, Nicaragua, and Panama (GEF CAC 2002), and in the soils of environmental reserves in Brazil (Rissato et al 2006). Surface waters and sediment contamination Published reports of endosulfan contamination of surface water between 1982 and 2003 contain 606 detections in Canada, USA, Sweden, Australia, South Africa, and Argentina (Schulz 2004), a far from complete record with many more reports since then. The US EPA (2007c) reported 667 detections between 1980 and 1999 for beta endosulfan in the USA alone. The contamination of surface waters has frequently occurred at levels above those reported to cause adverse effects on aquatic life (Schulz 2004). The US EPA (2007c) reports toxicity to estuarine/marine amphipods of endosulfan in sediment at concentrations as low as 1.5 ug/kg of pore water (water filling spaces between grains of sediment). Other estimations for hazardous concentrations for aquatic organisms include 0.2-11 ug/l for fish (Kennedy et al 2001); and a mean level of 0.02 ug/l for effects on 5% of aquatic organisms (Bollmohr et al 2007). All of the figures reported below exceed this level. Endosulfan reaches surface waters as a result of direct deposition and runoff from agricultural use. Reports of endosulfan concentrations in run-off include 100 ug/l in southern USA, 45 ug/l in Australia, 66.5 ug/l in India

(Menone et al 2008), 12,082 ug/kg of suspended particles in South Africa (Schulz 2001), and up to 1,530 ug/l in ditch water during application on adjacent fields in British Columbia, Canada (Schulz 2004). Kennedy et al (2001) found significant contamination of all runoff water throughout the entire cotton-growing season in New South Wales, Australia, with residue levels in the runoff reaching 10 ug/l seven days after aerial application, and typically being 2-10 ug/l. Major storms soon after application can cause runoff of 10% of the endosulfan applied. Concentrations of endosulfan in Australian rivers during the cotton growing season were reported to be in the range of 0.02 to 0.2 ug/l, significantly exceeding the 0.01 ug/l guideline for ecosystem protection set by the Australia and New Zealand Conservation Council in 1992due to nearly continuous low level input from spray drift and vapour transport representing a steady drizzle, and occasional higher input from run-off (Raupach et al 2001). Endosulfan has been found in 47% of surface water samples tested in an agricultural region of Western Cape, South Africa. 37% of the detections exceeded the EUs 26 limit of 0.1 ug/l, and reached levels of 3.6 ug/l. The main source was reported to be washout from the agricultural

soils after irrigation and rainfall (Dalvie et al 2003). A pervious study in Western Cape, reported by Dalvie et al (2003), found endosulfan in 26 out of 27 Elgin dams at levels up to 626 ug/l; Bollmohr et al (2007) report 12.7 ug/l particle-associated endosulfan. Levels of 1.85 ug/l of endosulfan were found in water samples from the Selangor River in Malaysia in 2003 (Leong et al 2007). It has also been found in water and sediment in rivers in Perak, Juru, Perai, Muda and Bernam (GEF SEA SP 2002). Other findings of surface water contamination: Indiain a number of rivers (Selvakumar et al 2005), including water from the Yamuna River at Delhi, India (Aleem & Malik 2005); streams, ponds and canals of the northern Indo-Gangetic alluvial plains (Singh et al 2007); water of the Gomti River, a tributary of the Ganges, at 0.09 ug/l (ES) (Malik et al 2008); water and sediment in prawn ponds (Amaraneni 2006); and levels of 2.4 ug/l in Kolleru Lake and 2.0 ug/l in Vellar River believed to be caused by a nearby endosulfan manufacturing facility (Sarkar et al 2008); Chinasediment of Lake Taihu at 50.5 ug/kg (ES) (Huang et al 2006); sediment of the Tonghui River (Zhang et al 2004); and water and sediment in the Minjiang River estuary 0.215 ug/l (BE) (Zhang et al 2003);

 sediment in the Wu-Shi River estuary, Taiwan, at 10.5 ug/l (ES) (Doong et al 2002); water and sediment of Uluabat Lake, Turkey (Barlas et al 2006); water and sediment in Ghana (Ntow 2001; Ntow et al 2008); lakes in Malawi (GEF SSA 2002); and in all samples of water and sediment from the Kafue River, Zambia (Syakalima et al 2006); In Benin, following its reintroduction into cotton growing in 1998-1999, endosulfan was found in almost all water samples taken in the W region transboundary biosphere reserve (which covers over one million hectares in Benin and Burkina Faso), and in Pendjari Reserve, at levels up to 0.46 ug/litre (Glin et al 2006); sediment in Lake Kinneret, Israel at 276 ug/kg (GEF M 2002); a number of rivers in the USA (Hinck et al 2008); the National Sediment Quality Survey found endosulfan stream sediments in 30 out of 76 watersheds it tested for residues (US EPA 2002); year-round detections of endosulfan in freshwater canals draining from agricultural areas in South Florida into the Everglades and Florida Bay, indicating chronic exposure to aquatic species (DeLorenzo et al 2002); 0.45 ug/kg (ES) in Florida surface waters and 120 ug/kg (ES) in

sediment (Pfeuffer & Rand 2004); 571 ug/kg in the sediment of tailwater ponds collecting runoff from lettuce fields in California and 17.7 ug/kg in a nearby creek (Weston et al 2004); frequent detections in the water of 30 lakes in Canada and the northeastern USA (Muir et al 2004); in the sediment of mountain lakes in the Rocky Mountain and Glacier National Parks in Western USA at up to 1.2 ug/kg (ES) (Mast et al 2007), with endosulfan sulphate steadily increasing in the sediment of the Rocky Mountains lakes since 1954, reaching a maximum in 2003 (Usenko et al 2007); Argentinain agricultural runoff, stream water, and river bottom sediments (Jergentz et al 2004, 2005; Menone et al 2008); endosulfan sulphate was the most frequent and abundant contaminant found in sediment of two creeks in south-eastern Argentina which contribute to coastal pollution (Miglioranza et al 2004a); sediment from a coastal wetland in Mexico at 0.814 ug/kg (AE) (Hernandez-Romero et al 2004); river water in Brazil (GEF SA 2002); and surface waters in Guatemala, Jamaica, Honduras and Colombia (GEF CAC 2002); suspended particles in small streams in Brittany, France (Schafer et al 2007); water from rivers

in Portugal (Cerejeira et al 2003), and Greece (Golfinopoulos et al 2003; Konstantinou et al 2006); water from the remote European mountain lakes in the Alps, Pyrenees and Caledonian mountains (Vilanova et al 2001); and Azerbaijansediment from a wetland adjacent to a wastewater treatment plant contained 31 ug/kg (BE) (Swartz et al 2003). Groundwater contamination There are many reports of groundwater contamination in India: Haryana - all samples of water from tube wells in agricultural areas were contaminated with endosulfan, with 83% of samples exceeding the EU maximum permissible level of 0.1 ug/l, with levels up to 0.405 ug/l (Kumari et al 2008); Hyderabad City - all the samples of domestic well water contained endosulfan, at levels up to 11.23 ug/l, assumed to derive from agriculture near the city (Shukla et al 2006); and Thiruvallur area of Tamil Nadu where endosulfan is used on rice - total endosulfan in open well and bore wells ranged from 1.1 to 19.2 ug/l (Jayashree & Vasudevan 2007a). It has also been found in 8% of samples from rural wells in 4 areas of Punjab, Pakistan (Tariq et al 2004).

The US reported endosulfan in 1.3% of wells tested and endosulfan sulphate in 0.3%, prior to 1990 (US EPA 2002). Endosulfan has recently been found in 38% of groundwater samples in Portugal, with 12% of them exceeding the European Unions limit of 0.1 ug/l (Goncalves et al 2007). In South Africa Dalvie et al (2003) found endosulfan in 32% of groundwater samples taken in farming areas in the Western Cape at levels up to 0.89 ug/l. It has been found in the groundwater in Morocco (El Bakouri 2007) and Guatemala (GEF CAC 2002).

Organochloride insecticides are chlorinated cyclic hydrocarbons that have molecular weights in the range of 300-550 Da(1). They have a long half-life in the human body, and cause moderate toxicity(2,3). One of such insecticides is endosulfan (6,7,8,9,10-10 hexachloro 1,5,5a,6,9,9a-hexahydro-6-methano2,4,3-hexadithioxanthiepin 3-oxide), which has been widely used in agriculture since 1960(4). The uncontrolled use of these compounds in developing countries have resulted in the deaths of animals and humans(3). Endosulfan is a polychlorinated hydrocarbon pesticide used in agriculture. Acute toxicity may result in permanent neurological impairment(5). The predominant toxicological effect is over-stimulation of the central nervous system (CNS), by inhibiting

Table I. The demographical and clinical characteristics of the cases. Characteristics n (%) Age (years) 17 4 17.4 18-44 15 65.2 45-65 3 13 65 1 4.3 Gender Male 18 78.3 Female 5 21.7 Mechanism Ingestion 23 100 Others* - Aetiology Accidental 23 100 Suicidal - Clinical CNS Seizure GTC 16 69.6 Focal motor 3 13 Agitation 1 4.3 Dizziness 4 17.4 Signs of meningial irritation - Lateralising sings - -

Abnormal-sized pupil - General Nausea 21 91.3 Vomiting 21 91.3 Diarrhoea 1 4.3 Hypotension - Hypertension - Jaundice - * Inhalation, injection, skin contamination GTC: Generalised tonic-clonic CNS: Central nervous system

KARATAS 2006sSingapore Med J 2006; 47(12) : 1031 Singapore Med J 2006; 47(12) : Ca- and Mg-ATPase and antagonising chloride ion transport in gamma-aminobutyric acid (GABA) receptors with little or no peripheral component(1). Characteristic clinical signs following acute exposure are indicative of CNS disturbances or overstimulation. These signs include seizures, nausea, vomiting, abdominal discomfort, hyperaesthesia of the mouth and face, tongue and extremities, headaches, agitation, hyperactivity, incoordination, confusion, dizziness, and myoclonus(1,3,6). Convulsions are a common and severe manifestation(7). Diarrhoea is

an unusual symptom, which was observed in one of our cases. The same symptom was also reported by Singh et al(7). In our study, nausea, vomiting and abdominal pain had been observed predominantly in up to twothirds of the cases, and these symptoms continued for six hours and then patients recovered spontaneously. Endosulfan is also toxic to the liver, kidney and lung, and can cause rhabdomyolysis in higher doses(8). Liver function tests in the form of AST or ALT could be abnormal(8). In our study, there were increased levels of AST and ALT in three patients. AST or ALT levels had been lowered with supporting treatment in two of these patients; however one case had been transferred for liver transplantation. There was no correlation with nausea, vomiting and liver function abnormality. Severe poisoning may result in death due to status epilepticus that can lead to asphyxia(3,8). None of our cases developed status epilepticus. Hypoxia may also be secondary to aspiration of vomitus or respiratory failure(1). The toxic effects generally seem to be completely reversible; hence, it is necessary to identify the poison and spare no effort to resuscitate the patient(8). Except for one case which develop liver function abnormality, in all of the other cases, the toxic effects were completely reversible. Gas

chromatography can detect endosulfan in the serum, tissue, and urine(3). Although a clinical diagnosis depends on a detailed history and suspicion, it may be necessary to measure concentrations of endosulfan for legal purposes(9,10). All of our cases had a history of exposure to endosulfan and the diagnosis was also confirmed by laboratory examination for endosulfan. In addition to general management, it has been reported that activated charcoal can be administered, although its ability to bind various organochlorines has never been adequately studied(1,3). However, it has been reported that haemoperfusion is ineffective(9). Seizures should be controlled with benzodiazepine, followed by phenobarbital if seizures persist. Phenytoin is probably less effective in these cases, given the effect of endosulfan on GABA receptors(1,3). Sood et al(11) reported a case of with endosulfan poisoning presenting with status epilepticus. Their case had a complete recovery with symptomatic treatment. In the present study, seizures were controlled with benzodiazepine, and none of the patients developed status epilepticus. It has been reported that in endosulfan poisoning, seizures are observed for a period of 30 minutes to six hours(4). In our study, seizures were seen within the first three hours in all affected patients.

