Whole-Genome Sequencing Reveals Germ Cell Mutagenicity of α-Endosulfan in Caenorhabditis elegans.

Endosulfan is an extensively used organochlorine pesticide around the world, which was classified as a persistent organic pollutant (POP) in 2009. Although previous studies have documented the reproductive toxicity of endosulfan in a variety of organisms, little is known about the influence of endosulfan on the genome stability of germ cells and nonexposed progeny. Here we applied whole-genome sequencing to explore the germ cell mutagenicity of α-endosulfan in Caenorhabditis elegans (C. elegans). We found that, although low doses of α-endosulfan exhibited a minor effect on the reproductive capacity of C. elegans, chronic exposure to 1 µM α-endosulfan significantly increased the mutation frequencies of nonexposed progeny. Further analysis of genome-wide mutation spectra demonstrated that α-endosulfan preferentially elicited A:T → G:C substitutions and clustered mutations. By using worms deficient in DNA damage response genes, our results suggest the involvement of translesion synthesis polymerase η in modulating α-endosulfan-induced mutations in germ cells. Together, these observations reveal the germ cell mutagenicity of α-endosulfan in C. elegans and the possible underlying mechanism. In addition, our findings implicate that germ cell mutagenicity might be a necessary consideration for the health risk assessment of environmental chemicals such as POPs.

Expression of estrogen-, progesterone-, and androgen-responsive genes in MCF-7 and MDA-MB-231 cells treated with o,p'-DDT, p,p'-DDT, or endosulfan.

Endocrine disruptors are a major concern due to their possible association with hormone-dependent carcinogenesis. Some examples of compounds with such properties are organochlorine pesticides (OCPs). OCPs are persistent pollutants with high lipophilicity, long half-life, and bioaccumulation potential. In the past, some of the most commonly used OCPs were dichlorodiphenyltrichloroethane (DDT) and endosulfan. Here, we investigated the effects of o,p'-DDT, p,p'-DDT, and endosulfan and of hormones estradiol, testosterone, and progesterone on the expression of estrogen, progesterone, and androgen receptors (ER, PR, and AR) and of their target genes (KLF4, VEGFA, CCND1, PRLR, CDKN1A, and BCL6) in MCF-7 and MDA-MB-231 cells. The results confirmed that under the action of the insecticides, there are dose- and time-dependent changes in the expression of these receptors and target genes. As corroborated by an experiment with ER, PR, and AR negative MDA-MB-231 cells, the change in the expression of KLF4, VEGFA, CCND1, and PRLR in MCF-7 cells treated with o,p'-DDT and the change in CDKN1A and PRLR expression in MCF-7 cells treated with p,p'-DDT are likely mediated by ER, PR, and AR pathways. In conclusion, we have identified some targets of DDT and endosulfan and confirmed that the effects of insecticides on the expression of these target genes differ for breast cancer cell lines with different receptor statuses.

RDL A301S alone does not confer high levels of resistance to cyclodiene organochlorine or phenyl pyrazole insecticides in Plutella xylostella.

Mutations in the GABA-gated chloride channel are associated with resistance to cyclodiene organochlorine and phenyl pyrazole insecticides. The best characterised of these is A301S, which was initially identified in a Dieldrin resistant strain of Drosophila melanogaster. The orthologous mutation has been found in a variety of different crop pests including the diamond back moth Plutella xylostella. However, the contribution of this mutation to resistance in this species remains unclear. We have used the CRISPR/Cas9 system in order to edit Plutella xylostella PxGABARalpha1 to Serine at the 301 orthologous position (282 in PxGABARalpha1) in an insecticide sensitive strain isolated from Vero Beach (VB) USA. In this edited line, no high level of resistance is conferred to Dieldrin, Endosulfan or Fipronil, rather only a subtle shift in sensitivity which could not confer commercially important resistance. We conclude that the high level of commercial resistance to cyclodiene organochlorine and phenyl pyrazole insecticides observed in some field isolates of Plutella xylostella cannot arise from A282S in PxGABARalpha1 alone.

Development and removal ability of non-toxigenic Aspergillus section Flavi in presence of atrazine, chlorpyrifos and endosulfan.

