Molecular insight into endosulfan degradation by Ese protein from Arthrobacter: Evidence-based structural bioinformatics and quantum mechanical calculations.

Endosulfan is an organochlorine insecticide widely used for agricultural pest control. Many nations worldwide have restricted or completely banned it due to its extreme toxicity to fish and aquatic invertebrates. Arthrobacter sp. strain KW has the ability to degrade α, ß endosulfan and its intermediate metabolite endosulfate; this degradation is associated with Ese protein, a two-component flavin-dependent monooxygenase (TC-FDM). Employing in silico tools, we obtained the 3D model of Ese protein, and our results suggest that it belongs to the Luciferase Like Monooxygenase family (LLM). Docking studies showed that the residues V59, V315, D316, and T335 interact with α-endosulfan. The residues: V59, T60, V315, D316, and T335 are implicated in the interacting site with ß-endosulfan, and the residues: H17, V315, D316, T335, N364, and Q363 participate in the interaction with endosulfate. Topological analysis of the electron density by means of the Quantum Theory of Atoms in Molecules (QTAIM) and the Non-Covalent Interaction (NCI) index reveals that the Ese-ligands complexes are formed mainly by dispersive forces, where Cl atoms have a predominant role. As Ese is a monooxygenase member, we predict the homodimer formation. However, enzymatic studies must be developed to investigate the Ese protein's enzymatic and catalytic activity.

Endosulfan induced kidney cell injury by modulating ACE2 through up-regulating miR-429 in HK-2 cells.

Endosulfan, a typical organochlorine pesticide, is widely used in agricultural countries and was detected in blood samples from the general population. Studies have shown a positive correlation between chronic kidney disease of unknown aetiology (CKDu) and endosulfan. CKDu has become endemic in agricultural countries, with clinical manifestations of tubulointerstitial fibrosis.The goal of this study was to investigate the effects of endosulfan in kidney cell injury in human renal tubular epithelial cells (HK-2), focusing on apoptosis, inflammatory response, and epithelial-mesenchymal transition (EMT). We found that endosulfan induced apoptosis in HK-2 cells by up-regulating the expression of BAX, APAF-1, Caspase-3 and mitochondrial Cytochrome c was released into the cytosol. Endosulfan caused an inflammatory response, showing the increase in the secretion and mRNA expression levels of IL-6/IL-8. Endosulfan triggered EMT, characterized by downregulation of E-cadherin and upregulation of Vimentin. Western blot results showed that p-Smad3 and Smad3 protein expression were elevated while the expression of Smad7 were decreased in endosulfan-exposed groups. Dual luciferase reporter assay confirmed the potential binding capacity of miR-429 to 3'-UTR of ACE2. Endosulfan causes upregulation of miR-429 and downregulation of ACE2 in HK-2 cells. Overexpression of miR-429 or silencing of ACE2 in HK-2 cells caused apoptosis, inflammation and EMT through TGF signaling pathway. These findings suggest that endosulfan can lead to kidney cell injury by modulating ACE2 through up-regulating miR-429, providing new evidence for the pathogenesis of CKDu.

Remediation of endosulfan-contaminated water by hairy roots: removal and phytometabolization assessment.

Although many countries banned the insecticide endosulfan, it is still an environmental pollutant. Plants metabolize the two diastereomers of the formulations known as technical grade endosulfan (TGE) by two phase I pathways: hydrolysis leading to less toxic derivatives and oxidation giving endosulfan sulfate which is as toxic as endosulfan itself. We assessed the removal, bioaccumulation and phase I metabolization of TGE from water matrices using hairy root clones (HRs) of three edible species, Brassica napus, Raphanus sativus and Capsicum annuum. B. napus and C. annuum HRs removed 86% of TGE from the bioreaction media in 2 and 96 h, respectively, whereas R. sativus HRs removed 91% of TGE within 6 h of biotreatment. In the experiments with B. napus, only endosulfan sulfate was detected in both biomass and medium, whereas R. sativus and C. annuum accumulated endosulfan sulfate and endosulfan alcohol. Besides, endosulfan lactone was detected in C. annuum reaction medium. Acute ichthyotoxicity assays toward Poecilia reticulata showed that media contaminated with TGE lethal levels did not produce mortality after the phytotreatments. This research highlights the feasibility of using HRs to evaluate plant enzymatic abilities toward xenobiotics and their potential for the design of ex situ decontamination processes.