Pulmonary toxicity associated with endosulfan poisoning has been reported as an important manifestation(8). We did not encounter pulmonary oedema in our cases. In our study, predominant symptoms due to endosulfan poisoning are due to the involvement of the CNS. Endosulfan poisoning should be suspected in the presence of primary CNS manifestations, with or without clinical or laboratory evidence of other organ dysfunctions such as liver failure. There was no difference among the age groups for the frequency of epileptic seizures. Endosulfan poisoning is often completely reversible with the appropriate management.

JAIN ET AL., 2011.Organochloride insecticides are chlorinated cyclic hydrocarbons that have molecular weights in the range of 300 to 550 dalton (Andrea and Edward, 2005). They have a long half-life in the human body and cause moderate toxicity. One of such insecticides is endosulfan (6,7,8,9,10-10hexachloro1,5,5a,6,9,9a-hexahydro-6methano-2,4,3-xadithioxanthiepin 3-oxide), which has been widely used in agriculture since 1960. The uncontrolled use of these compounds in developing countries has resulted in the deaths of animals and humans. Endosulfan is a polychlorinated hydrocarbon pesticide

used in agriculture. Endosulfan is a widely used insecticide that is associated with a high fatality rate in humans when ingested accidentally or with the aim of suicide. Amount ingested being greater than 35 g of endosulfan was the most independent variable that predicted patient mortality. In present case, patients consumed approximately 130 ml that is 48 g which is much greater then toxic dose. Patients with this risk factor must be treated aggressively during the early stage of endosulfan poisoning. Acute toxicity may result in permanent neurological impairment. The predominant toxicological effect is over-stimulation of the central nervous system (CNS), by inhibiting Ca- and Mg-ATPase and antagonising chloride ion transport in gammaaminobutyric acid (GABA) receptors with little or no peripheral component. Characteristic clinical signs after acute exposure are indicative of CNS disturbances or overstimulation. These signs include seizures, nausea, vomiting, abdominal discomfort, hyperaesthesia of the mouth and face, tongue and extremities, headaches, agitation, hyperactivity, incoordination, confusion, dizziness and myoclonus. Our patient was presented in a state of status epilepticus with agitation, hyperactivity and vomiting which is because of CNS stimulation as primary manifestation in acute poisoning. Endosulfan poisoning may be suspected in the presence of primary central

nervous system manifestations including seizures, with or without clinical or laboratory evidence of other organ dysfunction, such as liver failure (Karatas et al., 2006; Sood et al., 1994). Convulsions are a common and severe manifestation. Endosulfan is also toxic to the liver, kidney and lung and can cause rhabdomyolysis in higher doses. Liver function tests are abnormal in the form of deranged AST or ALT. In the present case too, acute hepatic impairment was reported as patients liver enzymes were found to be elevated 4 to 5 times of the normal serum level. But no signs and symptoms of fulminant hepatic failure were present. CPK level was 300 IU in our case which is attributed to mild rhabdomyolysis or due to persistent seizure activity but no myoglobinuria was seen. Renal functions assessed by serum levels of S. Creatinine and blood urea were within normal limit. No electrolyte abnormality was seen in our case. Severe poisoning may result in death due to status epilepticus that can lead to asphyxia. Status epilepticus refers to a condition in which there is a failure of the "normal" factors that serve to terminate a typical seizure. -Aminobutyric acid (GABA) receptor-mediated inhibition may be responsible for the normal termination of a seizure. In addition, the activation of the N-methyl-D aspartate (NMDA) receptor by the excitatory neurotransmitter glutamate may be required for

the propagation of seizure activity. Status epilepticus that is refractory to treatment may be the result of several processes and has been attributed to a mechanistic shift from inadequate GABAergic inhibitory receptor-mediated transmission to excessive NMDA excitatory receptormediated transmission. The toxic effects generally seem to be completely reversible; hence, it is necessary to identify the poison and spare no effort to resuscitate the patient. Seizures should be controlled with benzodiazepine, followed by phenobarbital if seizures persist (Murthy, 2006). Phenytoin is probably less effective in these cases, given the effect of endosulfan on GABA receptors. To date, no randomized controlled trials have been done for status epilepticus in these cases refractory to first- and second-line therapy. The most experience exists with continuous infusion (cIV) of pentobarbital, midazolam and propofol. Levetiracetam (LEV) is a new antiepileptic drug available for treatment of refractory epilepsy after endosulphan poisoning. The mean levetiracetam loading dose was 944 mg, usually given over 30 min followed by a mean maintenance dose of 2,166 mg/day. There were no serious adverse effects. Efficacy was impressive, with clinical seizure activity stopping in all patients and rarely recurring. Levetiracetam seems to desynchronize neuronal networks without affecting normal neuronal

transmission, thereby preventing burst firing. The most interesting discovery has been that levetiracetam binds to synaptic vesicle protein 2A, a regulator of vesicular traffic and therefore, of neurotransmitter release and the potency of binding seems to correlate with antiseizure efficacy. It may be preferable to utilize IV administration during acute seizure circumstances. Nonetheless, IV levetiracetam has many attractive features that will ensure

Table 2.1 Physical-Chemical Properties of Endosulfan Endosulfan Physical-Chemical Property Appearance

Reference(s)

Technical: cream to brown, Tomlin 2000 mostly beige

Common Name Trade Name

Endosulfan Thiodan,

Mackay et al.1997 Thionex, Mackay et al.1997

Endosan, Farmoz, Nufarm Class Chemical Name CAS: hexachloro-1, organochlorine 6,7,8,9,10,10- Tomlin 2000 5,5a,6,9,9a-

hexahydro-6,9-methano2,4,3-benzodioxathiepine-3oxide IUPAC: hexachloro-8,9, trinorborn-5-en-2,3(1,4,5,6,7,710-

ylenebismethylene) sulfite

Chemical Formula CAS Registry Number Molecular Weight Water Solubility

C9H6Cl6O3S 115-29-7 406.95 g/mol endosulfan 0.32, endosulfan 0.33 ( both in mg/l, 22 C)

Mackay et al.1997 Mackay et al.1997 Mackay et al.1997 Tomlin 2000

Melting Point Vapour Pressure

Technical: 70-100 C

Mackay et al.1997

0.83 mPa (20 C) for a 2:1 Tomlin 2000 mixture endosulfan of and

Partition Coefficient (log Kow) Soil Adsorption Coefficient (log Koc) Henrys Law Constant Stability

3.55

Mackay et al.1997

4.09 for soil, 20-25 C.

Mackay et al.1997

1.09 Pa.m3/mol Stable at

Mackay et al.1997 ambient Australian Stable to Registration National Review of

temperatures.

sunlight. Slowly hydrolysed Endosulfan 1998. in aqueous acids and alkalis, with the formation of the diol and sulfur dioxide. Tomlin

2.3 PRODUCTION AND USES

Endosulfan has been in commercial use since 1956 (Maier-Bode 1968). It is produced by a Diels-Alder addition of hexachloro-cyclo-pentadiene and cis-butene-1,4-diol in xylene. The adduct is then hydrolysed to form the cis-diol or di-alcohol. The reaction of this cis-diol with thionyl chloride forms the final product (German Federal Environment Agency 2007). Worldwide production of endosulfan was estimated at 10,000 metric tonnes in 1984 (WHO Endosulfan 1984). Current global production is likely to be higher as its use remains widespread (German Federal Environment Agency, 2004). Endosulfan is available as an emulsifiable concentrate (EC), water dispersible powder (WP), dispersion, dust or granules (IPCS 1988). Endosulfan is typically applied to crops using air-blast or ground boom sprayers, thus allowing for chance of drift and longer-range atmospheric transport (WHO 1984). Canadian Water Quality Guidelines for the Protection of Aquatic Life for Endosulfan 7

In Canada technical endosulfan is manufactured by Bayer and Makhteshim. It is registered in Canada to control a number of insect pests over a wide range of greenhouse food crops such as cucumber, tomato, lettuce and pepper as well as terrestrial food crops such as apple, pear, apricot, cherry, plum, peach, grapes, bean, broccoli, brussels sprouts, cabbage, lettuce, tomato, celery, corn, potato, strawberry and cauliflower. Brimble et al. (2005) state that 22,025.96 kg of endosulfan were sold in Canada. On an annual basis, the data were primarily taken from one of the years 2001 to 2003 for each of the provinces and territories and then summed across all of the provinces and territories using the data from the most recent year of data collection. The most frequent data year was 2003. As of 2007, endosulfan was banned in the European Union, the Philippines, Cambodia, and several other countries. It is still used extensively in many countries including the US and India. In July, the European Commission proposed to add endosulfan to the list of chemicals banned under the Stockholm Convention on Persistent Organic Pollutants. If approved, all use and manufacture of endosulfan would be banned globally. Canada also announced that endosulfan is under consideration for phase-out.

Routes of degradation of endosulfan in soil and water (German Federal Environment Agency 2007).

