Isotope Slopes Research Articles

Divergent dual C-H isotopic fractionation pattern during anaerobic biodegradation of toluene within Aromatoleum species under nitrate-reducing conditions

Toluene is a pollutant frequently detected in contaminated groundwater, mostly due to leakage from underground gasoline storage tanks and pipeline ruptures. Multi-element compound-specific isotope analysis provides a framework to understand transformation processes and design efficient remediation strategies. In this study, we enriched an anaerobic bacterial culture derived from a BTEX-contaminated aquifer that couples toluene and phenol oxidation with nitrate reduction and the concomitant production of carbon dioxide and biomass. The 16S rRNA gene amplicon data indicated that the toluene-degrading consortium was dominated by an Aromatoleum population (87 ± 2 % relative abundance), and metagenome sequencing confirmed that the genome of this Aromatoleum sp. encoded glycyl-radical enzyme benzylsuccinate synthase (BssABC) and phenylphospate synthase (PpsA1BC) homologous genes involved in the first step of toluene and phenol transformation, respectively. Carbon and hydrogen isotopic fractionation were εbulk, C = - 3.5 ± 0.6 ‰ and εrp, H = - 85 ± 11 ‰, respectively, leading to a dual C-H isotope slope of ΛH/C = 26 ± 2. This value fits with a previously reported value for a consortium dominated by an Azoarcus species (ΛH/C = 19 ± 5) but differs from that reported for Aromatoleum aromaticum (ΛH/C = 14 ± 1), both of which grow with toluene under nitrate-reducing conditions. Overall, this suggests the existence of different BssABC enzymes with different mechanistic motifs even within the same Aromatoleum genus.

Use of isotopic (C, Cl) and molecular biology tools to assess biodegradation in a source area of chlorinated ethenes after biostimulation with Emulsified Vegetable Oil (EVO)

Enhanced In Situ Bioremediation (EISB) using Emulsified Vegetable Oil (EVO) as a long-term electron donor has gained prominence for the treatment of groundwater contaminated with chlorinated ethenes (CEs). This study explores the potential of isotopic and molecular biology tools (MBT) to investigate the CEs (PCE, TCE and cis-DCE) bioremediation using EVO in a contaminated site. A multiple approach using C and Cl-CSIA, quantification of Dehalococcoides (Dhc) and specific reductive dechlorination (RD) gene population, and hydrochemical data in microcosm experiments and field samples was applied. Despite the high partitioning of CEs into the EVO phase, the carbon isotopic values of the remaining CEs fraction in the aqueous phase did not exhibit significant changes caused by phase partitioning in laboratory experiments. Both microcosm experiments and field data revealed a rapid RD of PCE and TCE, resulting in the transient accumulation of cis-DCE, which was slowly degraded to vinyl chloride (VC). These results agreed with the presence of Dhc populations and a shift to stronger reducing conditions in the field: i) RD functional genes (tceA, vcrA and bvcA) exhibited a trend to higher values and ii) a substantial increase in Dhc populations (up to 30% of the total bacterial populations) was observed over time. The dual-element isotope slope ΛC-Cl for RD of cis-DCE obtained from field data (ΛC – Cl = 5 ± 3) was similar to the one determined from the microcosm experiments under controlled anoxic conditions (ΛC – Cl = 4.9 ± 0.8). However, ΛC-Cl values differ from those reported so far for laboratory studies with Dhc strains and mixed cultures containing Dhc, i.e., between 8.3 and 17.8. This observation underscores the potential variety of reductive dehalogenases involved during cis-DCE RD and the importance of determining site-specific Λ and ɛ values in order to improve the identification and quantification of transformation processes in the field.