This study evaluated the in vitro effect of three concentrations of atrazine, chlorpyrifos and endosulfan on the growth parameters of four non-toxigenic Aspergillus section Flavi strains. The ability of the strains to remove these pesticides in a synthetic medium was also determined. Growth parameters were measured on soil extract solid medium supplied with 5, 10 and 20mg/l of each pesticide, and conditioned to -0.70, -2.78, -7.06 and -10.0 water potential (MPa). Removal assays were performed in Czapek Doc medium (CZD) supplied with 20mg/l of each pesticide under optimal environmental conditions (-2.78 of MPa and 25°C). The residual levels of each pesticide were detected by the reversed-phase HPLC/fluorescence detection system. The lag phases of the strains significantly decreased in the presence of the pesticides with respect to the control media. This result indicates a fast adaptation to the conditions assayed. Similarly, the mycelial growth rates in the different treatments increased depending on pesticide concentrations. Aspergillus oryzae AM 1 and AM 2 strains showed high percentages of atrazine degradation (above 90%), followed by endosulfan (56 and 76%) and chlorpyrifos (50 and 73%) after 30 days of incubation. A significant (p<0.001) correlation (r=0.974) between removal percentages and growth rate was found. This study shows that non-toxigenic Aspergillus section Flavi strains from agricultural soils are able to effectively grow in the presence of high concentrations of atrazine, chlorpyrifos and endosulfan under a wide range of MPa conditions. Moreover, these strains have the ability to remove high levels of these pesticides in vitro in a short time.

The effects of imidacloprid combined with endosulfan on IgE-mediated mouse bone marrow-derived mast cell degranulation and anaphylaxis.

Low levels of endosulfan are known to stimulate mast cells to release allergic mediators, while imidacloprid can inhibit IgE-mediated mast cell degranulation. However, little information about the effects of both pesticides together on mast cell degranulation is available. To measure the effects, IgE-activated mouse bone marrow-derived mast cells (BMMCs) were treated with imidacloprid and endosulfan, individually, and simultaneously at equi-molar concentrations in tenfold steps ranging from 10-4 to 10-11 M, followed by measuring several allergy-related parameters expressed in BMMCs: the mediator production and influx of Ca2+, the phosphorylation content of NF-κB in the FcεRI signaling pathway. Then, the effects of the mixtures on IgE-induced passive systemic anaphylaxis (PSA) of BALB/c was detectded. This study clearly showed that the application of equi-molar mixtures of both pesticides with 10-4-10-5 M significantly inhibited the IgE-mediated mouse bone marrow-derived mast cells degranulation in vitro and 10-4 M of them decreased IgE-mediated PSA in vivo, as the application of imidacloprid at the same concentration alone did. Morever endosulfan alone had no remarkable stimulatory effects on any of the factors measured. In conclusion, simultaneous application of equi-molar concentrations of both pesticides generally showed highly similar responses compared to the responses to imidacloprid alone, suggesting that the effects of the mixture could be solely attributed to the effects of imidacloprid.

Effects of glyphosate on the non-target leaf beetle Cerotoma arcuata (Coleoptera: Chrysomelidae) in field and laboratory conditions.

This study aimed to assess the glyphosate application effects on the Cerotoma arcuata Oliver (Coleoptera: Chrysomelidae) population in glyphosate-resistant soybean crops. Field studies were conducted with glyphosate and the insecticide endosulfan to observe the effects of these pesticides on C. arcuata, on its damages in the crop and on the populations of natural enemies in glyphosate-resistant soybean crops. Moreover, the lethal and behavioral sublethal response of C. arcuata to glyphosate and endosulfan was conducted in the laboratory. The results of the field and laboratory experiments showed that glyphosate caused moderate toxicity and high irritability in C. arcuata and that endosulfan caused high toxicity and irritability. Therefore, the direct effect of glyphosate on C. arcuata was negative and does not explain the population increases of this pest in glyphosate-resistant soybean. However, the glyphosate also decreased the density of predators. Thus, the negative effect of glyphosate on the predators may be related to population increases of C. arcuata in glyphosate-resistant soybean crops, however, more studies are needed to better evidence this relationship. This study suggests that glyphosate can impact other non-target organisms, such as herbivorous insects and natural enemies and that the use of this herbicide will need to be carefully stewarded to prevent potential disturbances in beneficial insect communities in agricultural systems.

