6.
7.
8 / ENCLOSURE – I
BRIEFRESUMEOF THE INTENDED WORK:
6.1 NEED FOR STUDY:
Endosulfan is a chlorinated hydrocarbon insecticide and acaricide belonging to class of chlorinated cyclodienes, a member of organochlorines. The molecular structures of its two stereochemicalisomers, are α-endosulfan and β-endosulfan. The α-isomer is asymmetric and exists as two twist chair forms. The β-isomer is symmetric. Isomerization was found to be favoured from β- to α-endosulfan.10, 15 Endosulfan is a broad-spectrum non-systemic insecticide and acaricide with contact and stomach action. It is used to control sucking, chewing, and boring insects on a wide variety of vegetables, fruits, grains, cotton, and tea, as well as ornamental shrubs, vines, and trees.
Endosulfan is not recommended for household use. Intentional misuse of endosulfan for killing fish and snailshas also been reported.1Endosulfan exhibits phytotoxicproperties.Toxic effects on plants like root growth inhibition, stunting, burning of tips and margins of leaves and affected root permeability have been reported. It is a prominent contaminant in vascular plants and lichen even in remote areas like the Arctic.
According to a report of World Health Organisation (WHO), about two million people have suffered from pesticide poisoning with about 2,00,000 deaths. Particularly, the people of developing countries have high levels of pesticides in their bodies (WHO, 1990). Pesticides exposure to environment is dependent on various factors like production formulation, processing and application doses.9
Endosulfan is highly toxic to soil micro arthropods,3 micro organisms, phytoplanktons, soil algae, actinomycetes, bacterial colonies etc.8It is also toxic to mammals like rabbits.13The disappearance of cats, frogs, bees, fresh water fishes etc. were reported form Kasaragod District in South India, where endosulfan was aerially sprayed.14Endosulfan is also highly poisonous to dogs. Reversible blindness and lack of muscle coordination has been reported in sheep and cattle, grazing on endosulfan contaminated grass target.
Many bacteria and fungi including Cornybacteriumspecies,Nocardiaspecies, Mycobacterium species, Pseudomonas luorescens, Penicilliumspecies, Aspergillusspecies, Phanerochaetechrysosporium have been reported to be endosulfan degraders.4 The present work deals with microbial degradation of endosulfan by Pseudomonas speciesthereby controlling its bioaccumulation in the environment.
6.2 REVIEW OF LITERATURE:
  1. Rainer Martens, (1976). 28 fungi, 49soilbacteria, and 10 Actinomycetes were tested for their ability to degrade the insecticide endosulfan. Using14C-labeled material, the qualitative as well as the quantitative formation of metabolites, as well as of 14CO2 could be followed. 16 fungi, 15 bacteria, and 3 Actinomyceteswere found capable of metabolizing more than 30ft area of applied endosulfan.The major metabolites detected were endosulfate, formed by oxidation of thesulfite group and endodiol, formed by hydrolysis of the ester bond. The majorityof highly active fungi formed endosulfate as the major metabolite, where as the majority of active bacteria formed endodiol. In addition to endosulfate andendodiol, individual cultures contained small quantities of endohydroxyether and two unidentified products. The very small quantities of 14CO2 evolved fromcultures indicated that an extensive mineralization of the carbon skeleton of endosulfan did not occur.
  1. Kim et.al., (2001) worked on chlorinated pesticide, endosulfan, and found that it could be degraded by Phanerochaetechrysosporium under non-ligninolyticconditions, and this did not require direct contact with mycelium. The major metabolites formed were endosulfan sulfate and endosulfandiol. The rate of degradation depended on the initial concentration. With 2.5 mg endosulfan, degradation was at 0.23 mg/day. The degradation could be described using a nonlinear rate expression that was similar to the Michaelis–Menten equation.
  1. Siddique.et,al.,(2003) reported that endosulfanis a cyclodieneorgano-chlorine currently used as an insecticide all over the world and its residues are posing a serious environmental threat. This study reports theisolation and identification of enriched microorganisms, capable ofdegrading endosulfan. Enrichment was achieved by using the insecticide as either the sole source of carbon or sulfur in parallel studies.Two strains each of fungi (F1 and F4) and bacteria (BF2 and B4) wereselected using endosulfan as a sole carbon source. A Pandoraeaspecies (Lin-3) previously isolated in their laboratory using lindane(-HCH)a carbon source was also screened for endosulfan degradation. F1and F4 (Fusariumventricosum) degraded endosulfan by as much82.2% and 91.1% and endosulfan by 78.5% and 89.9% respectively,within 15 days of incubation. Bacterial strains B4 and Lin-3 degraded endosulfan up to 79.6% and 81.8% and endosulfan up to 83.9% and86.8% respectively, in 15 days.
Among the bacterial strains isolated by providing endosulfan as a sulfur source, B4s and F4t degraded endosulfan by as much as 70.4% and 68.5% and endosulfan by 70.4% and 70.8% respectively, after15 days. Degradationof the insecticideoccurred concomitant with bacterial growth reaching an optical density (OD of 0.322 for B4 and Lin-3, respectively. High OD of 0.366was also notedwith the other bacterial strains utilizing endosulfan as a sulfur source.Fungal and bacterial strains significantly decreased the pH of the nutrient culture media while growing on endosulfan. The results of thisstudy suggest that these novel strains are a valuable source of potent endosulfan-degrading enzymes for use in enzymatic bioremediation.
