Jordi Palau

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 (Pseudomonas sp. strain DCA1) versus C-Cl bond cleavage by S(N)2 reaction (Xanthobacter autotrophicus GJ10 and Ancylobacter aquaticus AD20). Compound-specific chlorine isotope analysis of 1,2-DCA was performed for the first time, and isotope fractionation (ε(bulk)(Cl)) 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/Δδ37Cl), 0.78 ± 0.03 (oxidation) and 7.7 ± 0.2 (S(N)2), delineate the potential of the dual isotope approach to identify 1,2-DCA degradation pathways in the field. In contrast to different ε(bulk)(C) values [-3.5 ± 0.1‰ (oxidation) and -31.9 ± 0.7 and -32.0 ± 0.9‰ (S(N)2)], the obtained ε(bulk)(Cl) values were surprisingly similar for the two pathways: -3.8 ± 0.2‰ (oxidation) and -4.2 ± 0.1 and -4.4 ± 0.2‰ (S(N)2). Apparent kinetic isotope effects (AKIEs) of 1.0070 ± 0.0002 (13C-AKIE, oxidation), 1.068 ± 0.001 (13C-AKIE, S(N)2), and 1.0087 ± 0.0002 (37Cl-AKIE, S(N)2) 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.

Multi-isotope (carbon and chlorine) analysis for fingerprinting and site characterization at a fractured bedrock aquifer contaminated by chlorinated ethenes

The use of compound specific multi-isotope approach (C and Cl) in the characterization of a chlorinated ethenes contaminated fractured aquifer allows the identification of several sources and contaminant plumes, as well as the occurrence of biodegradation and mixing processes. The study site is located in Spain with contamination resulting in groundwater concentrations of up to 50mg/L of trichloroethene (TCE), the most abundant chlorinated ethene, and 7 mg/L of tetrachloroethene (PCE). The potential sources of contamination including abandoned barrels, an underground tank, and a disposal lagoon, showed a wide range in δ(13)C values from -15.6 to -40.5‰ for TCE and from -18.5 to -32.4‰ for PCE, allowing the use of isotope fingerprinting for tracing of the origin and migration of these contaminants in the aquifer. In contrast, there is no difference between the δ(37)Cl values for TCE in the contaminant sources, ranging from +0.53 to +0.66‰. Variations of δ(37)Cl and δ(13)C in the different contaminant plumes were used to investigate the role of biodegradation in groundwater. Moreover, the isotopic data were incorporated into a reactive transport model for determination of whether the isotope pattern observed downstream from the tanks source could be explained by the simultaneous effect of mixing and biodegradation. The results demonstrate that a multi-isotope approach is a valuable tool for characterization of complex sites such as fractured bedrock aquifer contaminated by multiple sources, providing important information which can be used by consultants and site managers to prioritize and design more successful remediation strategies.

Carbon and Chlorine Isotope Analysis to Identify Abiotic Degradation Pathways of 1,1,1-Trichloroethane

This study investigates dual C-Cl isotope fractionation during 1,1,1-TCA transformation by heat-activated persulfate (PS), hydrolysis/dehydrohalogenation (HY/DH) and Fe(0). Compound-specific chlorine isotope analysis of 1,1,1-TCA was performed for the first time, and transformation-associated isotope fractionation ε bulk C and ε bulk Cl values were -4.0 ± 0.2‰ and no chlorine isotope fractionation with PS, -1.6 ± 0.2‰ and -4.7 ± 0.1‰ for HY/DH, -7.8 ± 0.4‰ and -5.2 ± 0.2‰ with Fe(0). Distinctly different dual isotope slopes (Δδ13C/Δδ37Cl): ∞ with PS, 0.33 ± 0.04 for HY/DH and 1.5 ± 0.1 with Fe(0) highlight the potential of this approach to identify abiotic degradation pathways of 1,1,1-TCA in the field. The trend observed with PS agreed with a C-H bond oxidation mechanism in the first reaction step. For HY/DH and Fe(0) pathways, different slopes were obtained although both pathways involve cleavage of a C-Cl bond in their initial reaction step. In contrast to the expected larger primary carbon isotope effects relative to chlorine for C-Cl bond cleavage, ε bulk C < ε bulk Cl was observed for HY/DH and in a similar range for reduction by Fe(0), suggesting the contribution of secondary chlorine isotope effects. Therefore, different magnitude of secondary chlorine isotope effects could at least be partly responsible for the distinct slopes between HY/DH and Fe(0) pathways. Following this dual isotope approach, abiotic transformation processes can unambiguously be identified and quantified.