Bioconcentration and bioaccumulation

Bioconcentration data for endosulfan is available for several species of freshwater fish and invertebrates (Table 4.1). Estimates of a bioconcentration factors vary by almost four orders of magnitude ranging from 1.97 to 11583, likely due to the lipid content and the metabolic ability of the organisms tested. The endosulfan sulfate metabolite is generally included in the calculation of the bioconcentration factor, since it is just as toxic as the parent compound. Data for the white sucker exposed to endosulfan showed only a moderately low BCF ranging between 65 in muscle to 550 in liver (gill, kidney, gut and skin accumulation was also measured). Although endosulfan sulfate is typically the only metabolite included in bioconcentration calculations, investigators have detected many other metabolites; some have suggested that the sulfate is just an intermediate metabolic form in vertebrates (Schoettger 1970) and endosulfan ether is the main product of detoxification (Rao et al. 1981). Rao et al. (1981) reported finding ether, alcohol, a-hydroxy ether and lactone forms of endosulfan as well as two unidentified metabolites in tissues of the fish Macrognathus aculeatum. Endosulfan residues depurate rapidly in aquatic invertebrates and fish. Toledo and Jonsson (1992) reported depuration half-lives of 2.9 and 5.1 days for the and -isomers and 5.9 days for the endosulfan sulphate transformation product in zebra fish (Brachydanio rerio). Ernst (1977) reported a depuration half-life of 34 hours for the -isomer in marine mussels (Mytilus edulis).

6.0 ENVIRONMENTAL TOXICITY 6.1 Mode of Action Endosulfan acts as a toxic chemical to a wide variety of insects and mites on contact through the blockage of GABA-(gamma amino butyric acid) gated chlorine channels. GABA is an inhibitory neurotransmitter in the central nervous system that operates through membrane hyperpolarization as mediated by increased chloride flux into nerve cells. By impairing the inhibitory actions of this complex, and, thus, chloride influx into the nerve, hyper excitation results which, when prolonged, may lead to respiratory failure. External symptoms include depressed activity a few hours after exposure followed by hyper excitability, tremors and convulsions (Coats 1990). Convulsions can lead to death by interfering with pulmonary gas exchange and by generating severe metabolic acidosis (Coats 1990). Stimulation of the central nervous system, leading to convulsions, is the major characteristic of endosulfan toxicity (Ecobichon 1991). An alternate hypothesis of cyclodiene toxicity is based on the observation that endosulfan inhibits Ca+Mg-ATPase and Ca-ATPase (Coats 1990; Srikanth et al. 1989). This effect may be specific to the stereochemistry of the isomer, since the endosulfan did not elicit the same response in rat brain during in vitro studies (Srikanth et al. 1989). An energetic mode of action is further supported by the observation

that endosulfan impairs respiration in rat liver mitochondria, in vitro (Dubey et al. 1984) and in rainbow trout and catfish liver mitochondria, in vitro (Mishra and Shukla. 1994) and in vivo (Mishra and Shukla 1994; Arnold et al.1996). Exposure to endosulfan has resulted in both reproductive and developmental effects in nontarget animals. Endosulfan exposure resulted in impaired development in amphibians, reduced cortisol secretion in fish, impaired development of the genital tract in birds and reduced hormone levels and sperm production and produced testicular atrophy in mammals. Further, endosulfan has been shown to bind to the human estrogen receptor and exhibit significant estrogenic activity. Whether the toxicity endpoints are a result of endocrine disruption is not known. However, it is clear that organisms treated with endosulfan did exhibit some toxic effects that have historically been associated with endocrine disrupting chemicals, e.g., developmental and reproductive effects (U.S. EPA 2002). Solvents and/or emulsifiers used with endosulfan in formulated products may influence its absorption into the system through all routes. Technical endosulfan is slowly and incompletely absorbed into the body whereas absorption is more rapid in the presence of alcohols, oils, and emulsifiers (Gupta and Gupta 1979).

EPA RED 2002 Use Profile Endosulfan is a broad spectrum contact insecticide and acaricide registered for use on a wide variety of vegetables, fruits, cereal grains, and cotton, as well as ornamental shrubs, trees, vines, and ornamentals for use in commercial agricultural settings. Total average annual use of endosulfan is estimated at approximately 1.38 million pounds of active ingredient (lbs. ai), according to Agency and registrant estimates. Crops with the highest average percent drop treated are: squash (40%), eggplant (41%), cantaloupe (31%), sweet potato (31%), broccoli (26%), pears (20%), and pumpkins (20%). Crops with the highest sales in 2001 include: cotton (14.2%), cantaloupe (13.2%), tomatoes (12.2%), and potatoes (8.15%). Endosulfan is formulated as a liquid emulsifiable concentrate ( 9-34% ai)

and wettable powder (1-50% ai). The wettable powder formulation is frequently packaged in water soluble bags. Endosulfan can be applied by groundboom sprayer, fixed-wing aircraft, chemigation (potatoes only), airblast sprayer, rightsofway sprayer, low pressure handwand sprayer, high pressure handwand sprayer, backpack sprayer and dip treatment. Regulatory History Endosulfan was first registered as a pesticide in the U.S. in 1954 to control agricultural insect and mite pests on a variety of field, fruit, and vegetable crops. Human Health Assessment Toxicity Endosulfan generally has been shown to have high acute oral and inhalation toxicity as well as slightly toxic dermal toxicity. It is an irritant to the eyes and is not a dermal sensitizer. Endosulfan is neither mutagenic nor carcinogenic. Endosulfan primarily affects the nervous system. Toxic effects observed in animals from acute, subchronic, developmental neurotoxicity, and chronic/carcinogenic toxicity studies found that endosulfan causes neurotoxic effects, which are believed to result from over-stimulation of the central nervous system. Further, there is evidence (effects observed in a submitted chronic oral toxicity study in rats) that endosulfan acts as an endocrine disruptor. However, further investigation is necessary to determine the relevance and impact of such findings on public health. Dietary Exposure EPA has assessed dietary risk by estimating exposure to endosulfan residues from consumption of food and drinking water that can occur over a

single-day (acute) or longer (chronic). Generally, a dietary (food) risk estimate that is less than 100% of the acute or chronic Population Adjusted Dose does not exceed the Agencys risk concern. Acute risk estimates from exposures to food, associated with the use of endosulfan exceed the Agencys level of concern for some population subgroups. For example, for exposure resulting from applications of endosulfan, for the most exposed population subgroup, children 16 years old, the percent acute PAD value is 150% at the 99.9th percentile of exposure from consumption of food alone. The crops that contributed the most to the risks of concern are succulent beans and peas. Chronic dietary (food) exposure estimates are below the Agencys level of concern for all

Endosulfan, a dioxathiepin (broadly classified as an organochlorine), is a broad spectrum contact insecticide and acaricide that is used on a wide variety of vegetables, fruits, cereals, and cotton, as well as ornamental shrubs, trees, vines, and ornamental herbaceous plants in commercial agricultural settings. Technical grade endosulfan is composed of two stereochemical isomers: "-endosulfan and $-endosulfan, in concentrations of approximately 70% and 30%, respectively. Endosulfan was first registered in 1954 to control a broad spectrum of agricultural insect and mite pests on various crops. Use data from 1987 to 1997 indicate an average domestic use of approximately 1.38 million pounds of active ingredient per year.

MEMO FINDINGS Endosulfan is an insecticide used to kill a wide variety of insects infesting a range of crops. It is classified as a chlorinated hydrocarbon of the cyclodiene group, or organochlorine. Endosulfan exists in a and P isomeric forms. The a isomer is a more potent inhibitor of chloride flux in nerve cells (see Mechanisms of Toxicity below) and has been found at higher concentrations in air monitoring studies (see below). Usage and Reported Illnesses

1. . Today the crops most commonly treated with endosulfan are grapes, melons, lettuce, .tomatoes and cotton. 2. 3. 4. Environmental Pate 4. Endosulfan in the environment is subject to both hydrolysis and photolysis. Fungi and bacteria

degrade endosulfan under both aerobic and anaerobic conditions. Endosulfan adsorbs strongly to soil. Mechanisms of Toxicity

1. Endosulfan binds to the y-amino-butyric acid (GABA)-gated chloride channel receptor, thereby inhibiting chloride flux. This is thought to be the primary mechanism by which endosulfan causes generalized brain stimulation and neurotoxicity in mammals. Effects of endosulfan on developing male

12. The lowest oral LDsos for endosulfan were 7.38 mgllcg in male nlice and 9.58 mglkg in female rats (both by gavage)

Neurotoxicity Studies in Animals 17. A number of neurotoxicity studies were available, primarily in the rat. An acute l~eurotoxicity study in rats (gavage) showed a greater sensitivity of females compared to males (Bury, 1997). The female NOAEL was 1.5 mglkg and the male NOAEL was 12.5 mglkg, both based on mortality and clinical signs. The developmental neurotoxicity study in the rat (dietary) covered the dosing females from gestation day six through lactation day 21. The maternal LOAEL and pup developmental LOAEL were both 3.74 mglkg-day (lowest dose level tested), based on decreased bodyweights. No neurological effects were observed in either the dams or pups (highest dose level tested = 30 mglkg-day). In a recently published study (Cabaleiro et al., 2008, Effects of in utero and lactatiollal exposure to endosulfan in prefrontal cortex of male rats. Toxicol. Lett. 176:58-67), rat offspring were exposed to endosulfan via the dams during the last 13 days of gestation and 2 1 days of

lactation. Male offspring were sacrificed on post-natal days 15, 30 and 60, and the levels of small molecule neurotransmitters were measured in the prefrontal cortex of the brain. The lowest dose level tested of 0.6 mgllcg-day caused significant alterations in the content of a number of transmitting amino acids and amines. In contrast, bodyweights of dams and litter sizes were not significantly affected at this dose level, and bodyweights of the male offspring were not significantly different from controls at four of five time points. Dividing the study LOAEL by a factor of 10 yields an estimated NOAEL of 0.06 mglkg-day, which is below the critical inhalation NOAEL of 0.194 mglkg-day developed in the RCD. It should be noted that since these studies utilized oral exposures, their NOAELs and LOAELs may be significantly reduced if the route of exposure were changed to inhalation, due to the first pass effect in the liver (see Finding 11). A study in hens failed to detect any delayed neurotoxicity (Roberts and Phillips, 1983) females from gestation day six through lactation day 21. The maternal LOAEL and pup developmental LOAEL were both 3.74 mglkg-day (lowest dose level tested), based on decreased bodyweights. No neurological effects were observed in either the dams or pups (highest dose level tested = 30 mglkg-day). In a recently published study (Cabaleiro et al., 2008, Effects of in utero and lactatiollal exposure to endosulfan in prefrontal cortex of male rats. Toxicol. Lett. 176:58-67), rat offspring were exposed to endosulfan via the dams during the last 13 days of gestation and 2 1 days of lactation. Male offspring were sacrificed on postnatal days 15, 30 and 60, and the levels of small molecule neurotransmitters were measured in the prefrontal cortex of the brain. The lowest dose level tested of 0.6 mgllcg-day caused significant alterations in the content of a number of transmitting amino acids and amines. In contrast, bodyweights of dams and litter sizes were not significantly affected at this dose level, and bodyweights of the male offspring were not significantly different from controls at four of five time points. Dividing the study LOAEL by a factor of 10 yields an estimated NOAEL of 0.06 mglkg-day, which is below the critical inhalation NOAEL of 0.194 mglkg-day developed in the RCD. It should be noted that since these studies utilized oral exposures, their NOAELs and LOAELs may be significantly reduced if the route of exposure were changed to inhalation, due to the first pass effect in the liver (see Finding 11). A study in hens failed to detect any delayed neurotoxicity (Roberts and Phillips, 1983)

