Mechanistic Insights into Anaerobic Biotransformation of Tris(2-Chloroethyl) Phosphate by Compound-Specific Stable Isotope Analysis and Quantum Chemical Calculations

Tris(2-chloroethyl) phosphate (TCEP), a representative chlorinated organophosphate ester (Cl-OPE) widely used as a flame retardant, is frequently detected in aquatic environments and sediments due to its high water solubility and persistence. Anaerobic microbial transformation is a key process determining its environmental fate, in which <i>Dehalococcoides</i> plays an important role. A comprehensive understanding of the environmental fate of TCEP requires elucidating its degradation mechanisms and interactions with co-occurring contaminants. This study focuses on the anaerobic biotransformation mechanisms of TCEP mediated by a <i>Dehalococcoides</i>-containing enrichment culture (8E-N), also considering the transformation processes under co-contamination with polychlorinated biphenyl congeners (PCB85). The presence of PCB85 markedly inhibited TCEP degradation. In the absence of PCB85, TCEP transformation followed pseudo-zero-order kinetics (degradation rate = 0.1005 μM·h^-1), yielding bis(2-chloroethyl) phosphate and ethene in near-stoichiometric amounts (mass balance = 95.63 ± 3.43%). Significant carbon and chlorine isotope fractionation (ε_C = -1.10 ± 0.01‰, ε_Cl = -1.01 ± 0.01‰) indicated that C-Cl bond cleavage of TCEP was the initial and rate-limiting step. Quantum chemical calculations further supported this mechanism, revealing strong orbital overlap between the <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mn>3</mml:mn><mml:msub><mml:mi>d</mml:mi><mml:msup><mml:mrow><mml:mi>z</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:msub></mml:math> HOMO of cob(I)alamin and the lowest-energy unoccupied <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mo>(</mml:mo><mml:msubsup><mml:mrow><mml:mi>σ</mml:mi></mml:mrow><mml:mrow><mml:mi>C-Cl</mml:mi></mml:mrow><mml:mrow><mml:mo>*</mml:mo></mml:mrow></mml:msubsup><mml:mo>)</mml:mo></mml:mrow></mml:math> orbital of TCEP, enabling one-electron transfer and subsequent radical-mediated bond scission. Free energy analysis showed that the anaerobic transformation mechanism of TCEP involves an initial single-electron transfer followed by proton-coupled electron transfer, which is thermodynamically the most favorable pathway and consistent with the observed products. Overall, these findings reveal that PCB co-contamination inhibits TCEP degradation, present the first evidence of isotope fractionation during anaerobic TCEP transformation, and provide mechanistic insights into the environmental fate of Cl-OPEs.