HCH-Abbau

Hexachlorocyclohexane (HCH) was widely used as broad-spectrum insecticide in public health (malaria control), in agriculture against pests and rodents, in forestry, and as a wood preservative. This compound was first introduced in 1940’s in two commercial grades, technical HCH and lindane. Typically, technical HCH consisted of 60-70% a-HCH, 5-12% b-HCH, 10-15% g-HCH, 6-10% d-HCH and 3-4% e-HCH, and smaller amounts of other isomers and congeners Among all isomers, only g-HCH is considered to be the only isomer with insecticidal properties. Although the use of technical HCH is now virtually discontinued in most countries, pure g-HCH (lindane) is still used in some countries around the world.

During the wasteful production of technical HCH, and particularly the purification of lindane, significant amounts of waste (see picture 1) consisting of other inactive isomers (a-, b- and d-HCH) are formed. Nevertheless all isomers are highly toxic and carcinogenic.

Fig. 1: Antonie landfill site in Bitterfeld, Former Eastern Germany

In principal, manufacture of 1000kg of technical HCH produces 140kg of lindane and 860kg of muck containing other isomers. Although such waste HCH isomers had been at times used for production of other chemicals, they were often disposed in landfills, as chemical waste, and in dump sites. Estimates show that 4.8 millon tonnes of HCH are present worldwide. Subsequent leaching and run-off from such sites has resulted in heavily contaminated soils and sediments particularly with the chemically and environmentally more stable b- and d-HCH isomers.

Since developing an effective bioremediation technology requires basic understanding of the metabolic pathways, which provides the information on whether the toxic compound is completely or incompletely mineralized or converted to less or harmful metabolites under natural conditions.

HCH isomers differ structurally from each other not only with respect to the relative orientation (axial/equatorial) of the chlorine atoms on the cyclohexane ring (Fig. 1) but also with respect to physical and chemical properties and persistence. In general, degradation rates increase with increasing number of axial Cl’s in the thermodynamically most stable conformation of an HCH isomer in agreement with a mechanism of anti-periplanar dehydrohalogenation where leaving H and Cl are both axial and in antiparallel position ("trans HCl elimination”).

Fig. 2: Technical Hexachlorocyclohexane: Mixture of a-HCH, b-HCH, g-HCH, d-HCH and e-HCH showing relative orientation of chlorines. b-HCH and d-HCH have thermodynamically more stable configurations

Despite its toxicity and persistence in the environment HCHs are biodegradable and several micro-orgnisms, both anaerobic and aerobic have been identified. Among these three strains of the genus Sphingobium (previously Sphingomonas paucimobilis) that can degrade HCH under aerobic conditions have been identified. Although these strains have been isolated from different geographical locations like Japan (S. japonicum UT26), India (S. indicum B90A) and France (S. francense Sp+) slight variations in the degradation of various HCH isomers and differences in some “lin” genes and their organisation were observed (Sharma et al., 2006). These Sphingobium strains possess coinciding lin gene sequences and organization which make them interesting to study evolutionary metabolic pathways of HCH degradation.

Although the aerobic degradation pathway of g-HCH has been studied in some detail in S. japonicum UT26, much less is known on the pathways of other isomers, especially, the b- and d-HCH. Sphingobium indicum B90A (Kumari et al., 2002) was isolated from rice rhizosphere soil in India and it aerobically transforms all HCH isomers including the most recalcitrant b-HCH. Experimental evidence has suggested that aerobic degradation of a-and g-HCH releases 5-6 chlorides showing complete mineralization, whereas only 2-3 chlorides for b- and d-HCH indicate incomplete mineralization. Several hydroxylated metabolites were found and identified during transformation of b- and d-HCH by strain B90A. Additionally, these metabolites were found and later identified in the groundwater of a former HCH production site, which shows the environmental relevance of these metabolites (Figure 2, Raina et al., 2007).

Fig. 3 General reaction pathways for the formation of hydroxylated metabolites from b- and d-HCH in S. indicum B90A

Publications

Raina, V., Hauser, A., Buser H.R, Rentsch, D., Sharma, P., Lal, R., Holliger C, Poiger, T., Müller, M. D. and Kohler H. P. –E. 2007. Hydroxylated Metabolites of b- and d-Hexachlorocyclohexane: Bacterial Formation, Stereochemical Configuration, and Occurrence in Groundwater at a Former Production Site. Environ. Sci. Technol. 41:4292-4298.

Raina, V., Suar, M., Singh, A., Prakash, O., Dadhwal, M., Gupta, S.K., Dogra, C., Lawlor, K., Lal, S., van der Meer J.R., Holliger, C., Lal, R. 2007. Enhanced biodegradation of hexachlorocyclohexane (HCH) in contaminated soils via inoculation with Sphingobium indicum B90A. Biodegradation (In Press).

Sharma, P., Raina, V., Kumari, R., Malhotra, S., Dogra, C., Kumari, H., Kohler, H. P. -E., Buser, H.R., Holliger, C., and Lal, R. 2006. Haloalkane dehalogenase LinB is responsible for beta- and delta-hexachlorocyclohexane transformation in Sphingobium indicum B90A. Appl Environ Microbiol. 72:5720-7.

Suar, M., Dogra, C., Raina, V., Poiger, T., Hauser, A., Kohler, H. P. -E., Holliger, C., Vander Meer J. R., and Lal, R. 2005. Enantioselective transformation of chiral alpha HCH by HCH dehydrochlorinases (LinA1 and LinA2) from Sphingomonas paucimobilis B90A. Appl. Environ. Microbiol. 71:8514-8518.

Prakash, O., Suar, M., Raina V., Dogra, C., Pal, R., and Lal R. 2004. Residues of Hexachlorocyclohexane (HCH) Residues of hexachlorocyclohexane isomers in soil and water samples from Delhi and adjoining areas. Curr. Sci. 87:73-77.

Dogra, C., Raina, V., Pal, R., Suar, M. Lal, S. Gartemann., K.H., van der Meer, J.R., Holliger, C. and Lal, R. 2004. Organization of lin genes and IS6100 among different strains of Hexachlorocyclohexane degrading Sphingomonas paucimobilis: Evidence for Natural Horizontal Transfer. J. Bacteriol 186:2225-2235.

Kumari, R., Subudhi, S., Suar, M., Dhingra, G., Raina, V., Dogra, C., S, Lal, van der Meer, J.R., Holliger, C. and Lal, R. 2002. Cloning and characterization of lin genes responsible for the degradation of hexachlorocyclohexane isomers by Sphingomonas paucimobilis strain B90. Appl. Environ. Microbiol. 68:6021-6028.