TP41 WHAT IS ENDOSULFAN? Endosulfan is a manufactured pesticide. It is used to control a number of insects on food crops such as grains, tea, fruits, and vegetables and on nonfood crops such as tobacco and cotton. It is also used as a wood preservative. Endosulfan is sold as a mixture of two different forms of the same chemical (referred to as and -endosulfan). It is a cream- to brown-colored solid that may appear crystalline or in flakes. It has a distinct odor similar to turpentine. Endosulfan does not burn. ENDOSULFAN 2 1. PUBLIC HEALTH STATEMENT 1.2 WHAT HAPPENS TO ENDOSULFAN WHEN IT ENTERS THE ENVIRONMENT? Endosulfan enters air, water, and soil when it is manufactured or used as a pesticide. Endosulfan is often applied to crops using sprayers. Some endosulfan in the air may travel long distances before it lands on crops, soil, or water. Endosulfan on crops usually breaks down within a few weeks. Endosulfan released to soil attaches to soil particles. Endosulfan found near hazardous waste sites is usually found in soil. Some endosulfan in soil evaporates into air, and some endosulfan in soil breaks down. However, it may stay in soil for several years before it all breaks down. Rainwater can wash endosulfan that is attached to soil particles into surface water. Endosulfan does not dissolve easily in water. Most endosulfan in surface water is attached to soil particles floating in the water or attached to soil at the bottom. The small amounts of endosulfan that dissolve in water break down over time. Depending on the conditions in the water, endosulfan may break down within 1 day or it may take several months. Some endosulfan in surface water evaporates into air and breaks down. Because it does not dissolve easily in water, only very small amounts of endosulfan are found in groundwater (water below the soil surface; for example, well water). Animals that live in endosulfan-contaminated waters can build up endosulfan in their bodies. The amount of endosulfan in their bodies may be several

times greater than in the surrounding water. More information on the chemical and physical properties of endosulfan can be found in Chapter 3. More information on its occurrence and fate in the environment can be found in Chapter 5. 1.3 HOW MIGHT I BE EXPOSED TO ENDOSULFAN? The most likely way for people to be exposed to endosulfan is by eating food contaminated with it. Endosulfan has been found in some food products such as oils, fats, and fruit and vegetable products. You can also be exposed to low levels of endosulfan by skin contact with contaminated soil or by smoking cigarettes made from tobacco that has endosulfan residues on it. Well water and public water supplies are not likely sources of exposure to endosulfan. Workers can breathe in the chemical when spraying the pesticide on crops. If you are a farmer who works with vegetable or tobacco crops, or if you work in a greenhouse to grow flowers like chrysanthemums, you may use endosulfan to control insects. Exposure can occur by breathing ENDOSULFAN 3 1. PUBLIC HEALTH STATEMENT the dust or getting the pesticide on your skin if you do not follow all the safety and handling procedures. Accidental spills and releases to the environment at hazardous waste disposal sites are also possible sources of exposure to endosulfan. The most likely exposure to endosulfan for people living near hazardous waste sites is through contact with soils containing it. Endosulfan is usually not found in the air, and it is not found in soil and water very often. When endosulfan is found in soil and water, levels of less than 1 part of endosulfan in 1 billion parts of surface water (ppb) and less than 1 part of endosulfan in 1 million parts of soil (ppm) have been reported. For more information on human exposure to endosulfan, see Chapter 5. 1.4 HOW CAN ENDOSULFAN ENTER AND LEAVE MY BODY? If you breathe air containing dust contaminated with endosulfan, it can enter your body through your lungs and pass into the bloodstream. We do not know how much of the endosulfan will pass into your bloodstream or how fast this will happen. If you swallow food, water, or soil

contaminated with endosulfan, it will enter your body and some will pass from your stomach into the bloodstream, but we do not know how much or how fast this will occur. However, studies in animals show that endosulfan passes slowly through the stomach into the body tissues after it is taken in by mouth. If you touch soil containing endosulfan (for example, at a hazardous waste site), some endosulfan will pass through the skin into the bloodstream, but we do not know how much or how fast this will occur. Studies in animals also show that when endosulfan is applied to the skin, it passes slowly through the skin into the body tissues. If you have cuts on your skin or if you have covered your skin with oils or oily lotions, endosulfan will pass through the skin faster. Much of the endosulfan that you swallow leaves in the feces without actually entering the body tissues. For people living around waste sites or processing or storage facilities, the most likely way it will enter their bodies is from skin contact or breathing contaminated dusts. Once endosulfan is in the body, it may change in the liver and kidneys into other related chemicals called metabolites. Endosulfan and the metabolites leave your body in the urine and feces within a few days or a few weeks. Chapter 2 has more information on how endosulfan enters and leaves the body. ENDOSULFAN 4 1. PUBLIC HEALTH STATEMENT 1.5 HOW CAN ENDOSULFAN AFFECT MY HEALTH? Symptoms of endosulfan poisoning have been seen in some people who were exposed to very large amounts of this pesticide during its manufacture. Symptoms of endosulfan poisoning have also been seen in people who intentionally or accidentally ate or drank large amounts of endosulfan. Most of these people experienced convulsions or other nervous system effects. Some people who intentionally ate or drank large amounts of endosulfan died. The health effects in people exposed to smaller amounts of endosulfan for longer periods are not known. We do not know whether endosulfan has ever affected the ability of people to fight disease or has ever caused cancer in people. The Department of Health and Human Services (DHHS)

(National Toxicology Program), the International Agency for Research on Cancer (IARC), and EPA have not classified endosulfan as to its ability to cause cancer. To protect the public from the harmful effects of toxic chemicals and to find ways to treat people who have been harmed, scientists use many tests. One way to see if a chemical will hurt people is to learn how the chemical is absorbed, used, and released by the body; for some chemicals, animal testing may be necessary. Animal testing may also be used to identify health effects such as cancer or birth defects. Without laboratory animals, scientists would lose a basic method to get information needed to make wise decisions to protect public health. Scientists have the responsibility to treat research animals with care and compassion. Laws today protect the welfare of research animals, and scientists must comply with strict animal care guidelines. Results from animal studies show that exposure to very large amounts of endosulfan for short periods can cause adverse nervous system effects (such as hyperexcitability, tremors, and convulsions) and death. Because the brain controls the activity of the lungs and heart, lethal or near-lethal exposures in animals have also resulted in failure of these organs. Other effects seen in animals after short-term, high-level exposures include harmful effects on the stomach, blood, liver, and kidneys. One animal study suggested that after somewhat longer exposures, there is a possibility that the body's ability to fight infection may be impaired; however, this was not ENDOSULFAN 5 1. PUBLIC HEALTH STATEMENT directly demonstrated. The kidneys, testes, and possibly the liver are the only organs in laboratory animals affected by longer term exposure to low levels of endosulfan. The seriousness of these effects is increased when animals are exposed to higher concentrations of endosulfan. Because these effects occurred in animals, they might also occur in humans. Limited studies in animals show no evidence that endosulfan causes cancer in animals. Some studies in animals have shown that endosulfan causes damage to the genetic material within

cells. For more information on how endosulfan can affect your health, see Chapter 2. 1.6 HOW CAN ENDOSULFAN AFFECT CHILDREN? This section discusses potential health effects from exposures during the period from conception to maturity at 18 years of age in humans. Children can be exposed to endosulfan by eating food contaminated with the pesticide, by accidentally ingesting the pesticide if it is stored around the house, or by breathing air contaminated with the pesticide if it is sprayed on nearby fields. There are no known unique exposure pathways for children. We do not know if childrens intake of endosulfan per kilogram of body weight is different than that of adults. There have been no studies of health effects in young children exposed to endosulfan. Adults who accidentally or intentionally ingested amounts of endosulfan much greater than those found in the environment suffered convulsions and some died. The same adverse effects would probably occur in young children if they ingested large amounts of endosulfan. We do not know whether children differ from adults in their susceptibility to health effects from endosulfan exposure. We do not know whether endosulfan affects the ability of people to have children or whether it causes birth defects in children. Studies in animals showed no evidence that endosulfan affects the ability of animals to have babies. Some studies show that large amounts of endosulfan ENDOSULFAN 6 1. PUBLIC HEALTH STATEMENT damage the testes, but it is unknown whether such large amounts affect the ability of animals to reproduce. Pregnant animals given endosulfan by mouth had some offspring with low birth weight and length, and some with skeletal variations. Often, these effects were seen at doses where the pregnant animals themselves showed signs of poisoning by the endosulfan. Because these effects occurred in animals, they might also occur in humans. We do not know for certain

whether endosulfan or its breakdown products can cross the placenta, but it is likely that they can do so. Endosulfan has been found in human breast milk, but results of studies in animals that ate endosulfan while nursing their young suggest that only very small amounts of endosulfan can find their way into breast milk.