C, Cl and H compound-specific isotope analysis to assess natural versus Fe(0) barrier-induced degradation of chlorinated ethenes at a contaminated site.

Compound-specific isotopic analysis of multiple elements (C, Cl, H) was tested to better assess the effect of a zero-valent iron-permeable reactive barrier (ZVI-PRB) installation at a site contaminated with tetrachloroethene (PCE) and trichloroethene (TCE). The focus was on (1) using (13)C to evaluate natural chlorinated ethene biodegradation and the ZVI-PRB efficiency; (2) using dual element (13)C-(37)Cl isotopic analysis to distinguish biotic from abiotic degradation of cis-dichloroethene (cis-DCE); and (3) using (13)C-(37)Cl-(2)H isotopic analysis of cis-DCE and TCE to elucidate different contaminant sources. Both biodegradation and degradation by ZVI-PRB were indicated by the metabolites that were detected and the (13)C data, with a quantitative estimate of the ZVI-PRB efficiency of less than 10% for PCE. Dual element (13)C-(37)Cl isotopic plots confirmed that biodegradation was the main process at the site including the ZVI-PRB area. Based on the carbon isotope data, approximately 45% and 71% of PCE and TCE, respectively, were estimated to be removed by biodegradation. (2)H combined with (13)C and (37)Cl seems to have identified two discrete sources contributing to the contaminant plume, indicating the potential of δ(2)H to discriminate whether a compound is of industrial origin, or whether a compound is formed as a daughter product during degradation.

Identification of abiotic and biotic reductive dechlorination in a chlorinated ethene plume after thermal source remediation by means of isotopic and molecular biology tools

Thermal tetrachloroethene (PCE) remediation by steam injection in a sandy aquifer led to the release of dissolved organic carbon (DOC) from aquifer sediments resulting in more reduced redox conditions, accelerated PCE biodegradation, and changes in microbial populations. These changes were documented by comparing data collected prior to the remediation event and eight years later. Based on the premise that dual C-Cl isotope slopes reflect ongoing degradation pathways, the slopes associated with PCE and TCE suggest the predominance of biotic reductive dechlorination near the source area. PCE was the predominant chlorinated ethene near the source area prior to thermal treatment. After thermal treatment, cDCE became predominant. The biotic contribution to these changes was supported by the presence of Dehalococcoides sp. DNA (Dhc) and Dhc targeted rRNA close to the source area. In contrast, dual C-Cl isotope analysis together with the almost absent VC (13)C depletion in comparison to cDCE (13)C depletion suggested that cDCE was subject to abiotic degradation due to the presence of pyrite, possible surface-bound iron (II) or reduced iron sulphides in the downgradient part of the plume. This interpretation is supported by the relative lack of Dhc in the downgradient part of the plume. The results of this study show that thermal remediation can enhance the biodegradation of chlorinated ethenes, and that this effect can be traced to the mobilisation of DOC due to steam injection. This, in turn, results in more reduced redox conditions which favor active reductive dechlorination and/or may lead to a series of redox reactions which may consecutively trigger biotically induced abiotic degradation. Finally, this study illustrates the valuable complementary application of compound-specific isotopic analysis combined with molecular biology tools to evaluate which biogeochemical processes are taking place in an aquifer contaminated with chlorinated ethenes.

Carbon and Chlorine Isotope Fractionation Patterns Associated with Different Engineered Chloroform Transformation Reactions

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 E1CB elimination mechanism during alkaline hydrolysis) where a secondary AKIECl (1.00045 ± 0.00004) was observed for oxidation. The different dual carbon-chlorine (Δδ13C vs Δδ37Cl) 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.

Use of Environmental Isotopes (13C, 15N, and 18O) for Evaluating Sources and Fate of Nitrate and Tetrachloroethene in an Alluvial Aquifer

Environmental isotopes and geochemical parameters were used to evaluate the sources and fate of nitrate (NO− 3) and tetrachloroethene (PCE) in an alluvial contaminated aquifer. Isotope data and redox parameters data indicated that biodegradation does not play any role in the attenuation of these compounds along the groundwater flow system. Furthermore, nitrate isotopes and concentration data allowed the authors to distinguish two nitrate inputs and relate them to their possible sources. The primary source originates from septic waste and the secondary source is associated with nitrogenous fertilizer application. Regarding PCE, the δ13C values do not change for approximately 1000 m along the groundwater flow system despite large decreases of PCE concentration, thus confirming that physical processes do not alter the isotopic composition of organic compounds; this finding has a significant implication for the use of carbon isotope ratios for fingerprinting sources of PCE in VOC plumes. This study has shown that combined use of environmental isotopes and geochemical parameters is an efficient approach for water resource management studies in sites polluted by various types of contaminants.

