Pilot tests for the optimization of the bioremediation strategy of a multi-layered aquifer at a multi-focus site impacted with chlorinated ethenes.

A multi-layered aquifer in an industrial area in the north of the Iberian Peninsula is severely contaminated with the chlorinated ethenes (CEs) tetrachloroethylene, trichloroethylene, cis-1,2-dichloroethylene, and vinyl chloride. Both shallow and deep aquifers are polluted, with two differentiated north and south CEs plumes. Hydrogeochemical and isotopic data (δ13C of CEs) evidenced natural attenuation of CEs. To select the optimal remediation strategy to clean-up the contamination plumes, laboratory treatability studies were performed, which confirmed the intrinsic biodegradation potential of the north and south shallow aquifers to fully dechlorinate CEs to ethene after injection of lactate, but also the combination of lactate and sulfidized mZVI as an alternative treatment for the north deep aquifer. In the lactate-amended microcosms, full dechlorination of CEs was accompanied by an increase in 16S rRNA gene copies of Dehalococcoides and Dehalogenimonas, and the tceA, vcrA and bvcA reductive dehalogenases. Three in situ pilot tests were implemented, which consisted in injections of lactate in the north and south shallow aquifers, and injections of lactate and sulfidized mZVI in the north deep aquifer. The hydrogeochemical, isotopic and molecular analyses used to monitor the pilot tests evidenced that results obtained mimicked the laboratory observations, albeit at different dechlorination rates. It is likely that the efficiency of the injections was affected by the amendment distribution. In addition, monitoring of the pilot tests in the shallow aquifers showed the release of CEs due to back diffusion from secondary sources, which limited the use of isotopic data for assessing treatment efficiency. In the pilot test that combined the injection of lactate and sulfidized mZVI, both biotic and abiotic pathways contributed to the production of ethene. This study demonstrates the usefulness of integrating different chemical, isotopic and biomolecular approaches for a more robust selection and implementation of optimal remediation strategies in CEs polluted sites.

Novel extraction methods and compound-specific isotope analysis of methoxychlor in environmental water and aquifer slurry samples.

Multi-element compound-specific stable isotope analysis (ME-CSIA) allows monitoring the environmental behavior and transformation of most common and persistent contaminants. Recent advancements in analytical techniques have extended the applicability of ME-CSIA to organic micropollutants, including pesticides. Nevertheless, the application of this methodology remains unexplored concerning harmful insecticides such as methoxychlor, a polar organochlorine pesticide usually detected in soil and groundwater. This study introduces methods for dual carbon and chlorine compound-specific stable isotope analysis (δ13C-CSIA and δ37Cl-CSIA) of both methoxychlor and its metabolite, methoxychlor olefin, with a sensitivity down to 10 and 100 mg/L, and a precision lower than 0.3 and 0.5 ‰ for carbon and chlorine CSIA, respectively. Additionally, three extraction and preconcentration techniques suitable for ME-CSIA of the target pesticides at environmentally relevant concentrations were also developed. Solid-phase extraction (SPE) and liquid-solid extraction (LSE) effectively extracted methoxychlor (107 ± 27 % and 87 ± 13 %, respectively) and its metabolite (91 ± 27 % and 106 ± 14 %, respectively) from water and aquifer slurry samples, respectively, with high accuracy (Δδ13C and Δδ37Cl ≤ ± 1 ‰). Combining CSIA with polar organic chemical integrative samplers (POCISs) for the extraction of methoxychlor and methoxychlor olefin from water samples resulted in insignificant fractionation for POCIS-CSIA (Δδ13C ≤ ± 1 ‰). A relevant sorption of methoxychlor was detected within the polyethersulfones membranes of the POCISs resulting in temporary carbon isotope fractionation depending on the sorbed mass fraction during the first deployment days. This highlights the critical role of the interactions of polar analytes with POCIS sorbents and membranes in the performance of this method. Altogether, this study proposes a proof of concept for ME-CSIA of methoxychlor and its metabolites, opening the door for future investigations of their sources and transformation processes in contaminated sites.

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.

Quantifying the potential seepage from porphyry copper tailing impoundments using a multi-isotopic approach.