2.2.1.4 Neurological Effects Neurotoxicity is the primary effect observed in humans following occupational exposure to endosulfan. Convulsions were reported in nine individuals exposed to the endosulfan-containing insecticide Thiodan during bagging (Ely et al. 1967). Other effects noted in at least one of the subjects prior to the onset of convulsions included malaise, nausea, vomiting, dizziness, confusion, and/or weakness. In addition, a case of long-term, possibly permanent brain damage in an industrial worker was attributed by Aleksandrowicz (1979) to endosulfan exposure. This worker was exposed by cleaning vats that contained residues of endosulfan solution. The acute phase of the poisoning was manifested by repeated convulsions and impaired consciousness. After recovery from the repeated seizure episode, the patient became disoriented and agitated. Two years later, he exhibited cognitive and emotional deterioration, memory impairment, and impairment of visual-motor coordination manifested by an inability to perform small tasks. However, modest alcohol consumption (1L of wine consumed per week) may have been a contributing factor. Limitations associated with these reports include lack of quantitative exposure data, lack of data on the duration of exposure, the possibility of multiple routes of exposure (i.e., oral and dermal, as well as inhalation) and possible concurrent exposure to other chemicals. Therefore, this information can only provide qualitative evidence of neurotoxicity associated with inhalation exposure to endosulfan in humans. Evidence of neurotoxicity was also observed in animal studies. Nose-only exposure of rats to endosulfan at concentrations of 3.6 mg/m3 in females and 12.3 mg/m3 in males resulted in trembling and ataxia (Hoechst 1983a). At higher concentrations in both sexes, tremors, tonic-clonic convulsions, and reduced

corneal, pupillary, placing, shock, paw-pinch, and cutaneous reflexes were observed. Nose-only exposure of male and female rats to concentrations of endosulfan of up to 2 mg/m3 for 6 hours/day, 5 days/week for ENDOSULFAN 21 2. HEALTH EFFECTS a total of 21 out of 29 days resulted in no observed behavioral disturbances (Hoechst 1984c). In addition, routine gross and histopathologic examination of the cerebrum, cerebellum, brain stem, optic nerve, and pituitary demonstrated no treatment-related abnormalities.

2.2.2.4 Neurological Effects The most prominent signs of acute overexposure to endosulfan in both humans and animals are hyperactivity, tremors, decreased respiration, dyspnea, salivation, and tonic-clonic convulsions. Five ENDOSULFAN 70 2. HEALTH EFFECTS cases of acute lethal poisoning in humans resulting from accidental or intentional ingestion of Thiodan were reported in an early study by Terziev et al. (1974). The ingested doses were not specified. Initial clinical signs observed in all cases included nervous system effects such as agitation, writhing, and loss of consciousness. Autopsies performed in three of the cases revealed brain edema. Central nervous system stimulation also characterized the clinical syndrome displayed by a 20-year-old man who attempted suicide by ingesting 200 mL of a 30% endosulfan formulation (Thionax) (Shemesh et al. 1988). Although the patient's stomach contents were aspirated and he was given activated charcoal to reduce absorption, in the first 2 weeks following ingestion, the patient displayed recurrent convulsions. This stage was followed by a slow recovery phase, in which psychomotor function slowly returned. One year after his attempted suicide, his mental activity (presumably psychomotor activity) was still severely impaired, and he required medication to control his seizures. This case report demonstrates that long-term brain damage can occur following acute overexposure to endosulfan in humans. The brain damage may

have been a result of a direct action of endosulfan on the brain tissue or the hypoxia that accompanied the recurring seizures and respiratory insufficiency seen within the first 2 weeks of ingestion. It is also unclear whether the effects observed may have been due, in part, to other ingredients in the Thionax. Similarly, convulsive seizures and a sustained epileptic state persisted after stomach contents were pumped and activated charcoal and anticonvulsive medication were administered in a 43-year-old man who ingested approximately 260 mg/kg endosulfan (Boereboom et al. 1998). At 4 days after exposure, the man was pronounced brain dead, and autopsy revealed cerebral hernia from massive cerebral edema. Eight additional accidental and/or intentional cases of acute poisoning with endosulfan resulting in adverse neurological effects have been reported in more recent studies, six by Blanco-Coronado et al. (1992), one by Lo et al. (1995), and one by Pradhan et al. (1997); two out of the eight resulted in death. Tonic-clonic convulsions were seen in the Blanco-Coronado et al. (1992) cases, whereas Lo et al. (1995) reported the development of muscle fasciculations and episodes of convulsions in their case. In the case reported by Pradhan et al. (1997), the patient had consumed about 75 mL of liquid endosulfan (35% w/v). In this case, in addition to tonic-clonic seizures and myoclonic jerks, the patient developed psychosis, cortical blindness and limb rigidity. Magnetic resonance imaging showed reversible lesions of the basal ganglia and occipital cortex. The amount of endosulfan ingested in the Blanco-Coronado et al. (1992) and Lo et al. (1995) reports was unknown. Central nervous system stimulation is the hallmark of acute overexposure to endosulfan in experimental animals. The spectrum of effects includes hyperexcitability, tremors, decreased respiration, tonic-clonic convulsions, and ultimately, death (Boyd and Dobos 1969; Boyd et al. 1970; Ceron et al. 1995; FMC ENDOSULFAN 71 2. HEALTH EFFECTS 1958, 1959a, 1980a; Gilbert and Mack 1995; Hoechst 1970, 1975, 1984e; Kiran and Varma 1988; Terziev et al. 1974). Convulsions have been observed after single oral doses of 10 mg/kg in dogs (FMC 1959a), after a single dose of 5 mg/kg in rats (Gilbert and Mack 1995), after a single dose of 15.1 mg/kg in rabbits (Ceron et al. 1995), after 3 daily doses of 2.5 mg/kg/day in dogs (FMC 1959a), after 10 daily

doses of 1.8 mg/kg/day in pregnant rabbits (FMC 1981), and after 14 daily doses of 10 mg/kg/day in pregnant rats (FMC 1980a). At 2.5 mg/kg/day for 14 days, pregnant rats displayed poor muscle tone and head swaying (FMC 1980a); and at 6 mg/kg/day for 14 days, pregnant rats displayed face rubbing, flaccidity, and hyperactivity (FMC 1980b). Cerebral congestion and edema are often observed at necropsy in animals that die following acute ingestion of endosulfan (Boyd and Dobos 1969; Boyd et al. 1970; Terziev et al. 1974). A study designed to test anticonvulsants for their effectiveness in reducing the lethality of endosulfan found that phenobarbital, administered following administration of a lethal dose of endosulfan (80 mg/kg), significantly decreased the mortality and signs of neurotoxicity (e.g., convulsions and spasms) (Hoechst 1984e). Some of the severe central nervous system effects described above have not been described in some intermediate or chronic ingestion studies of endosulfan in experimental animals (FMC 1959a, 1965, 1967; Hoechst 1984b, 1988b, 1989a). For example, in rats given daily gavage doses of 5 mg/kg/day (males) and 1.5 mg/kg/day (females) of endosulfan for 30 days, signs of central nervous system stimulation were observed for the first 34 days only and subsided thereafter (Dikshith et al. 1984). However, dogs that ingested feed containing 30 ppm for 54 days, 45 ppm for 52 days, and 60 ppm for up to 40 days (a TWA dose of 2.9 mg/kg/day for males or 2.6 mg/kg/day for females) showed extreme sensitivity to noise, frightened reactions to optical stimuli, and tonic contractions of the muscles of the extremities, face, and jaw (Hoechst 1989c). Animals exhibiting these symptoms were sacrificed to prevent needless suffering. Prior to sacrifice, the reflexes of these animals were tested. The placing and righting reflexes were absent, but pupillary, flexor, patellar, oral, and cutaneous reflexes were unaffected. At autopsy, results of routine gross and microscopic examination of the cerebral cortex, brain stem, cerebellum, medulla, optic and sciatic nerves, and spinal cord were normal. At slightly lower doses in this study, approximately 2.56 hours after consuming 2 mg/kg/day (males) or 1.8 mg/kg/day (females), dogs showed convulsive spasms of the jaws and abdominal muscles without vomiting. Pathology of the gastrointestinal tract did not reveal any adverse effects on these tissues, suggesting that the nervous system may have been the cause of the spasms. However, no effects on reflexes were observed, and gross and microscopic

examination of central nervous system tissue revealed no abnormalities. Thus, it is unclear whether the effects observed at this dose were centrally mediated or were responses to gastrointestinal disturbances. Increased brain weights were observed in female rats following consumption of doses of 4.59 mg/kg/day ENDOSULFAN 72 2. HEALTH EFFECTS for 13 weeks (Hoechst 1985a) and in F0 parental females in a multigeneration reproduction study at doses of 0.75 mg/kg/day (Hoechst 1984a). However, similar results were not observed in other studies in rats at doses of 2.9 mg/kg/day (males) and 3.8 mg/kg/day (females) for up to 2 years (Hoechst 1989a) or in mice or dogs in intermediate- and chronic-duration studies (FMC 1959a, 1967; Hoechst 1984b, 1988b, 1989c). Thus, the significance of the increases in brain weight is unknown, but it could have been related to edema. A series of experiments were conducted in male Long-Evans rats to (a) assess the generality of an increased and persistent susceptibility to seizures following endosulfan treatment, (b) test the bidirectionality of kindling transfer induced by chemical and electrical means, and (c) determine whether chemical kindling reflects cumulative endosulfan toxicity (Gilbert 1992; Gilbert and Mack 1995). The findings can be summarized as follows: (1) a single gavage dose of 2.5 mg/kg of endosulfan reduced the threshold for seizure activity by electrical stimulation in amygdala kindled rats; (2) previous electrical stimulation reduced the threshold for convulsions by a single endosulfan dose; (3) repeated pretreatment with endosulfan followed by a 2-week drug-free period reduced the threshold for seizures by a challenge dose of endosulfan, arguing against cumulative toxicity; and (4) repeated pretreatment with endosulfan reduced the threshold for seizures by electrical stimulation. The positive transfer to electrical kindling suggested a commonality in the mechanism between seizures induced by repeated administration of endosulfan and those produced by repeated electrical stimulation. The effects of endosulfan on the concentration of neurotransmitter substances in various regions of the brain from rats has been examined (Lakshmana and Raju 1994). These authors found that, relative to controls, treatment of newborn rats by gavage with technical endosulfan (6 mg/kg) for 8 days resulted in

changes (increases and decreases) in the levels of noradrenaline, dopamine, and serotonin in the areas of the central nervous system that were examined (olfactory bulb, hippocampus, visual cortex, brainstem, and cerebellum). Treatment for 23 days also resulted in changes in neurotransmitter levels, but either of different magnitude or different direction than those observed in the animals exposed for 8 days, indicating that duration of exposure is an important parameter to consider when dealing with very young animals. Lakshmana and Raju (1994) also conducted a behavioral test in the rats treated for 23 days and found that treated rats took 29% more time to learn a task than the matched controls. The neurobehavioral effects of endosulfan have also been examined by others. Treatment of immature male rats with 2 mg technical endosulfan/kg/day by gavage for 90 days resulted in inhibition of learning and memory processes, and increased spontaneous motor activity (Paul et al. 1994). Since motor coordination was not significantly altered, Paul et al. (1994) suggested that the impairment in memory and learning ENDOSULFAN 73 2. HEALTH EFFECTS was due to a motivation deficit rather than to motor impairment. The learning process, but not the memory process, was reinstated by a serotonin depletor, suggesting that endosulfan produced a learning deficit by increasing serotonergic activity. In a subsequent study by the same group of investigators, in which both male and female rats were tested, it was found that a 30-day treatment with endosulfan in the diet (3 mg/kg/day) increased spontaneous motor activity to a greater degree in males than in females, but there was no sex difference regarding the impairment in memory and learning processes (Paul et al. 1995). The authors (Paul et al. 1995) speculated that the more marked effect in males may have been due to males preferentially metabolizing endosulfan to a more lipophilic metabolite, endosulfan sulfate, which could have reached the central nervous system. However, other factors cannot be ruled out, in particular since based on the chemical properties described in Chapter 3, endosulfan sulfate does not appear to be significantly more lipophilic than the parent compound. In summary, neurotoxic effects of endosulfan are usually apparent only after acute ingestion of relatively high doses. Cumulative neurotoxicity does not appear to be significant. If the animal survives the acute

toxic effects, then no long-term neurotoxic effects are evident from behavioral, gross, and microscopic observations. However, some impairment may occur that can be detected only by specialized neurobehavioral testing. The highest NOAEL values and all reliable LOAEL values for neurological effects in each species and duration category are recorded in Table 2-2 and plotted in Figure 2-2. In some studies, only the - or -isomer of endosulfan was tested. In such cases, a notation regarding the specific isomer tested is included in the effect description.