Dual C–Cl isotope fractionation offers potential to assess biodegradation of 1,2-dichloropropane and 1,2,3-trichloropropane by Dehalogenimonas cultures

1,2-dichloropropane (1,2-DCP) and 1,2,3-trichloropropane (1,2,3-TCP) are hazardous chemicals frequently detected in groundwater near agricultural zones due to their historical use in chlorinated fumigant formulations. In this study, we show that the organohalide-respiring bacterium Dehalogenimonas alkenigignens strain BRE15 M can grow during the dihaloelimination of 1,2-DCP and 1,2,3-TCP to propene and allyl chloride, respectively. Our work also provides the first application of dual isotope approach to investigate the anaerobic reductive dechlorination of 1,2-DCP and 1,2,3-TCP. Stable carbon and chlorine isotope fractionation values for 1,2-DCP (ƐC = −13.6 ± 1.4 ‰ and ƐCl = −27.4 ± 5.2 ‰) and 1,2,3-TCP (ƐC = −3.8 ± 0.6 ‰ and ƐCl = −0.8 ± 0.5 ‰) were obtained resulting in distinct dual isotope slopes (Λ12DCP = 0.5 ± 0.1, Λ123TCP = 4 ± 2). However direct comparison of ΛC-Cl among different substrates is not possible and investigation of the C and Cl apparent kinetic isotope effects lead to the hypothesis that concerted dichloroelimination mechanism is more likely for both compounds. In fact, whole cell activity assays using cells suspensions of the Dehalogenimonas-containing culture grown with 1,2-DCP and methyl viologen as electron donor suggest that the same set of reductive dehalogenases was involved in the transformation of 1,2-DCP and 1,2,3-TCP. This study opens the door to the application of isotope techniques for evaluating biodegradation of 1,2-DCP and 1,2,3-TCP, which often co-occur in groundwaters near agricultural fields.

Assessing Biodegradation Processes of Atrazine in Constructed Wetland Using Compound-Specific Stable Isotope Analysis

To bridge the gap between lab-scale microcosm research and field application in the compound-specific stable isotope analysis (CSIA) of atrazine, we studied the characteristics of carbon and nitrogen isotope fractionation in the atrazine degradation processes within a constructed wetland. In the wetland, we observed multiple element (C, N) isotope fractionation parameters, such as kinetic isotope effects and dual isotope slopes. These parameters are very consistent with those observed in the cultivation of AtzA- or TrzN-harboring strains, suggesting a similarity in the pathway and reaction mechanism of atrazine biodegradation between the two settings. However, we recorded variable carbon (εC: −3.2 ± 0.6‰ to −4.3 ± 0.6‰) and nitrogen isotope fractionation (εN: 1.0 ± 0.3‰ to 2.2 ± 0.3‰) across different phases. This variance could lead to an over- or underestimation of the biodegradation extent of atrazine when employing the large or small enrichment factor of the carbon isotope. Intriguingly, the estimation accuracy improved considerably when using the enrichment factor (−4.6‰) derived from the batch cultivation of the pore water. This study advances the application of CSIA in tracking atrazine biodegradation processes in ecosystems, and it also underlines the importance of the careful selection and application of the enrichment factor in quantifying the intrinsic biodegradation of atrazine in ecosystems.

Hydrogeochemical and isotopic evaluation of VOC commingled plumes in a weathered fractured bedrock aquifer treated with thermal and bioremediation

Chlorinated ethanes and ethenes isotopic analyses in groundwater and hydrogeochemical results from a former industrial area in Sao Paulo (Brazil) were used to confirm the existence and allow further characterization of source areas and their commingled plumes, both before and after thermal and bioremediation treatments. Prior to full scale remediation, a recently identified off-site source area with unknown history and limited access for further intrusive works presented lower δ13C values (−6.5‰ to −1.8‰ for 1,2-DCA) than the downgradient on-site source area (+8.6‰ to +20.0‰). Intermediate δ13C values for 1,2-DCA were identified further downgradient from the sources, within commingled plumes patterns. The isotope and concentration results show the typical degradation patterns associated with biotic reductive dechlorination for chlorinated ethenes and dihaloelimination for 1,2-DCA. Results following remediation treatments show further levels of isotopic enrichment, for chlorinated ethenes and chlorinated ethanes in the tropically weathered and deeper fractured bedrock (gneisses) groundwater. Hydrogeochemical results, isotopic mass balance and Carbon-Chlorine isotope slopes data are coherent with remediation treatment and a complex commingled plume setting. The results of this study confirmed the Temporal Conceptual Model proposed by Hart et al. (2021) and identified the need for further studies to evaluate isotopic dynamics under thermal remediation, including thermal-induced hydrolysis processes.