Study on the characterization of endosulfan-degrading bacterial strains isolated from contaminated rhizospheric soil.

In the present study, we have isolated endosulfan tolerant bacterial strains from the rhizosphere of plants growing in a pesticide-contaminated area. The tolerance capacities of these strains were tested up to 50,000 µg ml-1 of endosulfan. It was found that out of nineteen, four strains (EAG-EC-12, EAG-EC-13, EAG-EC-14, and EAG-EC-15) were capable of surviving up to 50,000 µg ml-1 endosulfan concentration in the media; thus, these four strains were selected for the characterization. Among four, two strains were identified as Serratia liquefaciens, while the other two strains were Bacillus sp. and Brevibacterium halotolerans. The result shows that growth of strain Serratia liquefaciens 1 and Serratia liquefaciens 2 in treated medium was statistically similar to that of control (cfu 6.8 × 107) after 24 h, while strains Bacillus sp. and Brevibacterium halotolerans have shown growth significantly less than the control. The degradation potential of these strains was analyzed against 100 to 250 µg ml-1 of endosulfan in a Minimal Broth Medium (MBM), and it was recorded that only 9, 2, 7, and 19% of endosulfan (100 µg ml-1) remain after a 72 h incubation period of Bacillus sp., Serratia liquefaciens 1, Serratia liquefaciens 2, and Brevibacterium halotolerans, respectively. This endosulfan removal potential of studied strains was decreased with an increase in concentration of endosulfan.

Biodegradation of Endosulfan-a Chlorinated Cyclodiene Pesticide by Indigenous Pseudomonas sp. MSCAS BT01.

Endosulfan remains as a lipophilic insecticide that causes serious medical problems because of biological stability and toxicity also found in air, water, soil sediments, and foodstuffs. Henceforward, the present study reveals a novel bacterial species isolated from pesticide-contaminated soil for enhanced endosulfan degradation. Next, isolated bacterial species was characterized with biochemical assays and 16S rRNA sequencing technique. Subsequently, the optimal conditions for endosulfan biodegradation such as pH, concentration of endosulfan, and bacterial growth were estimated with non-sulfur medium (NSM). Sequentially, the amount of endosulfan and compound degradation were analyzed through thin-layer chromatography and gas chromatography/mass spectrometry. Overall, the obtained results revealed the endosulfan acting as primary carbon source for bacterial growth. From the GC-MS analysis, the metabolic products released during endosulfan degradation by Pseudomonas sp. MSCAS BT01 were compared with standard GC-MS spectra. The highest (98%) endosulfan degradation was obtained at pH 7.0. The complete endosulfan degradation was achieved at 14th day of incubation and the less toxic endosulfan diol produced was observed via GC-MS. To conclude, the pesticide-contaminated isolate Pseudomonas sp. MSCAS BT01 emerged as a promising bioremediation tool and effectively employed to degrade endosulfan from contaminated soils, sediments, and wastewaters in the days yet to come.

Bioremediation of an agricultural saline soil contaminated with endosulfan and Escherichia coli by an active surface agent induced in a Penicillium crustosum culture.

This study evaluates the production of a biological active surface agent (BASA) through its surface tension (ST) and emulsifying activity (E24) for endosulfan degradation (ED) and Escherichia coli growth inhibition (EcGI) in an agricultural saline soil. The fungus, identified as Penicillium crustosum was isolated from the Citrus sinensis peel (CsP), then the surface properties were evaluated in 9 culture media through a Taguchi L9 experimental design. The culture conditions included: stirring speed, pH, carbon (C) and nitrogen (N) sources; being glucose, NH4N03, 120 rpm and pH of 5, the most significant parameters in the BASA production. The BASA identified as a lipopeptide type, showed a ST = 38 mN m-1 and E24=71%. Both properties were stable at 80 °C, while ST presented stability in the pH range of 2 - 12, and a saline concentration of 200 g L-1; E24 was also stable at a pH between 8-12. Further application of BASA and fungal inoculum to a contaminated agricultural saline soil presented an EcGI of 99.8% on the 8th day, and ED of 92.9 ± 4.7% in 30 days, respectively; being the first report that uses this fungus for pesticide and bacteria elimination from an agricultural saline soil.