Control of Fusarium verticillioides (Sacc.) Nirenberg and Fumonisins by Using a Combination of Crop Protection Products and Fertilization.

Fusarium verticillioides is the most common fungal pathogen associated with maize ear rot in Tanzania. In a two-year trial, we investigated the efficacy of crop protection (insecticide and/or fungicide) and fertilizer (nitrogen and/or phosphorus) treatments in reducing the occurrence of F. verticillioides and its mycotoxins in maize grown in Tanzania. Seasonal differences were seen to have a substantial influence on the incidence and severity of insect infestation, Fusarium ear and kernel rot, biomass of F. verticillioides and contamination with fumonisins. With regard to the application of fertilizers, it was concluded that the impact on maize stalk borer injury, Fusarium symptoms and fumonisin levels was not significant, whereas crop protection significantly reduced maize damage. The application of an insecticide was most effective in reducing insect injury and as a result of the reduced insect injury the insecticide treatment also resulted in a significant decrease in Fusarium symptoms. In 2014, fumonisin levels were also significantly lower in maize treated with an insecticide. Additionally, significant positive correlations between insect damage and Fusarium symptoms were observed. In conclusion, this study clearly shows that application of an insecticide alone or in combination with a fungicide at anthesis significantly reduces insect damage and consequently reduces F. verticillioides infection and associated fumonisin contamination.

Endosulfan poisoning: An overview.

Endosulfan, an organochlorine (OC) insecticide, is a widely used agricultural pesticide, despite its life threatening toxic effects. In this review, the pharmacokinetics of endosulfan, mechanism of endosulfan toxicity, clinical presentations and management, histopathological findings, and toxicological analysis are described, in addition to its environmental toxicity. The toxic effects of endosulfan can affect many organs and systems presenting in a wide array of signs and symptoms. Although termed a restricted OC-classed pesticide, it continues to be used, especially in the developing world, owing to its beneficial effects on agriculture. Several cases of endosulfan poisoning have been reported from different regions of the world. Whether accidental or intentional, endosulfan ingestion proves to be fatal unless immediate, aggressive treatment is initiated. Management is mainly supportive as no antidote exists for endosulfan poisoning as yet. The use of endosulfan needs to be strictly regulated and eventually banned worldwide altogether to lower the current morbidity and mortality resulting from this pesticide. Additionally, monitoring biological samples, using non-invasive techniques such as breast milk sampling, can provide an effective method of observing the elimination of this environmentally persistent organic pollutant from the general population.

Histomorphological changes in testes of broad-snouted caimans (Caiman latirostris) associated with in ovo exposure to endocrine-disrupting chemicals.

Studies regarding the effects of endocrine-disrupting chemicals (EDCs) on the reproductive functions of wild animals have raised increasing concern. Thus, here we evaluated the consequences of in ovo exposure to endosulfan (END) and bisphenol A (BPA) in testes from neonatal to juvenile (Juv) caimans (Caiman latirostris). Caiman eggs were collected from areas with low to moderate anthropogenic intervention and incubated at male-producing temperature. At stage 20 of embryonic development (previous to gonad sex determination), eggs were exposed to either END (20 ppm) or BPA (1.4 ppm) and male gonad histomorphology examined in 10-day-old, 90-day-old, and Juv caimans. The relative seminiferous tubular area (RTA) was measured in testes and the proliferation index and the expression of estrogen receptor alpha (ERα) were quantified in intratubular cells. Regardless of the treatment, all eggs resulted in male hatchlings. The testes of EDC-exposed caimans presented tortuous seminiferous tubules with empty tubular lumens. The RTA of 10-day-old caimans exposed to BPA was decreased. The percentage of cells expressing ERα was not different after in ovo treatment with EDCs (compared to the Control group), although caimans exposed to END showed a different ERα distribution pattern. The proliferation index was lower in 90-day-old caimans exposed to END, and higher in Juv caimans exposed to BPA. In ovo exposure to END or BPA modified sensitive parameters of C. latirostris male gonads. The alterations described here might compromise not only the sexual maturation but also the reproductive performance of adult caimans. J. Exp. Zool. 325A:84-96, 2016. © 2015 Wiley Periodicals, Inc.