A 2D CSIA-based math method to quantify degradation rate by C-H bond breaking.

Compound-specific isotope analysis has been demonstrated to be a powerful tool for the assessment of in situ pollutant degradation. Enrichment factor, an essential and prerequisite parameter, could be determined under simulated control laboratory in advance. However, different microbial community composition and substrate availability may significantly affect the accuracy of simulated enrichment factor. Here, a modified mathematic method of two dimensional is proposed to quantify the extent of pollutant degradation involving the break of carbon and hydrogen bond. In this new model, the laboratory cultures used to determine carbon or hydrogen enrichment factors in advance could be canceled and the key point to assess the extent of biodegradation is only determining the value of Λri (dual C-H isotope slope calculated with a self-modified model) in the field investigation. As a new and convenient method, this math model greatly facilitates the investigation of pollutant degradation extent under field conditions. Two approaches are applied to evaluate the proposed model. With our model, the estimated results based on C isotope are consistent with those measured values, while those based on H isotope are unsatisfactory. This can be attributed to the differences in accuracy of C-H isotope determinations. Overall, enrichment factors and biodegradation rates calculated with the proposed model are comparable with those measured figures.

Isotopic fractionation from deep roots to tall shoots: A forensic analysis of xylem water isotope composition in mature tropical savanna trees

Studies of plant water sources generally assume that xylem water integrates the isotopic composition (δ2H and δ18O) of water sources and does not fractionate during uptake or transport along the transpiration pathway. However, woody xerophytes, halophytes, and trees in mesic environments can show isotopic fractionation from source waters. Isotopic fractionation and variation in isotope composition can affect the interpretation of tree water sources, but most studies to date have been greenhouse experiments. Here we present a field-based forensic analysis of xylem water isotope composition for 12 Eucalyptus tetrodonta and Corymbia nesophila trees. We used a 25-tonne excavator to access materials from the trees' maximum rooting depth of 3 m to their highest canopies at 38 m. Substantial within-tree variation occurred in δ2H (−91.1‰ to −35.7‰ E. tetrodonta; −88.8‰ to −24.5‰ C. nesophila) and δ18O (−12.3‰ to −5.0‰ E. tetrodonta; −10.9‰ to −0.3‰ C. nesophila), with different root-to-branch isotope patterns in each species. Soil water δ2H and δ18O dual isotope slopes (7.26 E. tetrodonta, 6.66 C. nesophila) were closest to the Local Meteoric Water Line (8.4). The dual isotope slopes of the trees decreased progressively from roots (6.45 E. tetrodonta, 6.07 C. nesophila), to stems (4.61 E. tetrodonta, 5.97 C. nesophila) and branches (4.68 E. tetrodonta, 5.67 C. nesophila), indicative of fractionation along the xylem stream. Roots of both species were more enriched in 2H and 18O than soil water at all sampled depths. Bayesian mixing model analysis showed that estimated proportions of water sourced from different depths reflected the contrasting root systems of these species. Our study adds evidence of isotopic fractionation from water uptake and along the transpiration stream in mature trees in monsoonal environments, affecting the interpretation of water sources. We discuss the findings with view of interpreting aboveground xylem water isotopic composition, incorporating knowledge of root systems.

Substantial maturity influence on carbon and hydrogen isotopic composition of n-alkanes in sedimentary rocks

