Biodegradation of organochlorine pesticide, endosulfan, by a fungal soil isolate, Aspergillus niger
Introduction
In developing countries like India where the economy depends largely on agricultural products, one cannot afford to loose the harvest to pests. In India, 15–20% of the total harvest is destroyed by pests resulting in uncontrolled use of pesticides by the Indian farmers. India is the largest consumer of pesticides in South Asian countries where maximum (44.5%) consumption of the total pesticides is by cotton crop (Agnihotri, 1999). This results in rapid increase of soil contamination by pesticide residues.
Endosulfan (C,C′–(1,4,5,6,7,7–hexachloro–8,9,10–trinorborn 5–en–2,3–ylene) dimethyl sulfite) is a broad-spectrum insecticide. This is one of the few organochlorine insecticides, which is used extensively all over the world. Commercial endosulfan is synthesized as a mixture of two isomers approximately 70% α-endosulfan and 30% β-endosulfan. These have half-lives of only a few days in water but the toxic biological metabolite, endosulfan sulfate, has an aqueous half life of several weeks (Peterson and Batley, 1993).
Endosulfan gets sorbed to soil and sediments owing to its hydrophobic nature. This makes it persistent in soil and sediments (Rao and Murty, 1980; Leung et al., 1998; Sethunathan et al., 2002) and water (Witter et al., 1999; Chusaksri et al., 2006). Contamination and persistence of endosulfan in aquatic and soil environments lead to accumulation in crop wastes (Hernández-Rodriguez et al., 2006), macrophytes (Marzio et al., 2005), phytoplankton (DeLorenzo et al., 2002), fishes (Ramaneswari and Rao, 2000), vegetables, milk, and milk products (Kumari et al., 2005). Endosulfan is considered to be the priority pollutant (Keith and Telliard, 1979). It is extremely toxic to fish and aquatic invertebrates and has been reported for mammalian gonadal toxicity (Singh and Pandey, 1990), genotoxicity (Chaudhuri et al., 1999) and neurotoxicity (Paul and Balasubramaniam, 1997).
The chemical and physical properties of Endosulfan differ significantly from other cyclodiene insecticides and this affects both, its environmental and biological fates (Sutherland et al., 2000). Both the isomers, α-endosulfan and β-endosulfan, are degraded by attack at the sulphite group via either oxidation to form the toxic metabolite endosulfan sulfate, or by hydrolysis to form the nontoxic metabolite, endosulfan diol (Sutherland et al., 2002). Endosulfan sulfate is produced only through biological transformation, whereas, under alkaline conditions endosulfan is converted to diol (Martens, 1976). Natural transformation of endosulfan under alkaline conditions and photooxidation by UV light is insufficient for removal of endosulfan and endosulfan derivatives from the environment (Kwon et al., 2005).
Past investigations on the microbial degradation of endosulfan have revealed various intermediates of metabolism including endosulfan-sulfate, -diol, -ether, -lactone, -hydroxyether and -dialdehyde (Martens, 1976; Kwon et al., 2005). To date, production of endosulfan sulfate is the major concern of endosulfan degradation research, as this metabolite is more toxic, persists longer in soils and has bioaccumulation potential (Sutherland et al., 2002). Klebsiella oxytoca (Kwon et al., 2005), Bacillus spp. (Awasthi et al., 2003), Pandoraea sp. (Siddique et al., 2003), and Micrococcus sp. (Guha et al., 2000) are the bacteria reported to degrade endosulfan in solutions and soils. Many fungi have been tested for their ability to degrade endosulfan, including Aspergillus niger (Mukherjee and Gopal, 1994), Aspergillus terreus, Cladosporium oxysporum (Mukherjee and Mittal, 2005), Mucor thermohyalospora (Shetty et al., 2000), Fusarium ventricosum (Siddique et al., 2003), Phanerochaete chrysosporium (Kullman and Matsumura, 1996), Trichoderma harzianum (Katayama and Matsumura, 1993). Anabaena sp. (Lee et al., 2003), Chlorococcum sp., and Scenedesmus sp. (Sethunathan et al., 2004), are the photosynthetic microorganisms applied in endosulfan degradation studies.
The present study involves isolation and use of fungal cultures metabolizing endosulfan from soil having previous exposure to the pesticide. We describe here the isolation and characterization of Aspergillus niger, a fungal strain capable of metabolizing endosulfan.
Section snippets
Chemicals
Technical grade endosulfan, a 35% emulsified preparation (Excel Industries Ltd., Mumbai, India) was used in all studies. Standards α- and β-endosulfan, endosulfan sulfate and endosulfan diol (Fig. 1) were obtained from Sigma-Aldrich. All other reagents were of high purity and analytical grade. Working standard solutions of these compounds were prepared by appropriate dilution of stock solutions using n-hexane or acetone.
Microorganisms
Fungal isolates degrading endosulfan were obtained by enrichment culture in
Isolation and screening of fungal isolates
The soil enrichment samples yielded 16 fungal isolates selected on the basis of either different colony characteristics or source habitats. Successive transfer on agar plates purified the cultures, and stock cultures were stored on agar slopes containing endosulfan (100 mg l−1). Fungal isolates were characterized and identified from Agharkar Research Laboratory, Pune, India. Endosulfan gradient plate assay was applied to screen the isolates for highest tolerance to endosulfan. Growth performance
Acknowledgments
The authors are grateful to Hon’ble Vice-Chancellor, North Maharashtra University, and Prof. Chincholkar, Director, School of Life Sciences for providing necessary facilities for the study. We also thank Dr. K.P. Madhusudanan, Head, SAIF, Central Drug Research Institute, Lucknow, India for GC analysis and the Director, Agharkar Research Institute, Pune, India, for identification of fungal isolates.
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