Toxicological assessment of low-dose bisphenol A, lead and endosulfan combination: chronic toxicity study in male rats.

In the present study, toxic effects, both alone and combined, of bisphenol A (BPA), lead (Pb) and endosulfan (ES) in the low doses were investigated in rat liver and kidney functions. In the study, bisphenol A (BPA), lead (Pb) and endosulfan (ES) were chosen because although they are the chemicals people are most frequently exposed to, no combined toxic effect studies were conducted with these chemicals. Sixty-four male Wistar albino rats were used in the study, and they were randomly divided into eight groups (n = 8 per group); control, BPA (5 mg/kg), Pb (100 ppm), ES (0.61 mg/kg), BPA+Pb, BPA+ES, Pb+ES and BPA+P+ES. The rats were sacrificed after 65 days of treatment. Severe histopathological changes in the liver and kidney tissues were observed in the rats exposed to BPA+Pb+ES combination. Elevated malondialdehyde (MDA) in the liver and decreased superoxide dismutase activity (SOD) in the kidney tissue were detected in the BPA+Pb+ES group compared to those of the control group. It was found that serum alanine aminotransferase (ALT) and blood urea nitrogen (BUN) and creatinine (CREA) levels were higher in the BPA+Pb+ES combination group than the control group. Also, combined exposure of BPA, Pb and ES caused apoptotic cell numbers and inducible nitric oxide (iNOS) to increase in the liver and kidney tissues. The results of the present study suggested that the BPA, Pb and ES caused more dramatic changes to both histological architecture and cell apoptosis in the liver and kidney tissues when there was a combined exposure.

System biology analysis of endosulfan biodegradation in bacteria and its effect in other living systems: modeling and simulation studies.

Endosulfan is a broadly applied cyclodiene insecticide which has been in use across 80 countries since last 5 decades. Owing to its recalcitrant nature, endosulfan residues have been reported from air, water and soil causing toxicity to various non-target organisms. Microbial decontamination of endosulfan has been reported previously by several authors. In the current study, we have evaluated the pathways of endosulfan degradation and its hazardous impact on other living beings including insects, humans, plants, aquatic life and environment by in-silico methods. For establishment of the endosulfan metabolism in different ecosystems, cell designer was employed. The established model was thereafter assessed and simulated to understand the biochemical and physiological metabolism of the endosulfan in various systems of the network. Topological investigation analysis of the endosulfan metabolism validated the presence of 207 nodes and 274 edges in the network. We have concluded that biomagnification of the endosulfan generally occurs in the various elements of the ecosystem. Dynamics study of endosulfan degrading enzymes suggested the important role of monooxygenase I, II and hydrolase in endosulfan bioremediation. Endosulfan shows toxicity in human beings, fishes and plants, however it is biodegraded by the microbes. To date, there are no reports of in- silico analysis of bioremediation of endosulfan and its hazardous effects on the environment. Thus, this report can be important in terms of modelling and simulation of biodegradation network of endosulfan and similar compounds and their impact on several other systems.Communicated by Ramaswamy H. Sarma.

Isolation of lindane- and endosulfan-degrading bacteria and dominance analysis in the microbial communities by culture-dependent and independent methods.

　　Bioremediation for lindane and endosulfan removal is a cost-effective approach, but its effectiveness depends on the ability to isolate degrading functionalized microorganisms. Researchers have isolated many lindane and endosulfan degrading bacteria from enrichment cultures based on culture-dependent methods during the past decades. However, it is unknown whether the isolated bacteria can reflect the indigenous predominant degraders in enriching cultures. In this study, we compared the culture-dependent method with selective medium isolation with culture-independent method (PacBio SMRT sequencing of full-length 16S rRNA amplicon) to analyze the bacterial communities from four distinct lindane (LA1 and LC1) and endosulfan (EA1 and EC1) enrichment cultures. From all the isolates we harvested from lindane (63 isolates) and endosulfan (61 isolates) enrichment cultures, their BLAST alignment can only match 5.49 % and 4.32 % of the bacterial operational taxonomic units (OTUs), respectively. Rhodanbacter lindaniclasticus and Pandoraea thiooxydans were the rarely seen potential degrading representatives that were simultaneously enriched and isolated. This study is the first comparative analysis of microbial communities from lindane and endosulfan enrichment culture using culture-dependent and culture-independent methods. Our results suggested that developing a target-specific and efficient microbial isolation method is necessary to harvest and study representative degrading bacteria in the community.