2.4.2 Mechanisms of Toxicity The neurotoxic effects of endosulfan are well documented in both humans and animals, and extensive research has been conducted in recent years aimed at elucidating its mechanism of neurotoxicity. Although serious neurotoxic effects, including death, generally occur after acute exposure to concentrations much higher than those commonly found in the environment, there is concern about the possibility of accidental exposure of those occupationally exposed such as agricultural workers who apply the pesticide in the fields. In addition to neurotoxicity, exposure to endosulfan in animals has induced a wide array of effects including liver and kidney toxicity, hematological effects, alterations in the immune system, and alterations in the reproductive organs of males. The possible mechanisms of the effects on organ or systems other than the nervous system have not been as well studied as the mechanism of neurotoxicity. Speculation in this section on the mechanism(s) of action involved in effects that have not been well characterized and/or have been seen inconsistently in animal studies seems inappropriate at this time. Yet, where possible explanations for some effects have been suggested by the investigators ENDOSULFAN 112 2. HEALTH EFFECTS conducting the research, these are presented in the appropriate subsections in Section 2.5. Therefore, this section will focus mainly on the mechanism of neurotoxic effects of endosulfan. Acute exposure to large amounts of endosulfan results in frank effects manifested as hyperactivity,

muscle tremors, ataxia, and convulsions. Possible mechanisms of toxicity include (a) alteration of neurotransmitter levels in brain areas by affecting synthesis, degradation, and/or rates of release and reuptake, and/or (b) interference with the binding of those neurotransmitter to their receptors. Several studies have reported changes in neurotransmitter levels following exposure to endosulfan. For example, Gupta (1976) found that brain acetylcholinesterase activity was decreased following a single intraperitoneal injection of endosulfan in rats and postulated that the decreased activity of this enzyme resulted in an increase in brain levels of acetylcholine, which could, in turn, be responsible for the central nervous system stimulation observed. However, brain cholinesterase was increased in female rats that consumed 4.59 mg/kg/day and above for 13 weeks (Hoechst 1985a). Thus, it is unclear whether the decrease in brain acetylcholinesterase observed by Gupta (1976) was a representative finding. Neither Paul et al. (1994) nor Lakshmana and Raju (1994) found changes in the activity of acetylcholinesterase in the brain of rats treated with 2 mg endosulfan/kg/day for 90 days or with 6 mg/kg/day for 23 days, respectively. Ansari et al. (1987) also suggested that changes in neurotransmitter levels (specifically serotonin, GABA, and dopamine) in the brain may be partly responsible for the neurotoxicity of endosulfan in rats after observing hyperactivity, tremors, and convulsions following a single intraperitoneal injection of 40 mg/kg of endosulfan. More recently Paul et al. (1994) found significant increases in serotonin concentration in the cerebrum and midbrain of rats after 90 days of treatment with 2 mg/kg/day endosulfan, and in this study, spontaneous motor activity was significantly increased in the treated animals. Furthermore, Paul et al. (1994) also found a correlation between the increase in serotonin and inhibition of a learning paradigm. Lakshmana and Raju (1994) also reported changes in the concentrations of dopamine, noradrenaline, and serotonin in various brain areas of endosulfan-treated rats. In this case, treated rats took 29% more time to learn a behavioral task; however, it was not determined which neurotransmitter(s) change may have been responsible for the behavioral change. Studies have also examined the role of neurotransmitter receptors in endosulfan-induced neurotoxic

effects. For instance, a single intraperitoneal dose of 3 mg/kg of endosulfan or administration of 1 mg/kg/day for 30-days had no effect on frontal cortical 3H-serotonin binding or aggressive behavior in adult rats, but 30 daily injections of 3 mg/kg/day caused a significant increase in 3H-serotonin binding affinity and foot-shock-induced fighting (Agrawal et al. 1983). Serotonin may also play a role in the ENDOSULFAN 113 2. HEALTH EFFECTS increase in aggressive behavior (foot-shock-induced fighting) observed in rats following multiple exposures to endosulfan (Agrawal et al. 1983; Zaidi et al. 1985). Rat pups injected with 1 mg/kg/day for 25 days showed a significant increase in frontal cortical 3H-serotonin binding and exhibited a significant increase in foot-shock-induced fighting behavior (Zaidi et al. 1985). These effects were still observed 8 days after cessation of treatment. The authors concluded that endosulfan affects serotonergic function, which in turn induces neurotoxicity in both neonates and adults, as demonstrated by increased 3H-serotonin binding to the frontal cortex and aggressive behavior. A correlation between 3H-serotonin binding and aggressive behavior was also observed. These data also suggest that neonates show a greater sensitivity to endosulfan than adults. The results from several studies suggest the involvement of GABA receptors in endosulfan-induced neurotoxicity. In a series of in vitro experiments using 3H-dihydropicrotoxinin, Abalis et al. (1986), Cole and Casida (1986), Gant et al. (1987), and Ozoe and Matsumura (1986) showed that endosulfan acts as a noncompetitive GABA antagonist at the chloride channel within the GABA receptor in brain synaptosomes. Antagonism of GABAergic neurons within the central nervous system leads to generalized central nervous system stimulation. Binding of GABA to its receptor opens chloride-selective ion channels leading to influx of chloride into neurons through electrochemical gradient, resulting in hyperpolarization of the membrane and inhibition of cell firing. A reduced inhibitory drive translates into increased activity of the effector neurons. The studies mentioned above found that the ability of endosulfan to induce convulsions correlated with the potency to bind to this site and to inhibit GABA-induced chloride flux,

thus providing good evidence for this mechanism of action. A more recent study showed that -endosulfan blocked the chloride uptake induced by GABA in primary cultures of cortical neurones from 15-day old mice fetuses by interacting with the t-butylbicyclophosphorothionate (a GABA antagonist) binding site (Pomes et al. 1994). In a subsequent study, the same group of investigators found that -endosulfan had relatively low cytotoxicity (assessed by disruption of cell membrane integrity) in primary neuronal cultures of cerebellar granule cells, and that it did not increase the formation of intracellular oxidative radicals (Rosa et al. 1996). It did, however, increased mitochondrial transmembrane potential which, according to Rosa et al. (1996), could be linked to a detoxification process of the cell. The authors further stated that their findings were consistent with the view that in vivo neurotoxicity is mediated mainly by inhibition of GABAergic function and that other effects detected in vitro are less important. Currently, the GABA-antagonism mechanism of toxicity is the most widely accepted hypothesis. ENDOSULFAN 114 2. HEALTH EFFECTS As summarized in Section 2.3.3 (Metabolism), the biotransformation of endosulfan can give rise to a number of both polar and nonpolar metabolites. There is little and inconclusive information on whether the toxicological properties of endosulfan are due to the parent compound or to any of its metabolites. One could assume that the more lipophilic substances will cross cell membranes more easily than polar metabolites, accumulate to a greater extent, and perhaps be the most neurotoxic. Differential toxicity could also be related to differential affinity for the GABA receptor. What is known from oral acutelethality studies in rats and mice is that -endosulfan is approximately 3 times more toxic than -endosulfan (Dorough et al. 1978; Hoechst 1975, 1990; Maier-Bode 1968). In addition, in mice, the acute toxicity of endosulfan sulfate was comparable to that of -endosulfan (Dorough et al. 1978). Also in mice, the metabolites endosulfan -hydroxy ether, endosulfan lactone, and endosulfan ether had lethal doses 1020 times higher than the -or -isomers; the lethal dose for endosulfandiol was two orders of

magnitude higher than that of the -or -isomer (Dorough et al. 1978). Extrapolation of this information to possible potency differences in longer-term studies is clearly inappropriate since other factors, such as pharmacokinetics and possibly induction of biotransformation enzymes, play a role in longer-term studies. Evidence from some oral studies in rats suggests that there is a difference in susceptibility to some effects of endosulfan between males and females. For example, the LD50 values in females were 34 times lower than in males (Hoechst 1990), and similar observations had been made by others (Gupta 1976; Gupta and Chandra 1977). Also, in a 30-day feeding study, 3 out 10 females, but no males, died during the study and the female survivors experienced more pronounced liver toxicity than the males (Paul et al. 1995). The higher sensitivity of females is thought to be due to a greater accumulation and slower elimination of endosulfan residues than the males (Dikshith et al. 1984, 1988). Paul et al. (1995) also conducted a series of motor and neurobehavioral tests in both sexes and found that although endosulfan increased spontaneous motor activity in both sexes, the increase was significantly greater in males. They speculated that males may produce more lipophilic metabolites, such as endosulfan sulfate, than females, which could be responsible for the more marked stimulation of spontaneous activity in males. If this were the case, then endosulfan residues other than the sulfate would be responsible for the adverse liver effects. No consistent differential sensitivity has been observed in species other than the rat. Human data as well as studies in animals have provided negative evidence of carcinogenicity for endosulfan (Hack et al. 1995; Hoechst 1988b, 1989a). However, endosulfan promoted the development of altered hepatic foci in rats initiated with nitrosodiethylamine (Fransson-Steen et al. 1992). Although the mechanism of tumor promotion of endosulfan is not known, it has been suggested that it involves ENDOSULFAN 115 2. HEALTH EFFECTS inhibition of cellular communication (Kenne et al. 1994). Neurological Effects. The most prominent signs of acute exposure to high concentrations of