Effects of Pesticide Mixtures on Host-Pathogen Dynamics of the Amphibian Chytrid Fungus.

Anthropogenic and natural stressors often interact to affect organisms. Amphibian populations are undergoing unprecedented declines and extinctions with pesticides and emerging infectious diseases implicated as causal factors. Although these factors often co-occur, their effects on amphibians are usually examined in isolation. We hypothesized that exposure of larval and metamorphic amphibians to ecologically relevant concentrations of pesticide mixtures would increase their post-metamorphic susceptibility to the fungus Batrachochytrium dendrobatidis (Bd), a pathogen that has contributed to amphibian population declines worldwide. We exposed five anuran species (Pacific treefrog, Pseudacris regilla; spring peeper, Pseudacris crucifer; Cascades frog, Rana cascadae; northern leopard frog, Lithobates pipiens; and western toad, Anaxyrus boreas) from three families to mixtures of four common insecticides (chlorpyrifos, carbaryl, permethrin, and endosulfan) or herbicides (glyphosate, acetochlor, atrazine, and 2,4-D) or a control treatment, either as tadpoles or as newly metamorphic individuals (metamorphs). Subsequently, we exposed animals to Bd or a control inoculate after metamorphosis and compared survival and Bd load. Bd exposure significantly increased mortality in Pacific treefrogs, spring peepers, and western toads, but not in Cascades frogs or northern leopard frogs. However, the effects of pesticide exposure on mortality were negligible, regardless of the timing of exposure. Bd load varied considerably across species; Pacific treefrogs, spring peepers, and western toads had the highest loads, whereas Cascades frogs and northern leopard frogs had the lowest loads. The influence of pesticide exposure on Bd load depended on the amphibian species, timing of pesticide exposure, and the particular pesticide treatment. Our results suggest that exposure to realistic pesticide concentrations has minimal effects on Bd-induced mortality, but can alter Bd load. This result could have broad implications for risk assessment of amphibians; the outcome of exposure to multiple stressors may be unpredictable and can differ between species and life stages.

Pesticide- and sex steroid analogue-induced endocrine disruption differentially targets hypothalamo-hypophyseal-gonadal system during gametogenesis in teleosts - A review.

Pesticide-induced endocrine disruption often mimics sex steroidal action resulting in physiological functional disarray of hypothalamo-hypophyseal-gonadal (HHG) system at multiple levels. Among various group of pesticides, organochlorine and organophosphate family of pesticides are known to impart sex steroidal mimicking activity with slightly higher resemblance to estrogens when compared to androgenic action. This review will highlight the effects of organochlorine (for e.g. endosulfan) and organophosphate (for e.g. malathion) pesticides in comparison with sex-steroid analogue-induced changes on HHG axis during gametogenesis in few teleost fish models. Interestingly, the effects of these compounds have produced differential effects in juveniles and adults which also vary based on exposure dosage and duration. Further, the treatments had caused at times sexually dimorphic effects indicating that the action of these compounds bring out serious implications in sexual development. A comprehensive overview has been provided by considering all these aspects to recognize the adverse impacts of pesticide-induced endocrine disruption with special reference to endosulfan and malathion as those had been applied even today or used before for controlling agricultural pests in several Asian countries including India. This review also compares the effects of sex-steroid analogues where in sex reversal to reproductive dysfunction is evident, which may imply the extent of sexual plasticity in teleosts compared to other vertebrates.

Effect of alpha-naphthalene acetic acid and thidiazuron on seedling of economic crops grown in endosulfan sulfate-spiked sand.