Remedial potential of bacterial and fungal strains (Bacillus subtilis, Aspergillus niger, Aspergillus flavus and Penicillium chrysogenum) against organochlorine insecticide Endosulfan.

Endosulfan, an organochlorine insecticide, is known to cause detrimental effects to the environment and human health due to its excessive usage. Its highly toxic nature calls for an environmental-friendly approach for its detoxification. Environmental transformation of Endosulfan was assessed through biodegradation by isolated and cultured soil microbes (Bacillus subtilis (BS), Aspergillus niger (AN), Aspergillus flavus (AF) and Penicillium chrysogenum (PC)). Degradation of 10 mg/L Endosulfan was determined in aqueous solution at regular time intervals and analysed by gas chromatography-mass spectrometry for 35 days. BS and AN displayed substantial potential to degrade Endosulfan and subsequently transform it into its daughter products (95 and 77%, respectively). Endosulfan transformation followed first-order reaction kinetics. Chromatogram peaks revealed less toxic metabolites by Endosulfan transformation (Endosulfan diol, Endosulfan ether, Endosulfan hydroxyether and Endosulfan lactone). Half-life of Endosulfan obtained by various strains utilised in the experiments was in the order, PC (69) > AF (34.6) > AN (17.3) > BS (11.5) days. Statistical analysis was performed in MINITAB to evaluate the significance of results. Bioaugmentation of contaminated sites with such efficient microbes can facilitate rapid pesticide transformation and decontamination of the environment.

Metabolite identification and profile of endosulfan sulfate in three human liver preparations using liquid chromatography-high resolution mass spectrometry.

In this study, we performed the metabolism of endosulfan sulfate in human liver preparations (human liver microsomes, S9 fractions and hepatocytes) to identify new metabolites using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Endosulfan sulfate is a major oxidized metabolite of the organochlorine insecticide endosulfan, and it exhibits a similar toxicity to endosulfan. Six metabolites, including 5 novel metabolites of endosulfan sulfate, were identified in the three different human liver reaction mixtures and metabolic pathways of endosulfan sulfate were proposed. The phase I metabolites M1 and M2 were observed in human liver microsomes, S9 fractions and hepatocytes. M1 was suggested to be an endosulfan diol monosulfate and M2 was identified as (1,4,5,6,7,7-hexachloro-3-formylbicyclo[2,2,1]hept-5-en-2-yl)methyl hydrogen sulfate through the interpretation of the HRMS spectrum. The phase II metabolite M3 was produced as an endosulfan sulfate-GSH conjugate in those three liver preparations and transformed to M5 (dipeptide) in S9 fractions and hepatocytes. M3 was the most predominant metabolite identified in the three liver preparations. M4 was only detected in microsomes as an M2-GSH conjugate and was metabolized to M6 (monopeptide) in hepatocytes. These results are different from the metabolic pathway of endosulfan and suggest the possible detoxification metabolic reaction of endosulfan sulfate in living organisms.

Effect of endosulfan and bisphenol A on the expression of SUMO and UBC9.

This study was designed to investigate possible interference of Xenobiotics with SUMOylation in eukaryotic cells. To begin with, we docked 71 chemical structures from PubChem with human SUMO1 and UBC9 protein structures using Auto Dock 4.2 and Hex 6.3 and selected five compounds for binding studies in Surface Plasmon Resonance (SPR) with human SUMO1. In SPR studies, only endosulfan showed binding to SUMO1 (Kd1.313 × 10-4 M). Further, we treated HePG2 and differentiated 3T3-L1 cells with endosulfan/bisphenol A/perfluorooctanoic acid (PFOA) to test induction of oxidative stress and SUMO isoform/UBC9 expression. Treatment with these compounds resulted in higher levels of nitric oxide (NO), NOS2A mRNA, and reactive oxygen species (ROS) associated with decreased NADPH levels. Additionally, treatment with these chemicals resulted in elevated mRNA levels of IL-6 and IL-1ß in 3T3-L1 cells. In HePG2 cells, endosulfan treatment resulted in elevated mRNA levels of SUMO1, 3 and UBC9, whereas, treatment with bisphenol A resulted in increased mRNA of SUMO2, 3 and UBC9. Treatment with PFOA resulted in elevated mRNA levels of SUMO2. Apart from influencing the gene expression, endosulfan caused decrease in SUMO1-Sumoylation of few proteins. We propose that one reason for the severe health consequences of exposure to endosulfan/bisphenol could be due to induction of oxidative stress and modulation in SUMO and UBC9 gene expression.