endosulfan in humans are hyperactivity, tremors, decreased respiration, dyspnea, salivation, and tonicclonic convulsions. These effects have been observed in cases of occupational exposure as well as following intentional or accidental ingestion of large amounts of endosulfan. Autopsies performed in three cases of lethal exposure to endosulfan in humans revealed brain edema (Terziev et al. 1974), but a more recent study found no hemorrhagic areas in the brain of a patient who also died after ingestion of endosulfan (Blanco-Coronado et al. 1992). One year after an attempted suicide by ingestion of an endosulfan-containing pesticide, the mental activity (presumably psychomotor function) of a 20-year-old man was still severely impaired, and he required medication to control his seizures (Shemesh et al. 1988). Long-term brain damage has also been associated with occupational endosulfan intoxication (Aleksandrowicz 1979). These case reports suggest that long-term brain damage can occur following exposure to high concentrations of endosulfan in humans. The brain damage may have been a result of a direct action of endosulfan on the brain tissue or the hypoxia that accompanied seizures. However, interpretation of these studies is difficult because limited information was presented regarding neuropsychiatric status before exposure to endosulfan. It is possible that not all of the defects observed post-ingestion are attributable to the pesticide. Signs of acute endosulfan intoxication similar to those reported in humans have been observed in animals. Also, cerebral congestion and edema is often observed at necropsy in animals that die following acute ingestion of endosulfan (Boyd and Dobos 1969; Boyd et al. 1970; Terziev et al. 1974). The severe central nervous system effects described above have not been described in many intermediate or chronicduration ingestion studies of endosulfan in experimental animals. This may reflect lack of careful observation of the animals, administration of relatively low doses of endosulfan, or increased tolerance to endosulfan. ENDOSULFAN 128 2. HEALTH EFFECTS

Perhaps one of the most important findings in animal studies in recent years regarding the neurological effects of endosulfan is that low doses of endosulfan reduced the threshold for seizures produced by electrical stimulation in kindled animals (Gilbert and Mack 1995). Also, the increase in seizure susceptibility was long-lasting and transferred positively to electrical kindling up to 1 month following cessation of treatment. Keeping in mind that the endosulfan doses administered to the animals in these studies are well above those typically found in the environment, these results indicate that humans with a predisposition to seizure disorders through hereditary or environmental causes may be at higher risk to the adverse effects of endosulfan and related chemicals (Gilbert and Mack 1995). Recent studies in animals have also found that administration of endosulfan can induce changes in the levels of various neurotransmitter in different brain areas (Lakshmana and Raju 1994) and alter learning and memory processes (Paul et al. 1994, 1995). The possible mechanisms by which endosulfan can induce some of the neurotoxic effects that have been observed are briefly discussed in Section 2.4.2. In summary, the frank neurotoxic effects of endosulfan are apparent only after acute ingestion of relatively high doses in animals. However, long-term decreased psychomotor function, possibly resulting from acute endosulfan exposure, have been reported by two authors (Aleksandrowicz 1979; Shemesh et al. 1988). Such effects cannot be easily measured in animals. Hence, the fact that long-term neurotoxic effects have not been observed in animals does not mean that such effects cannot occur in humans. However, no information was located that indicated that persons exposed to low levels of endosulfan might experience any neurotoxicity.

The central nervous system is a major target of endosulfan-induced toxicity in both humans and animals (Blanco-Coronado et al. 1992; Boyd and Dobos 1969; Boyd et al. 1970; Garg et al. 1980; Kiran and Varma 1988; Terziev et al. 1974). Therefore, individuals with seizure disorders, such as epilepsy, may be particularly susceptible because exposure to endosulfan may reduce the threshold for tremors, seizures, and other forms of neurotoxicity, as demonstrated in studies in rats (Gilbert and Mack 1995; Gilbert 1992).

Neurotoxicity. Information indicates that the central nervous system is the major target of endosulfaninduced toxicity in humans and animals following acute exposure by any route (Aleksandrowicz 1979; Blanco-Coronado et al. 1992; Boereboom et al. 1998; Boyd and Dobos 1969; Boyd et al. 1970; Cable and Doherty 1999; Chugh et al. 1998; Ely et al. 1967; FMC 1958, 1959a, 1980a; Gilbert and Mack 1995; ENDOSULFAN 161 2. HEALTH EFFECTS Gupta and Chandra 1975; Hoechst 1970, 1975, 1983a, 1984e, 1989b; Kiran and Varma 1988; Lakshmana and Raju 1994; Lo et al. 1995; Nicholson and Cooper 1977; Pradhan et al. 1997; Shemesh et al. 1988; Terziev et al. 1974). The most prominent signs of acute exposure to endosulfan in humans (oral and occupational) and animals (by all routes) are hyperactivity, tremors, decreased respiration, dyspnea, salivation, and tonic-clonic convulsions, which can lead to death. Neurotoxic effects are not always seen following intermediate- or chronic-duration exposure; however, chronic effects were observed in dogs (Hoechst 1989c). Also, two reports indicated that persistent cognitive brain damage may result following acute exposure (Aleksandrowicz 1979; Shemesh et al. 1988). More recent studies in animals have shown changes in neurotransmitter levels and alterations in neurobehavioral processes after exposure to endosulfan (Lakshmana and Raju 1994; Paul et al. 1995). Since the limited information available on the effects of dermally administered endosulfan suggests that this chemical behaves similarly across both routes of exposure, and neurotoxicity has been observed following inhalation exposure as well, there is no reason to suspect that the neurological effects observed following oral exposure are route-specific. Further studies investigating the mechanism for endosulfan-induced neurotoxicity would be helpful since this information might help identify special populations at risk for such effects. Furthermore, although neurotoxic effects have not generally been observed in intermediate- or chronic-duration animal studies, sensitive neurological functional end points (e.g., various reflexes, grip strength, sensory function, motor activity, or nerve conduction velocity), extensive histologic neuropathological evaluations of brain, spinal cord, and peripheral nerves, or evaluations of higher functions such as learning and memory have not

been done for long-term exposures to endosulfan. This information would be useful to assess the potential for this chemical to cause permanent neurological damage.

Environmental Fate. Endosulfan partitions to the atmosphere and soils and sediments. It is transported in the atmosphere (Gregor and Gummer 1989; Strachan et al. 1980), but it is immobile in soils (Bowman et al. 1965; El Beit et al. 1981c; Hodapp and Winterlin 1989; Stewart and Cairns 1974). It is transformed in surface waters and soils via hydrolysis (Greve and Wit 1971; Schoetteger 1970) and biodegradation (Cotham and Bidleman 1989; El Beit et al. 1981c; Greve and Wit 1971; Martens 1976; Miles and Moy 1979; Stewart and Cairns 1974). Endosulfan sulfate persists in soils (Coleman and Dolinger 1982). Additional information is needed on the extent to which the compound undergoes photochemical oxidation in the atmosphere. This information would be helpful in establishing the atmospheric half-life of the compound. Bioavailability from Environmental Media. Endosulfan can be absorbed following inhalation of contaminated workplace air and ingestion of insecticide-contaminated food (Ely et al. 1967). Dermal contact with or ingestion of endosulfan that is tightly bound to soil particles is an exposure route of concern at hazardous waste sites. No information is available on the absorption of endosulfan in either adults or children following ingestion or dermal contact with contaminated soils. Therefore, additional information is needed on the uptake of endosulfan from contaminated soil following ingestion or dermal contact. This information would be useful in determining the bioavailability of soil-bound endosulfan. ENDOSULFAN 209 5. POTENTIAL FOR HUMAN EXPOSURE Food Chain Bioaccumulation. Endosulfan is bioconcentrated by aquatic organisms (Ernst 1977; Novak and Ahmad 1989; NRCC 1975; Roberts 1972; Schimmel et al. 1977) but not by plants or animals (EPA 1982a). The compound is metabolized by terrestrial (Coleman and Dolinger 1982; El Beit et al. 1981c; Martens 1977; NRCC 1975) and aquatic organisms (Cotham and Bidleman 1989), and it does not biomagnify to any great extent in terrestrial or aquatic food chains (HSDB 1999). No additional

information on the bioaccumulation of endosulfan is needed at this time. Exposure Levels in Environmental Media. Endosulfan and endosulfan sulfate have been detected in ambient air (Bidleman 1981; Kutz et al. 1976), surface water (EPA 1980b, 1982c; Frank et al. 1982b; Maguire et al. 1983; McFall et al. 1985; Miles and Harris 1971; Willis et al. 1987), rain water (Strachan and Huneault 1979), cropland soils (Carey et al. 1979a, 1979b), and some foodstuffs (Gartrell et al. 1986; Podrebarac 1984a). However, with the exception of the food concentrations, the data are not current. Estimates of human intake of endosulfan or endosulfan sulfate are limited to ingestion of contaminated foodstuffs. Additional information is needed on the current levels of these compounds in ambient air, surface water, and soils, particularly at the 162 NPL hazardous waste sites known to be contaminated with these compounds. This information would be helpful in estimating human exposure to these compounds via contact with contaminated media. Reliable monitoring data for the levels of endosulfan in contaminated media at hazardous waste sites are needed. This information could be used in combination with the known body burdens of endosulfan to assess the potential risk of adverse health effects in populations living in the vicinity of hazardous waste sites. Exposure Levels in Humans. Endosulfan and endosulfan sulfate can be measured in human blood, urine, and tissues following exposure to high levels in workplace environments or following accidental or intentional ingestion of insecticides containing endosulfan (Coutselinis et al. 1978; Demeter and Heyndrickx 1978; Demeter et al. 1977). However, no monitoring studies are available in which human fluids or tissues were used to assess occupational or general population exposure to endosulfan. Additional data on levels in human blood and urine are needed following occupational and general population exposure, particularly exposure at hazardous waste sites, in order to correlate concentrations in these media with those in environmental media and the subsequent development of health effects, if any.