Carbon (δ13C) and hydrogen (δ2H) compound-specific isotope analyses on sedimentary hydrocarbons are widely used for ecological reconstructions and oil-source rock or oil-oil correlations. However, the effects of thermal alteration on isotopic composition are not fully understood, potentially imparting a bias on interpretation of older and more mature sedimentary sequences. We measured δ13C and δ2H of n-alkanes in 23 extracted bitumens from the 1.64 Ga Paleoproterozoic Barney Creek Formation in the southern McArthur Basin, Australia. The samples cover a wide range of thermal maturities with calculated vitrinite reflectance (Rc) values from 0.4% to 1.3%. Our results illustrate that while δ13C of kerogen remains relatively constant, the δ13C and δ2H of n-alkanes have a strong positive correlation with thermal maturity. Average δ13Calk increase by 6.8‰ and δ2Halk by 69‰ among the samples in the analysed maturity range. At the same time, the carbon isotopic offset between n-alkanes and kerogen (Δδ13Calk–ker) climbs from 1.3‰ to 8.5‰ with increasing maturity. Therefore, the substantial maturity influence on stable carbon and hydrogen isotopes of n-alkanes must be considered in palaeoecological and petroleum correlation studies. In the initial stages of maturation, n-alkanes from the Barney Creek Formation display increasingly positive “isotope slopes” in plots of δ13Calk against carbon number. However, with further maturation, the slopes became increasingly negative. The isotope slope inversion indicates that the dominant mechanisms for n-alkane generation and degradation changed during the progression from early diagenesis to metagenesis. Numerical models suggest that the formation of positive and negative isotope slopes may be driven by the balance of the formation of n-alkanes from kerogen and their subsequent degradation, and by dependence of the degradation rate constant k on n-alkane chain length.

Multi-elemental C-Br-Cl isotope analysis for characterizing biotic and abiotic transformations of 1-bromo-2-chloroethane (BCE)

Multi-elemental C-Br-Cl compound-specific isotope analysis was applied for characterizing abiotic and biotic degradation of the environmental pollutant 1-bromo-2-chloroethane (BCE). Isotope effects were determined in the model processes following hydrolytic dehalogenation and dihaloelimination pathways as well as in a microcosm experiment by the microbial culture from the contaminated site. Hydrolytic dehalogenation of BCE under alkaline conditions and by DhaA enzyme resulted in similar dual isotope slopes (ɅC/Br 21.9 ± 4.7 and 19.4 ± 1.8, respectively, and ɅC/Cl ~ ∞). BCE transformation by cyanocobalamin (B12) and by Sulfurospirillum multivorans followed dihaloelimination and was accompanied by identical, within the uncertainty range, dual isotope slopes (ɅC/Br 8.4 ± 1.7 and 7.9 ± 4.2, respectively, and ɅC/Cl 2.4 ± 0.3 and 1.5 ± 0.6, respectively). Changes over time in the isotope composition of BCE from the contaminated groundwater showed only a slight variation in δ13C values and were not sufficient for the elucidation of the BCE degradation pathway in situ. However, an anaerobic microcosm experiment with the enrichment cultures from the contaminated groundwater presented dual isotope slopes similar to the hydrolytic pathway, suggesting that the potential for BCE degradation in situ by the hydrolytic dehalogenation pathway exists in the contaminated site.

Reductive Dehalogenation of Trichloromethane by Two Different Dehalobacter restrictus Strains Reveal Opposing Dual Element Isotope Effects.

Trichloromethane (TCM) is a frequently detected and persistent groundwater contaminant. Recent studies have reported that two closely related Dehalobacter strains (UNSWDHB and CF) transform TCM to dichloromethane, with inconsistent carbon isotope effects (ε13CUNSWDHB = -4.3 ± 0.45‰; ε13CCF = -27.5 ± 0.9‰). This study uses dual element compound specific isotope analysis (C; Cl) to explore the underlying differences. TCM transformation experiments using strain CF revealed pronounced normal carbon and chlorine isotope effects (ε13CCF = -27.9 ± 1.7‰; ε37ClCF = -4.2 ± 0.2‰). In contrast, small carbon and unprecedented inverse chlorine isotope effects were observed for strain UNSWDHB (ε13CUNSWDHB = -3.1 ± 0.5‰; ε37ClUNSWDHB = 2.5 ± 0.3‰) leading to opposing dual element isotope slopes (λCF = 6.64 ± 0.14 vs λUNSWDHB = -1.20 ± 0.18). Isotope effects of strain CF were identical to experiments with TCM and Vitamin B12 (ε13CVitaminB12 = -26.0 ± 0.9‰, ε37ClVitaminB12 = -4.0 ± 0.2‰, λVitaminB12 = 6.46 ± 0.20). Comparison to previously reported isotope effects suggests outer-sphere-single-electron transfer or SN2 as possible underlying mechanisms. Cell suspension and cell free extract experiments with strain UNSWDHB were both unable to unmask the intrinsic KIE of the reductive dehalogenase (TmrA) suggesting that enzyme binding and/or mass-transfer into the periplasm were rate-limiting. Nondirected intermolecular interactions of TCM with cellular material were ruled out as reason for the inverse isotope effect by gas/water and gas/hexadecane partitioning experiments indicating specific, yet uncharacterized interactions must be operating prior to catalysis.