Bioaccumulation and Distribution Behavior of Endosulfan on a Cichlid Fish: Differences Between Exposure to the Active Ingredient and a Commercial Formulation.

Persistent organic pollutants reach aquatic ecosystems during application and can bioconcentrate/biomagnify because of their lipophilic nature. Toxicological studies focus almost exclusively on the active ingredients of pesticides, instead of commercial formulations, whose toxicity can differ as a result of nonspecified ingredients. The intensive use of endosulfan as a wide-ranging insecticide over the last few decades makes it one of the most frequently detected contaminants in the aquatic environment, even after it has been restricted worldwide. The aim of the present study was to evaluate the bioaccumulation and organ distribution of waterborne endosulfan in the freshwater fish Cichlasoma dimerus, comparing the active ingredient and a commercial formulation. Males were exposed to 0.7 µg/L endosulfan for 2 wk, which was quantified (gas chromatography with an electron capture detector) in the liver, testes, gills, brain, and muscle. The results suggest rapid metabolism of α-endosulfan and ß-endosulfan isomers to endosulfan sulfate (endosulfan-S) in tissues. Isomer levels were highest in gills, indicative of recent uptake. Levels of endosulfan-S were highest in liver and testes for the active ingredient and testes and brain for the commercial formulation. For the active ingredient, endosulfan-S levels showed a positive correlation with organ-lipid percentage. No correlation was evident for the commercial formulation, indicating that the presence of adjuvants alters endosulfan distribution because gills and liver showed a higher uptake and mobilization of ß-endosulfan. These differences in organ distribution may alter tissue-specific toxicity; therefore, additives cannot be considered inactive even if nontoxic. Environ Toxicol Chem 2020;39:604-611. © 2019 SETAC.

Carbonyl reductase sniffer from the model organism daphnia: Cloning, substrate determination and inhibitory sensitivity.

Carbonyl reductases (CRs) represent a fundamental enzymatic defense mechanism against oxidative stress. While commonly two carbonyl reductases (CBR1 and CBR3) are found in mammalian genomes, invertebrate model organisms like Drosophila melanogaster express no CR but a functional homolog to human CBR1, termed sniffer. The importance of sniffer could be demonstrated in D. melanogaster where it protected against age-dependent neurodegeneration. Interestingly, the microcrustacean Daphnia harbors four copies of the CR gene (CR1, CR2, CR3, CR4) in addition to one sniffer gene. Due to this unique equipment Daphnia is an ideal model organism to investigate the function of sniffer. Recombinant sniffer from D. magna und D. pules were produced in E. coli, purified by Ni-affinity chromatography and tested with a variety of aliphatic and aromatic diketones, reactive aldehydes and precursors of advanced glycation end products (AGE). The highest catalytic activities were determined for sniffer from D. pulex with the aromatic dicarbonyls 9,10-phenanthrenequinone (kcat/Km = 2.6 s-1 x µM-1) and isatin (kcat/Km = 1.5 s-1 x µM-1). While sniffer from D. magna displayed preference for the same two substances, the respective catalytic activities were noticeably lower. Kinetic constants with aliphatic diketones were generally lower than those with aromatic dicarbonyls for both sniffer enzymes. The best aliphatic diketone as substrate for sniffer from D. magna and D. pulex was hexane-3,4-dione with kcat/Km = 0.23 s-1 µM-1 and kcat/Km = 0.35 s-1 µM-1, respectively. Poor or no detectable activity of the two sniffer enzymes was seen with the aliphatic diketones 2,5-hexanedione and 3,5-heptanedione, the aldehydes butanal, hexanal, decanal, crotonaldehyde, acrolein, trans-2-hexenal, and the AGE precursors glyoxal, methylglyoxal, furfural and glyceraldehyde, indicating no physiological function in the metabolism of short-chain aldehydes. Substrate inhibition for both sniffer enzymes was observed with the quinone substrates 1,4-naphthoquinone and 2-methyl-1,4-benzoquinone. From a variety of pesticides endosulfan turned out as an effective inhibitor of the sniffer enzymes (Ki = 9.2 µM for sniffer from D. magna, Ki = 12.0 µM for sniffer from D. pulex). In conclusion, the present results on sniffer from the protein superfamily of the short-chain dehydrogenases/reductases (SDR) in Daphnia ssp. complement earlier studies on carbonyl reductases in the same species and indicate that Daphnia is an interesting model to study the overall response to carbonyl stress.