IN TECH PATHOLOGY 2. Nervous system toxicity Clinical signs, such as depression, inappatence and slight nervous symptoms such as teeth grinding and hyperexcitability reported in the rabbits suffer from subacute endosulfan toxication (Mor and Ozmen, 2010a; Mor and Ozmen, 2010b). In acute toxication by endosulfan in cattle cause rapid and difficult breathing, foamy exudates in mouth, tremors, exophtalmos, coma and death (Mor and Ozmen, 2003). At the gross examination of the brains, marked hyperemia at the meningeal vessels and slight hemorrhages in brains and cerebellums in rabbits suffer from endosulfan poisoning were reported. The occurring of findings is prominent in rabbits that had shown clinical nervous symptoms (Mor and Ozmen, 2010b). 290 Pesticides in the Modern World Effects of Pesticides Exposure Histopathology of the central nervous system (CNS) lesions are commonly included hemorrhages, marked edema with enlargement of Virchow Robin spaces, degenerations, slight perivascular cuffing and slight gliosis in the rabbits. Immunohistochemistry of the CNS were revealed a strong apoptotic activity in neurons and microglial cells in rabbits in subacute endosulfan toxicity (Mor and Ozmen, 2010b). The main biochemical changes of CNS lesions revealed decreases in serum and tissue acetylcholinesterase activity and are commonly reported in the endosulfan treated animals (Gupta, 1976; Jia and Misra, 2007; Mor and Ozmen, 2010b). Excitations are the primary CNS symptom in human. Convulsions and seizures can occur suddenly after a massive overdose. Convulsions usually accompanied by confusion, incoordination, excitability, or, in some instances, coma. Syncope may be the earliest sign of endosulfan toxicity (Moon and Chun, 2009). Endosulfan can cause (lipid peroxidation) LPO was also increased in brain and it is the most sensitive organ to oxidative damage (Ballesteros et al., 2009). Endosulfan is also decreased

mitogen activated protein kinase activity (MAPK), gap junctional communication (GJIC) and connexin 43 in neuronal stem cells (Kang et al., 2011). Endosulfan had cytotoxic effects on rat glial and neuronal cell cultures as well as on human glial and neuronal cells in an in vitro study in tissue cultures (Chan et al., 2006)

WU DESSERTATION 1.2.1.1.3. Endosulfan Endosulfan is a broad spectrum chlorinated hydrocarbon insecticide used to control insects in agriculture and as a wood preservative. It is also released into the environment during its manufacture and use. Residues on crops usually break down within a few weeks, but it may take years to degrade endsulfan bound to soil. Human exposure to endosulfan is usually from eating contaminated food, occupational exposure, inhalation, and skin contact with contaminated soil. Endosulfan is rapidly metabolized and does not bioaccumulate. Animal studies show that long-term exposure can damage the kidney, liver, testis, central nervous and immune system. An unintentional exposure to endosulfan shows neurological manifestations, liver toxicity, and required mechanical ventilation and emergent hemodialysis in human [268]. Endosulfan is also a xenoestrogen that causes reproductive and developmental damage. Studies in a village in northern Kerala, India where endosulfan is widely used for insect control shows that high exposure delayed sexual maturity, lowered testosterone levels, decreased penile length in young males and caused birth defects in males [269]. However, a three-day rat uterotrophic assay with endosulfan does not change uterine weight [270, 271]. Endosulfan is weakly estrogenic in transactivation assays, but is a potent activator of the MAPK pathway in a rat prolactinoma cell line [272]. At relatively low

concentrations (1~100 nM), endosulfan enhances ERK1/2 phosphorylation via multiple ER nongenomic pathways and promotes cell proliferation [272, 273].

ARMOUR THESIS 2.2.0 Endosulfan 2.2.1 Chemical Composition and Introduction of Use Endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano2,4,3-benzodioxathiepin-3-oxide) is a non-systemic organochlorine pesticide (insecticide/acaricide) commercially known as Thiodan (EPA, 2002; Bernab et al., 2008; Bayer Cropscience, 2008). Technical grade endosulfan is composed of two biologically active stereo-chemical isomers alpha and beta in a ratio of 70 % to 30 % respectively (Garret, 2004; EPA, 2002). Despite the introduction of organochlorine pesticides in the 1950s (endosulfan in 1957) and many of them being banned in the 1970s (Siang et al., 2007), endosulfan is still used commercially worldwide in developed and developing countries including Canada (Siang et al., 2007, Garret, 2004, and Tuduri et al., 2006). Endosulfan was thought to be a safer alternative than other organochlorine pesticides like DDT (dichlorodiphenyltrichloroethane) because it had not been shown to cause eggshell thinning, and has a much shorter half life in the environment (Bernab et al., 2008; Benguira and Hontela, 2000). 2.2.2 Applications of Endosulfan As previously mentioned endosulfan is a non-systemic (does not affect the body as a whole) insecticide, namely an acaricide (aphids, ticks and mites). Besides being used specifically for these organisms endosulfan can also be used for a wide range of other 9 organisms (Presibella et al., 2005). A short list of organisms that endosulfan is capable of

controlling include; meadow spittlebug, cucumber beetle, green stink bug, corn earworm, squash bug, thrips, grape phyloxera, grape leafhopper, leather leaf fern borer, aphids, rose chafer, lilac borer, and douglas fir needle midge (EPA, 2002; PMRA, 2007; Bayer Cropscience, 2008). The list of applicable crops and non crop vegetation include: barley, beans, brussels sprouts, cauliflower, collards, kale, corn, eggplants, oats, melons, rye, potatoes, squash, tomoatos, apples, almonds, nectarines, peaches, wheat, alfalfa, kohlrabi, Christmas trees, shade trees, and ornamental plants and shrubs (EPA, 2002; PMRA, 2007; Naqvi and Vaishnavi, 1993; Bayer Cropscience, 2008). With a large range of crop and non crop uses, it is quite realistic to assume a large potential for endosulfan to come into contact with non-target organisms and environments. 2.2.3 Mode of Action Endosulfans mode of action has not been conclusively proven, with two postulates being currently tested. The first postulate is that endosulfan affects the central nervous system by binding to the picrotoxinin site in the g-aminobutyric acid (GABA) chloride ionophore complex (Harris et al., 2000; Markey et al., 2007). GABA is an inhibitory neurotransmitter that operates through membrane polarization which is regulated through increased chloride flux (Harris et al., 2000; Gant et al., 1987; Markey et al., 2007). The second postulate is similar in fashion but deals with the inhibition of Ca+ and Mg-ATPase (Naqvi and Vishnavi, 1993; Yu, 2008). It is thought that this is only attributable to the stereo chemical specificity of -endosulfan (Harris et al., 2000). In both cases a negative after potential is created which prevents the axon from recovering (Harris et al., 2000; Naqvi and Vaishnavi, 1993; Yu, 2008). This causes continual firing of neurotransmitters from a single stimulus leading to a depressed state followed by hyper activity, tremors, convulsions, rigid paralysis, respiratory failure and eventually death in non target organisms (Harris et al., 2000; Naqvi; Vaishnavi, 1993 and Yu, 2008).

2.2.7 Persistence in the Environment Endosulfan has been shown to be moderately-highly persistent in the environment, depending on the media that it is in (EPA, 2002; Tuduri et al., 2006; Siang et al., 2007). In all media endosulfan will break down to its daughter compounds (endosulfan sulfate, diol, ether, lactone, and hydroxyether (Figure 2.3)) (Deger et al., 2003). The breakdown of the parent compound is predominantly associated with oxidative and hydrolytic mechanisms with the predominant metabolite being endosulfan sulphate which can be equally toxic as endosulfan itself (Garret, 2004; Hose et al., 2003). 2.3.5 Acetylcholine Esterase Acetylcholine esterase (AChE) is an enzyme found on the post-synaptic membrane that hydrolyzes the neurotransmitter acetylcholine (ACh), which provides a pivotal role in the maintenance of normal nerve function (Sandhal and Jenkins, 2002; Liu et al., 2007). ACh is synthesized in the pre-synaptic cleft (composed of acetyl-CoA and choline), and released into the synaptic cleft where it then reacts with receptors on the post-synaptic cleft which in turn causes a neurological response (Pope et al., 2005; Malomouzh and Nikolskii, 2007). In the case of AChE inhibition, there is a build up of ACh in the synaptic cleft, and continual firing at the receptors (overabundance of neurotransmitter) (Liu et al., 2007). In muscle fibres this causes hyper activity, tetany, paralysis, convulsion and eventually death (Liu et al., 2007; Siang et al., 2007). This is important because these effects are commonly seen in fish following exposure to organophosphate and carbamate pesticides (Liu et al., 2007; Cannard, 2006; Pope et al., 23 2005). Thus AChE inhibition is an excellent biomarker for monitoring aquatic systems in agricultural areas.

JAYSHREE 2006 The use of pesticides in agriculture has increased substantially during the past few decades, to increase the crop production and to meet the needs of the growing population. The broad spectrum insecticide endosulfan (6, 7, 8, 9, 10-hexachlor-1, 5, 5a, 6, 9, 9ahexahydro6, 9-methano-2, 3, 4-benzo dioxathiepin-3oxide), currently used throughout the world on a variety of vegetables, fruits, cereals and cotton as well as shrubs, trees, vines and ornamentals for use in commercial agricultural region (Kullman and Matsumura, 1996). Endosulfan is currently registered to control insects, mites on 60 crops. Total average annual use of endosulfan is estimated to be 1.38 million pounds of active ingredients. Endosulfan is a Persistent Organic Pollutant (POP) that enters the air, water and soil during its use and manufacture. Endosulfan and its break down products are persistent in the environment with an estimated half-life of 9 months to 6 years. Endosulfan has been ubiquitously detected in atmosphere, soil, sediments and ground water (Turner et al., 1997). It is one of the most commonly detected pesticides in ground and surface waters of India. Technical grade endosulfan comprises of two stereo isomers, and -endosulfan in a ratio of 7:3, low soluble in water and both of the isomers are

extremely toxic to fish and aquatic organisms (Sunderam et al., 1992). Endosulfan affects the central nervous system, kidney, liver, blood chemistry and parathyroid gland and has reproductive tetragenic and mutagenic effects (Paul and Balasubramaniam, 1997). Bioavailability of endosulfan was limited by its sequestration in soil organic matter (SOM) and incorporation in the soil micropores due to its long contact with soil matrix. Therefore it is important to find a way to enhance the release of the aged endosulfan from long term contaminated soils. Surfactants (surface active agents) increase the concentration of a hydrophobic compound in the aqueous phase by emulsification and solubilization. Surfactants are amphiphilic compound powering both hydrophobic and hydrophilic moieties. A phenomenon unique to surfactants is the self-assembly of molecules into dynamic clusters called micelles. Micelle formation occurs above a critical concentration of surfactant monomers referred to as the critical micelle concentration (CMC). Incorporation of hydrophobic compound in the micelles is termed as solubilization. Solubilization and lowering of the surface and interface tension are thought to be the main reasons for Int. J. Environ. Sci. Tech., 3 (3): 251-259, 2006 ISSN: 1735-1472

 Summer 2006, IRSEN, CEERS, IAU