Distinct Dual C–Cl Isotope Fractionation Patterns during Anaerobic Biodegradation of 1,2-Dichloroethane: Potential To Characterize Microbial Degradation in the Field

This study investigates, for the first time, dual C-Cl isotope fractionation during anaerobic biodegradation of 1,2-dichloroethane (1,2-DCA) via dihaloelimination by Dehalococcoides and Dehalogenimonas-containing enrichment cultures. Isotopic fractionation of 1,2-DCA (εbulkC and εbulkCl) for Dehalococcoides (-33.0 ± 0.4‰ and -5.1 ± 0.1‰) and Dehalogenimonas-containing microcosms (-23 ± 2‰ and -12.0 ± 0.8‰) resulted in distinctly different dual element C-Cl isotope correlations (Λ = Δδ13C/Δδ37Cl ≈ εbulkC/εbulkCl), 6.8 ± 0.2 and 1.89 ± 0.02, respectively. Determined isotope effects and detected products suggest that the difference on the obtained Λ values for biodihaloelimination could be associated with a different mode of concerted bond cleavage rather than two different reaction pathways (i.e., stepwise vs concerted). Λ values of 1,2-DCA were, for the first time, determined in two field sites under reducing conditions (2.1 ± 0.1 and 2.2 ± 2.9). They were similar to the one obtained for the Dehalogenimonas-containing microcosms (1.89 ± 0.02) and very different from those reported for aerobic degradation pathways in a previous laboratory study (7.6 ± 0.1 and 0.78 ± 0.03). Thus, this study illustrates the potential of a dual isotope analysis to differentiate between aerobic and anaerobic biodegradation pathways of 1,2-DCA in the field and suggests that this approach might also be used to characterize dihaloelimination of 1,2-DCA by different bacteria, which needs to be confirmed in future studies.

Compound-Specific Chlorine Isotope Analysis of Tetrachloromethane and Trichloromethane by Gas Chromatography-Isotope Ratio Mass Spectrometry vs Gas Chromatography-Quadrupole Mass Spectrometry: Method Development and Evaluation of Precision and Trueness

Compound-specific chlorine isotope analysis of tetrachloromethane (CCl4) and trichloromethane (CHCl3) 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 CCl3, CCl2, CCl (of CCl4) and CHCl3, CHCl2, CHCl (of CHCl3), respectively. Lowest amount dependence (good linearity) was obtained (i) in H-containing fragment ions where interference of 35Cl- to 37Cl-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‰ (CHCl3) and 0.4‰ (CCl4), 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 CCl4 and CHCl3 degradation experiments were analyzed and calibrated against isotopically different reference standards for both CCl4 and CHCl3 (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.

Hydrogen Isotope Fractionation during the Biodegradation of 1,2-Dichloroethane: Potential for Pathway Identification Using a Multi-element (C, Cl, and H) Isotope Approach

Even though multi-element isotope fractionation patterns provide crucial information with which to identify contaminant degradation pathways in the field, those involving hydrogen are still lacking for many halogenated groundwater contaminants and degradation pathways. This study investigates for the first time hydrogen isotope fractionation during both aerobic and anaerobic biodegradation of 1,2-dichloroethane (1,2-DCA) using five microbial cultures. Transformation-associated isotope fractionation values (εbulkH) were -115 ± 18‰ (aerobic C-H bond oxidation), -34 ± 4‰ and -38 ± 4‰ (aerobic C-Cl bond cleavage via hydrolytic dehalogenation), and -57 ± 3‰ and -77 ± 9‰ (anaerobic C-Cl bond cleavage via reductive dihaloelimination). The dual-element C-H isotope approach (ΛC-H = Δδ2H/Δδ13C ≈ εbulkH/εbulkC, where Δδ2H and Δδ13C are changes in isotope ratios during degradation) resulted in clearly different ΛC-H values: 28 ± 4 (oxidation), 0.7 ± 0.1 and 0.9 ± 0.1 (hydrolytic dehalogenation), and 1.76 ± 0.05 and 3.5 ± 0.1 (dihaloelimination). This result highlights the potential of this approach to identify 1,2-DCA degradation pathways in the field. In addition, distinct trends were also observed in a multi- (i.e., Δδ2H versus Δδ37Cl versus Δδ13C) isotope plot, which opens further possibilities for pathway identification in future field studies. This is crucial information to understand the mechanisms controlling natural attenuation of 1,2-DCA and to design appropriate strategies to enhance biodegradation.