Influence of plant growth promoting rhizobacterial strains Paenibacillus sp. IITISM08, Bacillus sp. PRB77 and Bacillus sp. PRB101 using Helianthus annuus on degradation of endosulfan from contaminated soil.

Endosulfan is a broad spectrum insecticide used in agriculture for protection of various food and non-food crops. It is persistent in nature and hence found in soil, air and water. The potential use of plants and microorganisms for the removal of endosulfan from soil was studied. Helianthus annuus plant was grown in soil spiked with 5, 10, 25 and 50 mg kg-1 concentrations of endosulfan and inoculated with plant growth promoting rhizobacterial strains Paenibacillus sp. IITISM08, Bacillus sp. PRB77 and Bacillus sp. PRB101 for 40, 80 and 120 days. Potential of plant for endosulfan uptake was evaluated by investigating the endosulfan levels in plant tissues (root and shoot). The results indicated that endosulfan accumulation followed the pattern of root > shoot as well as decrease in uptake of endosulfan in root and shoot of a plant grown in bacterial inoculated soil as compared to un-inoculated soil. Bacterial inoculation had a positive effect on endosulfan degradation. Maximum degradation of 92% at 5 mg kg-1 of endosulfan in soil was observed on inoculation with PRB101 after 120 days of inoculation. The results showed that plant growth promoting bacteria enhances plant biomass production. Lipid peroxidation was also estimated by determining the malondialdehyde (MDA) production, which is a biomarker of oxidative damage. Decrease in MDA formation by root and leaves of plants grown in the bacteria inoculated plant was also observed. The results suggested the effectiveness of plant growth promoting rhizobacteria to boost accumulation potential, biomass production and enhance remediation of endosulfan contaminated soil.

Identification of enzyme(s) capable of degrading endosulfan and endosulfan sulfate using in silico techniques.

Endosulfan is one of the most widely used organochlorine cyclodiene insecticides. Microbial oxidation of endosulfan forms endosulfan sulfate, which is more or less toxic and persistent as endosulfan. Due to lack of specificity and efficiency of microbial bioremediation technique in the field conditions, enzymatic bioremediation is receiving huge attention to clean-up the environment. In the present study, X-ray crystal structures of enzymes from Brookhaven Protein Data Bank were screened for their potential to degrade endosulfan and endosulfan sulfate using molecular docking and molecular dynamics simulation techniques. A phenol hydroxylase, 1PN0 from Trichosporon cutaneum was found to have the potential to degrade both α-endosulfan and endosulfan sulfate while a bacterial CotA laccase, 3ZDW from Bacillus subtilis has the potential to degrade α-endosulfan. The in silico result correlate with in vitro degradation study using two different strains of Trichosporon cutaneum. In vitro degradation study found that the fungal strain was capable of degrading 60.36% α-endosulfan, 70.73% ß-endosulfan, and 52.08% endosulfan sulfate. The presence of phenol hydroxylase inhibitor in the sulfur-free medium with endosulfan and endosulfan sulfate as sole sulfur source inhibits the growth of both the fungal strains. Such in silico techniques can provide an easy and reliable way to speed up the development of bioremediation processes through rapid identification of potential enzymes and microbes to counter the ever-increasing number of toxic compounds in the environment.

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.

A newly isolated Pseudomonas sp. can degrade endosulfan via hydrolytic pathway.