Use of C–Cl CSIA to elucidate origin and fate of DCM in complex contaminated field sites

We used C-Cl dual isotope analysis and microcosm studies for elucidating the origin and fate of the common groundwater pollutant dichloromethane (DCM) in two different multi-contaminant field sites in Catalonia, Spain; where DCM contamination could be the result of direct solvent releases and/or chloroform (CF) transformation. Known commercial solvents isotopic compositions as well as characteristic C-Cl dual isotope slopes from our anaerobic enrichment culture containing Dehalobacterium sp., capable of fermenting DCM, and other bacteria from the literature were used for field data interpretation.

The discovery of chemically produced mass independent isotope effects: The physical chemistry basis and applications to the early solar system, planetary atmospheres, and the origin of life

The discovery of chemically produced mass independent isotope effects: The physical chemistry basis and applications to the early solar system, planetary atmospheres, and the origin of life

Dual carbon - chlorine isotope fractionation during dichloroelimination of 1,1,2-trichloroethane by an enrichment culture containing Dehalogenimonas sp

Chlorinated ethanes are frequent groundwater contaminants but compound specific isotope analysis (CSIA) has been scarcely applied to investigate their degradation pathways. In this study, dual carbon and chlorine isotope fractionation was used to investigate for the first time the anoxic biodegradation of 1,1,2-trichloroethane (1,1,2-TCA) using a Dehalogenimonas-containing culture. The isotopic fractionation values obtained for the biodegradation of 1,1,2-TCA were ɛC = −6.9 ± 0.4‰ and ɛCl = −2.7 ± 0.3‰. The detection of vinyl chloride (VC) as unique byproduct and a closed carbon isotopic mass balance corroborated that dichloroelimination was the degradation pathway used by this strain. Combining the values of δ13C and δ37Cl resulted in a dual element C-Cl isotope slope of Λ = 2.5 ± 0.2‰. Investigation of the apparent kinetic isotope effects (AKIEs) expected for cleavage of a CCl bond showed an important masking of the intrinsic isotope fractionation. Theoretical calculation of Λ suggested that dichloroelimination of 1,1,2-TCA was taking place via simultaneous cleavage of two CCl bonds (concerted reaction mechanism). The isotope data obtained in this study can be useful to monitor natural attenuation of 1,1,2-TCA via dichloroelimination and provide insights into the source and fate of VC in contaminated groundwaters.

Unravelling long-term source removal effects and chlorinated methanes natural attenuation processes by C and Cl stable isotopic patterns at a complex field site

The effects of contaminant sources removal in 2005 (i.e. barrels, tank, pit and wastewater pipe sources) on carbon tetrachloride (CT) and chloroform (CF) concentration in groundwater were assessed at several areas of a fractured multi-contaminant aquifer (Òdena, Spain) over a long-term period (2010–2014). Changes in redox conditions, in these chlorinated methanes (CMs) concentration and in their carbon isotopic compositions (δ13C) were monitored in multilevel wells. δ13C values from these wells were compared to those obtained from sources (barrels, tank and pit before their removal, 2002–2005) and to commercial solvents values in literature. Additionally, CMs natural attenuation processes were identified by C-Cl isotope slopes (Λ).Analyses revealed the downstream migration of the pollutant focus and an efficient removal of DNAPLs in the pit source's influence area. However, the removal of the contaminated soil from former tank and wastewater pipe was incomplete as leaching from unsaturated zone was proved, evidencing these areas are still active sources. Nevertheless, significant CMs degradation was detected close to all sources and Λ values pointed to different reactions. For CT in the tank area, Λ value fitted with hydrogenolysis pathway although other possible reduction processes were also uncovered. Near the wastewater pipe area, CT thiolytic reduction combined with hydrogenolysis was derived. The highest CT degradation extent accounted for these areas was 72 ± 11% and 84 ± 6%, respectively. For CF, the Λ value in the pit source's area was consistent with oxidation and/or with transport of CF affected by alkaline hydrolysis from upstream interception trenches. In contrast, isotope data evidenced CF reduction in the tank and wastewater pipe influence areas, although the observed Λ slightly deviates from the reference values, likely due to the continuous leaching of CF degraded in the non-saturated zone by a mechanism different from reduction.

