Enantioselective Carbon Stable Isotope Fractionation of Hexachlorocyclohexane during Aerobic Biodegradation by Sphingobium spp. Safdar Bashir, † Anko Fischer, †,‡ Ivonne Nijenhuis, †, * and Hans-Hermann Richnow † † Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research - UFZ, Permoserstraße 15, 04318, Leipzig, Germany ‡ Isodetect - Company for Isotope Monitoring, Permoserstraße 15, 04318 Leipzig, Germany * S Supporting Information ABSTRACT: Carbon isotope fractionation was investigated for the biotransformation of γ- and α- hexachlorocyclohexane (HCH) as well as enantiomers of α-HCH using two aerobic bacterial strains: Sphingobium indicum strain B90A and Sphingobium japonicum strain UT26. Carbon isotope enrichment factors (ε c ) for γ-HCH (ε c = -1.5 ± 0.1‰ and -1.7 ± 0.2‰) and α-HCH (ε c = -1.0 ± 0.2‰ and -1.6 ± 0.3‰) were similar for both aerobic strains, but lower in comparison with previously reported values for anaerobic γ- and α-HCH degradation. Isotope fractionation of α-HCH enantiomers was higher for (+) α-HCH (ε c = -2.4 ± 0.8 ‰ and -3.3 ± 0.8 ‰) in comparison to (-) α- HCH (ε c = -0.7 ± 0.2‰ and -1.0 ± 0.6‰). The microbial fractionation between the α-HCH enantiomers was quantified by the Rayleigh equation and enantiomeric fractionation factors (ε e ) for S. indicum strain B90A and S. japonicum strain UT26 were -42 ± 16% and -22 ± 6%, respectively. The extent and range of isomer and enantiomeric carbon isotope fractionation of HCHs with Sphingobium spp. suggests that aerobic biodegradation of HCHs can be monitored in situ by compound-specific stable isotope analysis (CSIA) and enantiomer-specific isotope analysis (ESIA). In addition, enantiomeric fractionation has the potential as a complementary approach to CSIA and ESIA for assessing the biodegradation of α-HCH at contaminated field sites. ■ INTRODUCTION About 25% of worldwide applied organic chemicals, for example, pharmaceuticals or pesticides, are chiral and were applied as mixtures of isomers and/or enantiomers. 1 Despite the almost similar molecular structure and identical physical properties, however, enantiomers may have different character- istics related to (bio)chemical reactions governing their persistence and toxicity in the environment. 2-4 This motivates our investigation for studying the behavior of enantiomers combined with stable isotope techniques to gain further information for tracing their fate in the environment. For this purpose, we selected the hexachlorocyclohexanes (HCH) of which α-HCH is chiral. HCHs, comprising mainly α, β, γ, and δ-HCH, were among the most produced and applied insecticides, as technical HCH or Lindane (γ-HCH), between 1950 and 2000. 5-7 Technical HCH, containing 60-70% of the chiral isomer α-HCH, 5-12% of β-HCH, 10-12% of γ-HCH, 6-10% δ-HCH, and 3-4% ε- HCH of which only γ-HCH has specific insecticidal activity, was extensively used mainly in developing countries. 6,8 In addition to technical HCH, the γ-HCH isomer known as Lindane was intensively used as a pure component in insecticide formulations. 9 The large amount of byproducts produced during the Lindane production (one ton of purified γ-HCH results in 8-12 tons of HCH byproducts containing all other isomers) were often disposed improperly resulting in point source contamination of soil and groundwater at recent and former HCH production sites. 10 Between 4 and 7 million tons of technical HCH waste was estimated to be produced around the globe during 60 years of Lindane synthesis. These residual of HCHs are toxic, persistent, and potentially bioaccumulative 11-14 resulting in the recent inclusion as new persistent organic pollutants (POPs) in the Stockholm convention. 10 Therefore, concepts to trace the fate of HCH in the environment are essential. Biodegradation is a major process removing HCHs in soil, aquifers and surface water bodies. 15 Anaerobic transformation of HCH results in monochlorobenzene and benzene as accumulating metabolites via reductive beta-elimination, 16 whereas aerobic degradation proceeds via dehydrochlorination. The aerobic transformation of γ-HCH is characterized by two Received: May 16, 2013 Revised: September 4, 2013 Accepted: September 5, 2013 Published: September 5, 2013 Article pubs.acs.org/est © 2013 American Chemical Society 11432 dx.doi.org/10.1021/es402197s | Environ. Sci. Technol. 2013, 47, 11432-11439