The effect of two plant growth regulators, alpha-naphthalene acetic acid (NAA) and thidiazuron (TDZ) on the growth of sweet corn (Zea mays), cowpea (Vigna sinensis) and cucumber (Cucurmis sativus) seedling planted in 1-100 mg kg(-1) of endosulfan sulfate spiked sand was investigated. Endosulfan sulfate had no apparent toxicity as seedlings of these crop plants grew normally in endosulfan sulfate spiked sand. Concentration of endosulfan sulfate in sand affected the response of seedling induction by NAA or TDZ. Induction of crop seeds by NAA or TDZ did not promote growth of sweet corn, cowpea and cucumber to an appreciable extent. Both plant regulators at concentration of 10 mg l(-1) seemed to exert adverse effect on crop seedling. TDZ decreased shoot length, root length and chlorophyll contents in leaves of sweet corn and cowpea growing in endosulfan sulfate spiked sand. In contrast, NAA was not toxic and promoted growth of sweet corn and cowpea seedling. However, cucumber was affected by NAA and TDZ more than other plants. TDZ significantly decreased biomass and root length of cucumber. Also, NAA significantly decreased cucumber root length and tended to increase cucumber root dried weight when grown in 100 mg kg(-1) of endosulfan sulfate spiked sand.

Dietary lecithin potentiates thermal tolerance and cellular stress protection of milk fish (Chanos Chanos) reared under low dose endosulfan-induced stress.

Endosulfan is an organochlorine pesticide commonly found in aquatic environments that has been found to reduce thermal tolerance of fish. Lipotropes such as the food additive, Lecithin has been shown to improve thermal tolerance in fish species. This study was conducted to evaluate the role of lipotropes (lecithin) for enhancing the thermal tolerance of Chanos chanos reared under sublethal low dose endosulfan-induced stress. Two hundred and twenty-five fish were distributed randomly into five treatments, each with three replicates. Four isocaloric and isonitrogenous diets were prepared with graded levels of lecithin: normal water and fed with control diet (En0/L0), endosulfan-treated water and fed with control diet (En/L0), endosulfan-treated water and fed with 1% (En/L1%), 1.5% (En/L 1.5%) and 2% (En/L 2%) lecithin supplemented feed. The endosulfan in treated water was maintained at the level of 1/40th of LC50 (0.52ppb). At the end of the five weeks, critical temperature maxima (CTmax), lethal temperature maxima (LTmax), critical temperature minima (CTmin) and lethal temperature minima (LTmin) were Determined. There was a significant (P<0.01) effect of dietary lecithin on temperature tolerance (CTmax, LTmax, CTmin and LTmin) of the groups fed with 1, 1.5 and 2% lecithin-supplemented diet compared to control and endosulfan-exposed groups. Positive correlations were observed between CT max and LTmax (R(2)=0.934) as well as between CTmin and LTmin (R(2)=0.9313). At the end of the thermal tolerance study, endosulfan-induced changes in cellular stress enzymes (Catalase, SOD and GST in liver and gill and neurotansmitter enzyme, brain AChE) were significantly (p<0.01) improved by dietary lecithin. We herein report the role of lecithin in enhancing the thermal tolerance and protection against cellular stress in fish exposed to an organochlorine pesticide.

Impact of pesticides on plant growth promotion of Vigna radiata and non-target microbes: comparison between chemical- and bio-pesticides.

To compare the target and non-target effects of two chemical-pesticides (chlorpyrifos and endosulfan) with that of a bio-pesticide (azadirachtin), Vigna radiata (mung bean) was grown in a randomized pot experiment with recommended and higher application rates of pesticides. Colony counts enumerating specific microbial populations, viz. fungi, Pseudomonas, nitrogen-fixing bacteria, and phosphate-solubilizing microorganisms, were performed. In addition, several plant growth parameters such as root and shoot lengths were also monitored. It was observed that the pesticides exerted a suppressive effect on different microbial communities under study in the initial 30 days period. The bacterial and fungal populations in chlorpyrifos treated plants increased thereafter. Endosulfan resulted in enhancement of fungi and nitrogen-fixing bacteria, although phosphate-solubilizing microorganisms were suppressed at higher application rates. Azadirachtin, which is gaining popularity owing to its biological origin, did not result in enhancement of any microbial populations; on the other hand, it had a deleterious effect on phosphate-solubilizing bacteria. This study is the first to evaluate the non-target effects of pesticides with a comparison between chemical- and bio-pesticides, and also stresses the importance of critical investigation of bio-pesticides before their wide spread application in agriculture.

Discovery of the Rdl mutation in association with a cyclodiene resistant population of horn flies, Haematobia irritans (Diptera: Muscidae).

