Chiral organic contaminants, like α-hexachlorocyclohexane (α-HCH), showed isotope fractionation and enantiomer fractionation during biodegradation. This study aims to understand the correlation between these two processes. Initial tests of α-HCH degradation by six Sphingobium strains (with different LinA variants) were conducted. Results showed variable enantiomer selectivity over the time course. In contrast, constant enantiomer selectivity was observed in experiments employing (i) cell suspensions, (ii) crude extracts, or (iii) LinA1 and LinA2 enzymes of strain B90A for α-HCH degradation in enzyme activity assay buffer. The average value of enantioselectivity (ES) were −0.45 ± 0.03 (cell suspensions), −0.60 ± 0.05 (crude extracts), and 1 (LinA1) or −1 (LinA2). The average carbon isotope enrichment factors (εc) of (+)­α- and (−)­α-HCH were increased from cells suspensions (−6.3 ± 0.1‰ and −2.3 ± 0.03‰) over crude extracts (−7.7 ± 0.4‰ and −3.4 ± 0.02‰) to purified enzymes (−11.1 ± 0.3‰ and −3.8 ± 0.2‰). The variability of ES and the εc were discussed based on the effect of mass transport and degradation rates. Our study demonstrates that enantiomer and isotope fractionation of α-HCH are two independent processes and both are affected by underlying reactions of individual enzymes and mass transport to a different extent.

Technical hexachlorocyclohexane (HCH) mixtures and Lindane (γ-HCH) have been produced in Bitterfeld-Wolfen, Germany, for about 30 years until 1982. In the vicinity of the former dump sites and production facilities, large plumes of HCHs persist within two aquifer systems. We studied the natural attenuation of HCH in these groundwater systems through a combination of enantiomeric and carbon isotope fractionation to characterize the degradation of α-HCH in the areas downstream of a former disposal and production site in Bitterfeld-Wolfen. The concentration and isotope composition of α-HCH from the Quaternary and Tertiary aquifers were analyzed. The carbon isotope compositions were compared to the source signal of waste deposits for the dumpsite and highly contaminated areas. The average value of δC at dumpsite was -29.7 ± 0.3 ‰ and -29.0 ± 0.1 ‰ for (-) and (+)α-HCH, respectively, while those for the β-, γ-, δ-HCH isomers were -29.0 ± 0.3 ‰, -29.5 ± 0.4 ‰, and -28.2 ± 0.2 ‰, respectively. In the plume, the enantiomer fraction shifted up to 0.35, from 0.50 at source area to 0.15 (well T1), and was found accompanied by a carbon isotope enrichment of 5 ‰ and 2.9 ‰ for (-) and (+)α-HCH, respectively. The established model for interpreting isotope and enantiomer fractionation patterns showed potential for analyzing the degradation process at a field site with a complex history with respect to contamination and fluctuating geochemical conditions.

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Dehydrochlorination is one of the main (thus far discovered) processes for aerobic microbial transformation of hexachlorocyclohexane (HCH) which is mainly catalyzed by LinA enzymes. In order to gain a better understanding of the reaction mechanisms, multi-element compound-specific stable isotope analysis was applied for evaluating αand γ-HCH transformations catalyzed by LinA1 and LinA2 enzymes. The isotopic fractionation (ε E ) values for particular elements of (), whereas the dual-isotope fractionation patterns were almost identical for both enantiomers (Λ C−Cl = 2.4 ± 0.4 and 2.5 ± 0.2, Λ H−C = 12.9 ± 2.4 and 14.9 ± 1.1). The ε E of γ-HCH transformation by LinA1 and LinA2 were −7.8 ± 1.0‰ and −7.5 ± 0.8‰ (ε C ), −2.7 ± 0.3‰ and −2.5 ± 0.4‰ (ε Cl ), −170 ± 25‰ and −150 ± 13‰ (ε H ), respectively. Similar Λ C−Cl values (2.7 ± 0.2 and 2.9 ± 0.2) were observed as well as similar Λ H−C values (20.1 ± 2.0 and 18.4 ± 1.9), indicating a similar reaction mechanism by both enzymes during γ-HCH transformation. This is the first data set on 3D isotope fractionation of αand γ-HCH enzymatic dehydrochlorination, which gave a more precise characterization of the bond cleavages, highlighting the potential of multi-element compound-specific stable isotope analysis to characterize different transformation processes (e.g., dehydrochlorination and reductive dehalogenation).