  1. Shivaramaiah and Kennady, (2006) studied a bacterium capable of metabolizing endosulfan. It was isolated from cotton-growing soil and was effectively shown to degrade endosulfan intoendosulfan sulfate. The bacterium degraded 50% of the compound within 3 days of incubation. Endosulfan sulfate was the only terminal product and no other metabolites were formed during the incubation. Endosulfan and its metabolites were analyzed by gas chromatography. The metabolites formed indicated that the organism follows an oxidative pathway for metabolism of this pesticide. Therefore, the present study deals with microbial degradation of endosulfan by a soil bacterium, may provide a basis for the development of bioremediation strategies toremediate the pollutants in the environment.
  1. Jayshree and Vasudevan, (2009) have studied the effect of tween 80 and moisture regimes on Endosulfan degradation by Pseudomonas aeruginosa. Bioremediation ofendosulfan is poor solubility and restriction of appropriate biocatalyst. One promisingapproach in increasing the bioavailability of this organic compound is by addition ofsurfactants. The synthetic surfactant Tween 80 was non-toxic to soil microorganisms andinert to the soil matrix and had the additional benefit of causing an enhanced dissolutionrate for single compounds. The degradation of alpha and beta endosulfan byPseudomonas aeruginosa with Tween 80 and different moisture regimes (flooded andnon-flooded conditions) was studied. The rate of degradation was maximum (92 %) innon-flooded and Tween 80 added soil; the bacterial count was also maximum. Theaddition of synthetic surfactant Tween 80 enhanced the solubility and degradation ofendosulfan. The degradation of both the isomers were observed and accompanied withformation of endodiol and endosulfan sulfate.
6.3 OBJECTIVES:
  • Isolation and identification of biodegrading pesticide organism.
  • Estimation of degradation.
  • Optimization, and characterization of degradation.
ENCLOSURE: II
MATERIALS AND METHODS:
1. Isolation and identification of biodegrading pesticide organism.
  • Collection of soil samples of agricultural fields from different places.
  • Isolate organism by serial dilution method or by using modified method.
  • Identification of organism by Bergyal’s manual.
  • Isolation of microbial strains capable of utilizing endosulfan by using Enrichment culture technique.
2. Estimation of degradation.
  • Estimation of endosulfan residues by HPLC analysis.
3. Optimization and characterization of degradation.
  • Based on effect of soil texture on biodegradation of endosulfan isomers by pseudomonas aeruginosa.
  • Based on effect of pH on biodegradation of endosulfan in broth and slurry.
  • Based on incubation temperature on biodegradation of endosulfan in broth and soil slurry.
  • Based on incubation period.
7.1 SOURCE OF DATA:
  1. Bharathi college of Pharmacy library, BharathiNagara.
  2. E-Library from Bharathi college of Pharmacy.
  3. IISC Library, Bangalore.
  4. GKVK Library, Bangalore.
  5. Indian Institute of Horticulture Research Centre, Hesaraghatta. Bangalore
7.2METHOD OF COLLECTION OF DATA:
  • Internet
  • Scientific abstract
  • Scientific journals
  • Relevant books on application of Bio technology
7.3 Does the study require any investigation or interventions to beconducted on patients or other humans or animals?
-No-
7.4 7.4 Has ethical clearance been obtained from your institution in caseof 7.3?
-Not applicable-
ENCLOSURE: III
LIST OF REFERENCES:
  1. Anon. Death in small doses. Combodias pesticide problems and solutions 2002.
  2. Jayashree, Vasudevan. Applied ecology and Environmental research 2009; 7(1):35-44.
  3. Joy VC, Chakravarthy PP. Impact of insecticide on non target micro arthropod fauna in agricultural Soil. Ecotoxicol EnvironSaf 1991; 22(1):8-27.
  4. Kullmann SW, Matsumura F. Metabolic pathway utilizedPhanerochaetechrysosporium for degradation of thecyclodine pesticide endosulfan. Applied Environmental Microbiology 1996; 62(1):593-600.
  5. Kim YK, Kim SC,Choi.Kinetics of endosulfan degradation by PhanerochaetechrysosporiumBiotechnology Letters 2001;23(1):163-166.
  6. Naqvi SM,Vaishnavi C. Bio Accumulative potential and toxicity of Endosulfan insecticide to non target animals. Comp BiochemPhysiol C 1993; 105(3):347-61.
  7. Rener martens. Degradation of (8,9- 14C) Endosulfan by soil microorganisms. Applied and Environmental Microbiology 1976; 853-859.
  8. Romeo F, Quijano MD. Risk Assessment in a third world reality. An Endosulfan case History. International Journal of Occupational and Environment Health 2000; 6(1)4.
  9. Hussain S. Accelerated Biodegradation of Endosulfan in soil and water environments 2008
  10. Schmidt WF, Bilboulian S, Rice CP, Fettinger JC, McConnell LL, Hapeman CJ. Thermodynamic, spectroscopic, computational evidence for the irreversible conversion of β- to α-Endosulfan. Journal of Agricultural and Food Chemistry 2001;9(5):53-76.
  11. Shivaramaiah HM, Kennedy IR . Journal of Environmental Science and Health Part B2006;41(1):895-905.
  1. Siddique.Endosulfan degrading micro organisms in JEnviron Qual 2003; 32:47-54 .
  2. Susan S, Sania P. Endosulfan review of its toxicity and its effects on the Endocrine system. World Wild Life Fund – Canada 199
  3. Thanal. Conservation action, information network Thiruvananthapuram. Preliminary findings of the survey on the impact of aerial spraying on the people and the ecosystem: Long Term Monitoring – The Impact of Pesticides on the People and Ecosystem 2001.
  4. Walse SS, Scott GI, Ferry JL. Stereoselective degradation of aqueous Edosulfanin modular estuarine mesocosms formation of Endosulfanhydroxycarboxylate. J Environ Monit 2003; 5:373-37.