Multi-Element (C, N, Cl) Stable Isotope Fractionation During Reductive Dechlorination of Chloroanilines.

Compound-specific isotope analysis (CSIA) is widely used to verify in situ biodegradation of organic contaminants. Chloroanilines are persistent groundwater pollutants known to undergo reductive dechlorination under environmental conditions, yet their isotope fractionation remains unclear. This study reports multi-element isotopic fractionation (ε) associated with reductive dechlorination of 2,3-dichloroaniline (2,3-DCA) and 2-chloroaniline (2-CA) using a Dehalobacter-dominated enrichment culture. During 2,3-DCA dechlorination, significant C (εC = -8.1 ± 0.3‰) and Cl (εCl = -7.5 ± 0.1‰) isotope fractionation was observed (ΛC-Cl = 1.12 ± 0.09), while N isotope fractionation was insignificant (Δδ15N < 1‰). Similarly, reductive dechlorination of 2-CA yielded εC = -8.9 ± 0.3‰ and εCl = -18.1 ± 0.4‰ (ΛC-Cl = 0.48 ± 0.03), with negligible changes in δ15N. The large and comparable apparent kinetic isotope effects for C and Cl may suggest that both reactions share similar transition-state characteristics, with C-Cl bond cleavage as the rate-limiting step. In contrast to isotope fractionation reported for aerobic 2,3-DCA biodegradation, the distinct isotope patterns observed here enable differentiation between aerobic and anaerobic pathways. Using groundwater samples from a chloroaniline-impacted aquifer, we show that multi-element CSIA can help distinguish reductive dechlorination from aerobic biodegradation under spatially and temporally variable redox conditions.