The horn fly, Haematobia irritans irritans, is an obligate blood-feeding parasite of cattle that causes significant economic impact in many countries. We investigated the resistance of a horn fly population from Louisiana/USA to endosulfan, a cyclodiene insecticide. Bioassays were performed in 2010 and 2011 in order to determine the resistance ratio of the population to endosulfan and a PCR assay was developed to detect the Rdl mutation which is the replacement of an alanine with a serine at the GABA receptor locus that has been associated with resistance to cyclodienes in other insect species. Endosulfan tags had provided 8 weeks of effective control in 2010 but only 1 week in 2011. After only one summer (June-September/2010) of exposure to the endosulfan tagged cattle, there was a significant increase in the resistance ratio for endosulfan in the fly population. Most flies surveyed by the PCR diagnostic assay were homozygous susceptible at the Rdl locus, the resistant (R) allele was mainly present in the heterozygous state and there was no difference in the frequency of the R allele between female and male flies. After the first year's exposure of the horn flies to the endosulfan tags, the frequency of the R allele increased significantly. However, after one year without endosulfan treatment (2011-2012), the frequency of the R allele significantly dropped. These results indicate that target site resistance was responsible, at least in part, for the resistance and that a fitness cost is possibly associated with the Rdl mutation.

Resurgence of the cotton bollworm Helicoverpa armigera in northern Greece associated with insecticide resistance.

Helicoverpa armigera has been controlled effectively with chemical insecticides in the major cotton crop production areas of northern Greece for many years. However, a resurgence of the pest was observed in 2010, which significantly affected crop production. During a 4-year survey (2007-2010), we examined the insecticide resistance status of H. armigera populations from two major and representative cotton production areas in northern Greece against seven insecticides (chlorpyrifos, diazinon, methomyl, alpha-cypermethrin, cypermethrin, gamma-cyhalothrin and endosulfan). Full dose-response bioassays on third instar larvae were performed by topical application. Lethal doses at 50% were estimated by probit analysis and resistance factors (RF) were calculated, compared to a susceptible laboratory reference strain. Resistance levels were relatively moderate until 2009, with resistance ratios below 10-fold for organophosphates and carbamates and up to 16-fold for the pyrethroid alpha-cypermethrin. However, resistance rose to 46- and 81-fold for chlorpyrifos and alpha-cypermethrin, respectively in 2010, when the resurgence of the pest was observed. None of the known pyrethroid resistance mutations were found in the pyrethroid-resistant insects. The possible association between resistance and H. armigera resurgence in Greece is discussed.

Cardiotoxicity and apoptotic activity in subacute endosulfan toxicity and the protective effect of vitamin C in rabbits: a pathological study.

Cardiovascular disease is one of the most significant causes of mortality in humans and animals, and its etiology is usually unknown. The aim of this study was to investigate the cardiac pathology of endosulfan toxicity and the protective effect of vitamin C in rabbits. Twenty-four rabbits were divided into 4 groups: (1) the END group was given a daily sublethal dose of endosulfan in corn oil by oral gavage for 6 weeks; (2) the END + C group received the endosulfan as well as vitamin C over the same 6-week period; (3) the OIL + C group received corn oil daily and vitamin C every other day; and (4) the OIL group received only corn oil daily. We observed microscopic hemorrhages, single-cell necrosis, inflammatory reactions, and fibrotic changes in the myocardium in the END group. Small hemorrhages and single-cell necrosis also were seen in some hearts in the END + C group, but no inflammation was observed. Caspase-3 immunoreactivity was more significant in myocardial cells in the END group compared with the others. A protective effect of vitamin C on lesions was observed in the END + C group. These results showed that endosulfan resulted in toxic changes in the hearts of rabbits, but this toxicity could be decreased with vitamin C treatment.

Endosulfan-induced biomarkers in Japanese rice fish (Oryzias latipes) analyzed by SELDI-TOF-MS.