Stage-specific distribution of Japanese sea cucumber Apostichopus japonicus in Maizuru Bay, Sea of Japan, in relation to environmental factors

The Japanese sea cucumber Apostichopus japonicus plays important ecological and economic roles in the coastal areas of Japan. Here, we aim to reveal the relation between habitat characteristics and density of juvenile and adult sea cucumbers in Maizuru Bay. Density of adult sea cucumbers was estimated by underwater visual surveys and bottom trawls and that of newly settled individuals by seedling collectors distributed in the bay. The physical environment of the sea bottom was evaluated from median diameter, total organic carbon, total nitrogen, carbon-to-nitrogen ratio, carbon isotope ratio, nitrogen isotope ratio, depth, slope, and distance from the coastline. Adult sea cucumber density was higher in the east side than the west side of Maizuru Bay, the former having a no-take zone for sea cucumbers. Juvenile sea cucumbers were most abundantly collected in the central part of the bay. High organic matter from the sea, short distance from the coastline, and high seabed gradient (steepness) had a positive effect on the density of adult sea cucumbers, and depth had positive effects on their body size. We suggest that sustainable management of sea cucumbers can be achieved by a combination of the establishment of no-take zones and increased protection of nursery habitats.

Vitamin B12 effects on chlorinated methanes-degrading microcosms: Dual isotope and metabolically active microbial populations assessment

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 B12 was added (B12/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. B12 catalyzed CF and CT biodegradation without the accumulation of dichloromethane, carbon disulphide, or CF. The carbon isotopic fractionation value of CF (ƐCCF) with B12 was −14±4‰, and the value for chlorine (ƐClCF) was −2.4±0.4‰. The carbon isotopic fractionation values of CT (ƐCCT) were −16±6 with B12, and −13±2‰ without B12; and the chlorine isotopic fractionation values of CT (ƐClCT) 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=Δδ13C/Δδ37Cl) for CF biodegradation (only detected with B12, 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 B12 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 B12 (5±1 vs 6.1±0.5), respectively. Thus, positive B12 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 B12 on the degradation of chlorinated ethenes were proved, creating a basis for remediation strategies in multi-contaminant polluted sites.

Inconsistent carbon and nitrogen isotope fractionation in the biotransformation of atrazine by Ensifer sp. CX-T and Sinorihizobium sp. K

Compound-specific stable isotope analysis (CSIA) has been used to quantify and identify biodegradation of organic pesticides in the environment. In this work, the kinetic isotope fractionation of carbon and nitrogen was investigated during rapid atrazine degradation by two bacterial isolates, CX-T (Ensifer sp.) and K (Sinorihizobium sp.), under aerobic conditions. Significant isotope fractionation (εcarbon = -2.40 ± 0.07‰, εnitrogen = 1.30 ± 0.13‰, strain CX-T; εcarbon = -1.40 ± 0.07‰, εnitrogen = 2.5 ± 0.5‰, strain K) was observed for both isolates. The same pattern of normal carbon and inverse nitrogen isotope effects were obtained, confirming the same reaction mechanisms of atrazine. The calculated, apparent kinetic isotope effects of strain CX-T (AKIEcarbon = 1.020; AKIEnitrogen = 0.994) and strain K (AKIEcarbon = 1.011; AKIEnitrogen = 0.988) were observed, implying that carbon-chlorine bonds weakened and additional bonds appeared on the triazine ring nitrogen at the transition state. However, isotope enrichment factors and dual isotope slopes (Δ = δ15N/δ13C) of both isolates (Δ = -0.55 ± 0.05, strain CX-T; Δ = -1.07 ± 0.14, strain K) were, nevertheless, inconsistent even for the same compound and same transformation pathway.

