Hydroxylated Metabolites of β- and δ-Hexachlorocyclohexane:  Bacterial Formation, Stereochemical Configuration, and Occurrence in Groundwater at a Former Production Site

Abstract

Although the use of hexachlorocyclohexane (HCH), one of the most popular insecticides after the Second World War, has been discontinued in many countries, problems remain from former production and waste sites. Despite the widespread occurrence of HCHs, the environmental fate of these compounds is not fully understood. In particular, environmental metabolites of the more persistent beta-HCH and delta-HCH have not been fully identified. Such knowledge, however, is important to follow degradation and environmental fate of the HCHs. In the present study, several hydroxy metabolites that formed during incubation of beta- and delta-HCH with the common soil microorganism Sphingobium indicum B90A were isolated, characterized, and stereochemically identified by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR). The metabolites were identified as isomeric pentachlorocyclohexanols (B1, D1) and tetrachlorocyclohexane-1,4-diols (B2, D2); delta-HCH additionally formed a tetrachloro-2-cyclohexen-1-ol (D3) and a trichloro-2-cyclohexene-1,4-diol (D4), most likely by hydroxylation of delta-pentachlorocyclohexene (delta-PCCH), initially formed by dehydrochlorination. The dehydrochlorinase LinA was responsible for conversion of delta-HCH into delta-PCCH, and the haloalkane dehalogenase LinB was responsible for the transformation of beta-HCH and delta-HCH into B1 and D1, respectively, and subsequently into B2 and D2, respectively. LinB was also responsible for transforming delta-PCCH into D3 and subsequently into D4. These hydroxylations proceeded in accordance with SN2 type reactions with initial substitution of equatorial Cls and formation of axially hydroxylated stereoisomers. The apparently high reactivity of equatorial Cls in beta- and delta-HCH toward initial hydroxylation by LinB of Sphingobium indicum B90A is remarkable when considering the otherwise usually higher reactivity of axial Cls. Several of these metabolites were detected in groundwater from a former HCH production site in Switzerland. Their presence indicates that these reactions proceed under natural environmental conditions and that the metabolites are of environmental relevance.

Localization of HCH catabolic genes (lin genes) in Sphingobium indicum B90A

“…Additionally, a linX gene, encoding a protein that has activity similar to that of linC (but probably is not involved in the degradation pathway of γ-HCH), was also characterized 7 . Recent studies have shown that both LinA and LinB are also involved in the degradation of the recalcitrant β-and δ-HCH isomers [12][13][14] .…”

Pseudomonas sp. to Sphingobium indicum: a journey of microbial degradation and bioremediation of Hexachlorocyclohexane

“…However, these metabolites were not formed when δ-HCH was incubated in presence of LinB. Further studies revealed that D3 and D4 were not formed in δ-HCH degradation via D1 and D2 respectively but from δ-PCCH (formed by dehydrochlorination of δ-HCH by LinA) as a result of hydrochlorination reaction by LinB [9] (Fig. 7).…”

“…Studies on the degradation of β-and δ-HCH with purifi ed LinB from E. coli revealed that these isomers are hydroxylated to form pentachlorocyclohexanols (B1 and D1) and tetrachlorocyclohexandiols (B2 and D2) [62]. Subsequent studies have confi rmed the formation of B1 and B2 from β-HCH and D1 and D2 from δ-HCH in a resting cell assay of strain B90A [9] (Figs. 6, 7).…”

“…This was the fi rst report on microbial degradation of the most recalcitrant β isomer of HCH. Gradually, the strain was extensively studied and has formed the basis of several novel discoveries such as the association of mobile genetic element (IS6100) with the catabolic lin genes [8]; the elucidation of HCH degradation pathway intermediates [9] as well as application-based bioremediation studies [10]. In this article we describe the alarming situation of environmental contamination by HCH, the function and genetic organization of lin genes in S. indicum B90A and the possibilities of decontamination of HCH through bioremediation by bioaugmentation.…”

The effect of distribution processes on the isomeric composition of hexachlorocyclohexane in a contaminated riverine system

“…Aerobic biodegradations of HCHs in soil are well investigated for the bacteria Sphingomonadaceae (Pal et al 2005). Degradation of HCHs is enabled by the exoenzymes LinA and LinB (Nagata et al 1999;Suar et al 2005;Raina et al 2007;Bala et al 2012;Geueke et al 2013). Degradation thereby forms isomer-specific metabolization products such as pentachlorocyclohexene for a-HCH and c-HCH and pentachlorocyclohexane-1-ol for b-HCH and d-HCH.…”