The objective of this study was to find and validate estrogen-related biomarkers from plasma proteins in Oryzias latipes after exposure to an estrogen disrupting compound, α-endosulfan. The acute toxicity of α-endosulfan on O. latipes after 96 h of exposure was 13.72, 16.18, and 22.18 µg L(-1) for the LC10, LC20, and LC50 values, respectively. To confirm estrogenic disturbance by α-endosulfan, the expression level of vitellogenin in the liver of male fishes was measured at the LC10 value, and it was found to be significantly different from the reference group, confirming the estrogenic effect of endosulfan in this concentration range. Proteinchip® array techniques using a weak cation exchange (CM10) and a strong anion exchange proteinchip (Q10) in conjunction with surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) were used to determine plasma proteins of O. latipes differently expressed in response to endosulfan exposure at LC10 and LC20 concentrations. Analysis of protein profiling of the male fish exposed to α-endosulfan detected 48 significantly different protein peaks and the proteins at m/z 2819, 8462, 8860, and 9462 were significantly different (p<0.05). The protein peaks at m/z 2819, 8860, and 9462 were up-regulated and the peak at m/z 8462 was down-regulated. Therefore, these four differentially expressed proteins could be used as biomarkers to rapidly determine a possible risk of endosulfan on aquatic ecosystems, although these are not necessarily produced as a result of endocrine disruption.

Effects of a sublethal pesticide exposure on locomotor behavior: a video-tracking analysis in larval amphibians.

Organochlorine pesticides such as endosulfan have been shown to have both lethal and sublethal effects on amphibians. In this context, behavioral endpoints have proved their usefulness in evidencing impacts of such chemicals at environmental concentrations that do not necessarily cause mortality. The recent development of video-tracking technologies now offers the possibility of accurately quantifying locomotor behaviors. However, these techniques have not yet been applied to evaluating the toxicity of pesticides in amphibians. We therefore aimed at determining the potential toxicity of endosulfan on endpoints associated with locomotion after short-term environmental endosulfan exposure in Rana temporaria tadpoles and at using these data as warning systems for survival alterations after a longer exposure. To this end, we analyzed video-tracks of 64 tadpoles (two pesticide treatments: 5 and 50 µg L(-1), one control and one solvent-control) with Ethovision XT 7 software. The highest endosulfan concentration had a significant effect on all four behavioral endpoints. Contaminated tadpoles traveled shorter distances, swam less often, at a lower mean speed, and occupied a less peripherical position than control tadpoles. The lowest endosulfan concentration had similar but lower effects, and did not affect mean speed during swimming. Survival was reduced only after a long-term exposure to endosulfan and was associated with short-term behavioral dysfunctions. These results show that endosulfan strongly affects the behavioral repertory of amphibian tadpoles, but in different ways depending on concentration, thus suggesting that the pesticide has complex modes of action. Given the importance of locomotion and space use in tadpole success in their aquatic environment, these results confirm the toxic action of endosulfan. By highlighting effects before mortality markers, video-tracking systems also show their potential as sentinels of sublethal effects of pesticides.

Organochlorine pesticide, endosulfan induced cellular and organismal response in Drosophila melanogaster.

The effect of endosulfan (0.02-2.0µgmL(-1)) to Drosophila melanogaster (Oregon R(+)) at the cellular and organismal levels was examined. Third instar larvae of D. melanogaster and the strains transgenic for hsp70, hsp83 and hsp26 were exposed to endosulfan through food for 12-48h to examine the heat shock proteins (hsps), reactive oxygen species (ROS) generation, anti-oxidant stress markers and xenobiotic metabolism enzymes. We observed a concentration- and time-dependent significant induction of only small hsps (hsp23>hsp22) in the exposed organism in concurrence with a significant induction of ROS generation, oxidative stress and xenobiotic metabolism markers. Sub-organismal response was to be propagated towards organismal response, i.e., delay in the emergence of flies and decreased locomotor behaviour. Organisms with diminished locomotion also exhibited significantly lowered acetylcholinesterase activity. A significant positive correlation observed among ROS generation and different cellular endpoints (small hsps, oxidative stress markers, cytochrome P450 activities) in the exposed organism indicate a modulatory role of ROS in endosulfan-mediated cellular toxicity. The study thus suggests that the adverse effects of endosulfan in exposed Drosophila are manifested both at cellular and organismal levels and recommends Drosophila as an alternative animal model for screening the risk caused by environmental chemicals.