Endosulfan an organochlorinated pesticide was used extensively throughout the world. Its enormous and inadequate use creates environmental as well as health problems. A bacterial strain capable to utilize endosulfan as a sole source of sulfur was isolated from pesticide contaminated soil and identified as Pseudomonas sp. on the basis of 16S rRNA. Batch experiments were conducted at various initial concentrations of endosulfan, i.e. 5, 25, 50, 75 and 100 mg/l to study its rate of degradation. After three days of incubation, 70-80% of each initial concentration was degraded by the isolated strain as compared to the control. Degradation of endosulfan increased with the time of incubation and maximum degradation was observed after 5 days of incubation. GC-MS revealed that the major metabolite was endosulfan lactone, which accumulated after 5 days of incubation. Kinetic studies at various initial concentrations also revealed that the bacterium has very promising attitude to utilize endosulfan as sole source of sulfur. It was observed that the addition of auxiliary sulfur Fe(SO4)3 in any concentration (0.05, 0.01 and 0.1%) decreased the rate of degradation of endosulfan. The ratio of µmax/ Ks was high (0.03 mg/l) when endosulfan was single sulfur source as compared to the value recorded when Fe(SO4)3 was added alongwith the endosulfan. This indicates that the newly isolated bacterium attacks sulfur moiety for its degradation.

Determination of Endosulfan Concentrations in Biological Samples by GC-MS/MS.

OBJECTIVES: To establish an analytical method of the endosulfan concentrations （α-endosulfan and ß-endosulfan） in biological samples by GC-MS/MS. To observe the distribution of endosulfan in aquatic animals and provide experimental evidence for forensic identification of relevant cases. METHODS: Acetonitrile was added to the blood and muscle samples for precipitating the protein. The endosulfan concentrations were determined by GC-MS/MS in multiple reaction monitoring mode. Qualitative analysis was performed according to the retention time and ion rate, and quantitative analysis was performed by external standard working curve method. RESULTS: In blood samples, the calibration curves of α-endosulfan and ß-endosulfan ranging from 0.062 5 to 10 µg/mL had good linear relationship, the correlation coefficients （r） of which were >0.99. The limits of detection （LOD） were 1 ng/mL and 2 ng/mL and the limits of quantification （LOQ） were 4 ng/mL and 8 ng/mL, respectively. In muscle samples, the calibration curves of α-endosulfan and ß-endosulfan ranging from 0.062 5 to 10 µg/g, the r of which were >0.98. The LOD were 1 ng/g and 4 ng/g and the LOQ were 4 ng/g and 16 ng/g, respectively. The accuracy of α-endosulfan and ß-endosulfan was 90.76%-108.91% both in blood and muscle samples, the interday and intraday precision were 2.35%-8.71% and 5.44%-10.29%, respectively. In poisoning cases, endosulfan were detected in all parts of fish and crab and the content difference was statistically significant. CONCLUSIONS: The endosulfan detection method based on GC-MS/MS established in the present study is rapid, sensitive and accurate, which can be applied to the endosulfan detection in traces biological samples. The distribution of endosulfan in fish and crab was different, which can provide evidence to the sample collection and analysis for toxicological analysis in relevant forensic identification.

Influence of isolated bacterial strains on the in situ biodegradation of endosulfan and the reduction of endosulfan- contaminated soil toxicity.

The recently discovered endosulfan-degrading bacterial strains Pusillimonas sp. JW2 and Bordetella petrii NS were isolated from endosulfan-polluted water and soil environments. The optimal conditions for the growth and biodegradation activity of the strains JW2 and NS were studied in detail. In addition, the ability of the strains JW2 and NS to biodegrade endosulfan in soils during in situ bioremediation experiments was investigated. At a concentration of 2 mg of endosulfan per kilogram of soil, both JW2 and NS had positive effects on the degradation of endosulfan; JW2 degraded 100% and 91.5% of α- and ß-endosulfan, respectively, and NS degraded 95.1% and 90.3% of α- and ß-endosulfan, respectively. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) of soil samples showed the successful colonization of JW2 and NS, and the toxicity of the soil decreased, as determined by single-cell gel electrophoresis (SCGE) assays of Eiseniafetida and micronucleus (MN) assays of Viciafaba root tip cells. Furthermore, the metabolic products of the bacterially degraded endosulfan from the in situ experiments were identified as endosulfan ether and lactone. This study provided potentially foundational backgrounds information for the remediation of endosulfan-contaminated soil.