Contrasting dual (C, Cl) isotope fractionation offers potential to distinguish reductive chloroethene transformation from breakdown by permanganate

cis-1,2-Dichloroethene (cis-DCE) and trichloroethene (TCE) are persistent, toxic and mobile pollutants in groundwater systems. They are both conducive to reductive dehalogenation and to oxidation by permanganate. In this study, the potential of dual element (C, Cl) compound specific isotope analyses (CSIA) for distinguishing between chemical oxidation and anaerobic reductive dechlorination of cis-DCE and TCE was investigated. Well-controlled cis-DCE degradation batch tests gave similar carbon isotope enrichment factors εC (‰), but starkly contrasting dual element isotope slopes Δδ13C/Δδ37Cl for permanganate oxidation (εC=−26‰±6‰, Δδ13C/Δδ37Cl≈−125±47) compared to reductive dechlorination (εC=−18‰±4‰, Δδ13C/Δδ37Cl≈4.5±3.4). The difference can be tracked down to distinctly different chlorine isotope fractionation: an inverse isotope effect during chemical oxidation (εCl=+0.2‰±0.1‰) compared to a large normal isotope effect in reductive dechlorination (εCl=−3.3‰±0.9‰) (p≪0.05). A similar trend was observed for TCE. The dual isotope approach was evaluated in the field before and up to 443days after a pilot scale permanganate injection in the subsurface. Our study indicates, for the first time, the potential of the dual element isotope approach for distinguishing cis-DCE (and TCE) concentration drops caused by dilution, oxidation by permanganate and reductive dechlorination both at laboratory and field scale.

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 13C depletion in comparison to cDCE 13C 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.

Use of dual carbon–chlorine isotope analysis to assess the degradation pathways of 1,1,1-trichloroethane in groundwater

Compound-specific isotope analysis (CSIA) is a powerful tool to track contaminant fate in groundwater. However, the application of CSIA to chlorinated ethanes has received little attention so far. These compounds are toxic and prevalent groundwater contaminants of environmental concern. The high susceptibility of chlorinated ethanes like 1,1,1-trichloroethane (1,1,1-TCA) to be transformed via different competing pathways (biotic and abiotic) complicates the assessment of their fate in the subsurface. In this study, the use of a dual C–Cl isotope approach to identify the active degradation pathways of 1,1,1-TCA is evaluated for the first time in an aerobic aquifer impacted by 1,1,1-TCA and trichloroethylene (TCE) with concentrations of up to 20 mg/L and 3.4 mg/L, respectively. The reaction-specific dual carbon–chlorine (C–Cl) isotope trends determined in a recent laboratory study illustrated the potential of a dual isotope approach to identify contaminant degradation pathways of 1,1,1-TCA. Compared to the dual isotope slopes (Δδ13C/Δδ37Cl) previously determined in the laboratory for dehydrohalogenation/hydrolysis (DH/HY, 0.33 ± 0.04) and oxidation by persulfate (∞), the slope determined from field samples (0.6 ± 0.2, r2 = 0.75) is closer to the one observed for DH/HY, pointing to DH/HY as the predominant degradation pathway of 1,1,1-TCA in the aquifer. The observed deviation could be explained by a minor contribution of additional degradation processes. This result, along with the little degradation of TCE determined from isotope measurements, confirmed that 1,1,1-TCA is the main source of the 1,1-dichlorethylene (1,1-DCE) detected in the aquifer with concentrations of up to 10 mg/L. This study demonstrates that a dual C–Cl isotope approach can strongly improve the qualitative and quantitative assessment of 1,1,1-TCA degradation processes in the field.