To use compound-specific isotope analysis for confidently assessing organic contaminant attenuation in the environment, isotope fractionation patterns associated with different transformation mechanisms must first be explored in laboratory experiments. To deliver this information for the common groundwater contaminant chloroform (CF), this study investigated for the first time both carbon and chlorine isotope fractionation for three different engineered reactions: oxidative C-H bond cleavage using heat-activated persulfate, transformation under alkaline conditions (pH ∼ 12) and reductive C-Cl bond cleavage by cast zerovalent iron, Fe(0). Carbon and chlorine isotope fractionation values were -8 ± 1‰ and -0.44 ± 0.06‰ for oxidation, -57 ± 5‰ and -4.4 ± 0.4‰ for alkaline hydrolysis (pH 11.84 ± 0.03), and -33 ± 11‰ and -3 ± 1‰ for dechlorination, respectively. Carbon and chlorine apparent kinetic isotope effects (AKIEs) were in general agreement with expected mechanisms (C-H bond cleavage in oxidation by persulfate, C-Cl bond cleavage in Fe(0)-mediated reductive dechlorination and E1 elimination mechanism during alkaline hydrolysis) where a secondary AKIE (1.00045 ± 0.00004) was observed for oxidation. The different dual carbon-chlorine (ΔδC vs ΔδCl) isotope patterns for oxidation by thermally activated persulfate and alkaline hydrolysis (17 ± 2 and 13.0 ± 0.8, respectively) vs reductive dechlorination by Fe(0) (8 ± 2) establish a base to identify and quantify these CF degradation mechanisms in the field.

Compound-specific chlorine isotope analysis of tetrachloromethane (CCl) and trichloromethane (CHCl) was explored by both, gas chromatography-isotope ratio mass spectrometry (GC-IRMS) and GC-quadrupole MS (GC-qMS), where GC-qMS was validated in an interlaboratory comparison between Munich and Neuchâtel with the same type of commercial GC-qMS instrument. GC-IRMS measurements analyzed CCl isotopologue ions, whereas GC-qMS analyzed the isotopologue ions CCl, CCl, CCl (of CCl) and CHCl, CHCl, CHCl (of CHCl), respectively. Lowest amount dependence (good linearity) was obtained (i) in H-containing fragment ions where interference of Cl- toCl-containing ions was avoided; (ii) with tuning parameters favoring one predominant rather than multiple fragment ions in the mass spectra. Optimized GC-qMS parameters (dwell time 70 ms, 2 most abundant ions) resulted in standard deviations of 0.2‰ (CHCl) and 0.4‰ (CCl), which are only about twice as large as 0.1‰ and 0.2‰ for GC-IRMS. To compare also the trueness of both methods and laboratories, samples from CCl and CHCl degradation experiments were analyzed and calibrated against isotopically different reference standards for both CCl and CHCl (two of each). Excellent agreement confirms that true results can be obtained by both methods provided that a consistent set of isotopically characterized reference materials is used.

A dual element CCl isotopic study was performed for assessing chlorinated methanes (CMs) abiotic transformation reactions mediated by iron minerals and Fe(0) to further distinguish them in natural attenuation monitoring or when applying remediation strategies in polluted sites. Isotope fractionation was investigated during carbon tetrachloride (CT) and chloroform (CF) degradation in anoxic batch experiments with Fe(0), with FeCl(aq), and with Fe-bearing minerals (magnetite, Mag and pyrite, Py) amended with FeCl(aq), at two different pH values (7 and 12) representative of field and remediation conditions. At pH 7, only CT batches with Fe(0) and Py underwent degradation and CF accumulation evidenced hydrogenolysis. With Py, thiolytic reduction was revealed by CS yield and is a likely reason for different Λ value (ΔδC/ΔδCl) comparing with Fe(0) experiments at pH 7 (2.9 ± 0.5 and 6.1 ± 0.5, respectively). At pH 12, all CT experiments showed degradation to CF, again with significant differences in Λ values between Fe(0) (5.8 ± 0.4) and Fe-bearing minerals (Mag, 2 ± 1, and Py, 3.7 ± 0.9), probably evidencing other parallel pathways (hydrolytic and thiolytic reduction). Variation of pH did not significantly affect the Λ values of CT degradation by Fe(0) nor Py. CF degradation by Fe(0) at pH 12 showed a Λ (8 ± 1) similar to that reported at pH 7 (8 ± 2), suggesting CF hydrogenolysis as the main reaction and that CF alkaline hydrolysis (13.0 ± 0.8) was negligible. Our data establish a base for discerning the predominant or combined pathways of CMs natural attenuation or for assessing the effectiveness of remediation strategies using recycled minerals or Fe(0).

Field-derived anoxic microcosms were used to characterize chloroform (CF) and carbon tetrachloride (CT) natural attenuation to compare it with biostimulation scenarios in which vitamin B was added (B/pollutant ratio of 0.01 and 0.1) by means of by-products, carbon and chlorine compound-specific stable-isotope analysis, and the active microbial community through 16S rRNA MiSeq high-throughput sequencing. Autoclaved slurry controls discarded abiotic degradation processes. B catalyzed CF and CT biodegradation without the accumulation of dichloromethane, carbon disulphide, or CF. The carbon isotopic fractionation value of CF (ƐC) with B was -14±4‰, and the value for chlorine (ƐCl) was -2.4±0.4‰. The carbon isotopic fractionation values of CT (ƐC) were -16±6 with B, and -13±2‰ without B; and the chlorine isotopic fractionation values of CT (ƐCl) were -6±3 and -4±2‰, respectively. Acidovorax, Ancylobacter, and Pseudomonas were the most metabolically active genera, whereas Dehalobacter and Desulfitobacterium were below 0.1% of relative abundance. The dual C-Cl element isotope slope (Λ=ΔδC/ΔδCl) for CF biodegradation (only detected with B, 7±1) was similar to that reported for CF reduction by Fe(0) (8±2). Several reductive pathways might be competing in the tested CT scenarios, as evidenced by the lack of CF accumulation when B was added, which might be linked to a major activity of Pseudomonas stutzeri; by different chlorine apparent kinetic isotope effect values and Λ which was statistically different with and without B (5±1 vs 6.1±0.5), respectively. Thus, positive B effects such as CT and CF degradation catalyst were quantified for the first time in isotopic terms, and confirmed with the major activity of species potentially capable of their degradation. Moreover, the indirect benefits of B on the degradation of chlorinated ethenes were proved, creating a basis for remediation strategies in multi-contaminant polluted sites.

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