C and Cl Isotope Fractionation of 1,2-Dichloroethane Displays Unique δ¹³C/δ³⁷Cl Patterns for Pathway Identification and Reveals Surprising C–Cl Bond Involvement in Microbial Oxidation

This study investigates dual element isotope fractionation during aerobic biodegradation of 1,2-dichloroethane (1,2-DCA) via oxidative cleavage of a C−H bond (<i>Pseudomonas</i> sp. strain DCA1) versus C−Cl bond cleavage by S_N2 reaction (<i>Xanthobacter autotrophicus</i> GJ10 and <i>Ancylobacter aquaticus</i> AD20). Compound-specific chlorine isotope analysis of 1,2-DCA was performed for the first time, and isotope fractionation (ε^Cl_bulk) was determined by measurements of the same samples in three different laboratories using two gas chromatography−isotope ratio mass spectrometry systems and one gas chromatography−quadrupole mass spectrometry system. Strongly pathway-dependent slopes (Δδ¹³C/Δ δ³⁷Cl), 0.78 ± 0.03 (oxidation) and 7.7 ± 0.2 (S_N2), delineate the potential of the dual isotope approach to identify 1,2-DCA degradation pathways in the field. In contrast to different ε^C_bulk values [−3.5 ± 0.1‰ (oxidation) and −31.9 ± 0.7 and −32.0 ± 0.9‰ (S_N2)], the obtained ε^Cl_bulk values were surprisingly similar for the two pathways: −3.8 ± 0.2‰ (oxidation) and −4.2 ± 0.1 and −4.4 ± 0.2‰ (S_N2). Apparent kinetic isotope effects (AKIEs) of 1.0070 ± 0.0002 (¹³C-AKIE, oxidation), 1.068 ± 0.001 (¹³C-AKIE, S_N2), and 1.0087 ± 0.0002 (³⁷Cl-AKIE, S_N2) fell within expected ranges. In contrast, an unexpectedly large secondary ³⁷Cl-AKIE of 1.0038 ± 0.0002 reveals a hitherto unrecognized involvement of C−Cl bonds in microbial C−H bond oxidation. Our two-dimensional isotope fractionation patterns allow for the first time reliable 1,2-DCA degradation pathway identification in the field, which unlocks the full potential of isotope applications for this important groundwater contaminant.