Porphyry-style copper deposits are characterized by low Cu grades and high tonnages, resulting in large mine tailing volumes disposed in impoundments. Due to the mining tailing sizes, waterproofing techniques cannot be applied along the dam base. Therefore, to minimize seepage towards the aquifers, pumping wells are usually installed as hydraulic barriers. Currently, there is a controversy over whether or not the water extracted from hydraulic barriers should be counted as the use of new water rights. Consequently, a growing interest to develop tools to trace and quantify the tailing impacts in groundwater and to determine the water pumped amount subjected to water rights exist. In the present study, isotope data (δ2H-H2O, δ18O-H2O, δ34S-SO42- and δ18O-SO42-) are proposed as a tool to quantify tailings seepage towards groundwater and to assess hydraulic barriers effectiveness. To illustrate this approach usefulness, the Quillayes porphyry Cu tailing impoundment (Chile) case study is presented. The multi-isotopic approach revealed that tailing waters are highly evaporated showing high SO42- content (~1900 mg L-1) derived from primary sulfate ore dissolution, whereas freshwaters, derived from recharge water, have low SO42- contents (10-400 mg L-1) resulting from the interaction with geogenic sulfides from barren host rock. The δ2H and δ18O values of groundwater samples collected downstream from the impoundment suggest a mixing at different proportions of highly evaporated water from the mine tailing waters and non-evaporated regional fresh groundwater. Cl-/SO42-, δ34S-SO42-/δ18O-SO42-, δ34S-SO42-/ln(SO42-) and δ2H-H2O/δ18O-H2O mixing models allowed to determine that groundwater located closer to the impoundment had a mine tailing water contribution from 45 to 90 %, whereas those located farther away had lower contribution (5-25 %). Results confirmed the stable isotope usefulness to determine the water origin and to calculate the hydraulic barrier efficiencies and the pumped water proportions unrelated to the mining tailing subject to the water rights.

Trichloromethane dechlorination by a novel Dehalobacter sp. strain 8M reveals a third contrasting C and Cl isotope fractionation pattern within this genus.

Trichloromethane (TCM) is a pollutant frequently detected in contaminated aquifers, and only four bacterial strains are known to respire it. Here, we obtained a novel Dehalobacter strain capable of transforming TCM to dichloromethane, which was denominated Dehalobacter sp. strain 8M. Besides TCM, strain 8M also completely transformed 1,1,2-trichloroethane to vinyl chloride and 1,2-dichloroethane. Quantitative PCR analysis for the 16S rRNA genes confirmed growth of Dehalobacter with TCM and 1,1,2-trichloroethane as electron acceptors. Carbon and chlorine isotope fractionation during TCM transformation was studied in cultured cells and in enzymatic assays with cell suspensions and crude protein extracts. TCM transformation in the three studied systems resulted in small but significant carbon (εC = -2.7 ± 0.1‰ for respiring cells, -3.1 ± 0.1‰ for cell suspensions, and - 4.1 ± 0.5‰ for crude protein extracts) and chlorine (εCl = -0.9 ± 0.1‰, -1.1 ± 0.1‰, and - 1.2 ± 0.2‰, respectively) isotope fractionation. A characteristic and consistent dual CCl isotope fractionation pattern was observed for the three systems (combined ΛC/Cl = 2.8 ± 0.3). This ΛC/Cl differed significantly from previously reported values for anaerobic dechlorination of TCM by the corrinoid cofactor vitamin B12 and other Dehalobacter strains. These findings widen our knowledge on the existence of different enzyme binding mechanisms underlying TCM-dechlorination within the genus Dehalobacter and demonstrates that dual isotope analysis could be a feasible tool to differentiate TCM degraders at field studies.

Triple-Element Compound-Specific Stable Isotope Analysis (3D-CSIA): Added Value of Cl Isotope Ratios to Assess Herbicide Degradation.

Multielement isotope fractionation studies to assess pollutant transformation are well-established for point-source pollution but are only emerging for diffuse pollution by micropollutants like pesticides. Specifically, chlorine isotope fractionation is hardly explored but promising, because many pesticides contain only few chlorine atoms so that "undiluted" position-specific Cl isotope effects can be expected in compound-average data. This study explored combined Cl, N, and C isotope fractionation to sensitively detect biotic and abiotic transformation of the widespread herbicides and groundwater contaminants acetochlor, metolachlor, and atrazine. For chloroacetanilides, abiotic hydrolysis pathways studied under acidic, neutral, and alkaline conditions as well as biodegradation in two soils resulted in pronounced Cl isotope fractionation (εCl from -5.0 ± 2.3 to -6.5 ± 0.7‰). The characteristic dual C-Cl isotope fractionation patterns (ΛC-Cl from 0.39 ± 0.15 to 0.67 ± 0.08) reveal that Cl isotope analysis provides a robust indicator of chloroacetanilide degradation. For atrazine, distinct ΛC-Cl values were observed for abiotic hydrolysis (7.4 ± 1.9) compared to previous reports for biotic hydrolysis and oxidative dealkylation (1.7 ± 0.9 and 0.6 ± 0.1, respectively). The 3D isotope approach allowed differentiating transformations that would not be distinguishable based on C and N isotope data alone. This first data set on Cl isotope fractionation in chloroacetanilides, together with new data in atrazine degradation, highlights the potential of using compound-specific chlorine isotope analysis for studying in situ pesticide degradation.

Using a multi-disciplinary approach to characterize groundwater systems in arid and semi-arid environments: The case of Biskra and Batna regions (NE Algeria).

This study presents a multi-disciplinary approach for the hydrogeological assessment and characterization of water resources in typical arid and semi-arid areas with high anthropogenic pressure, and where environmental conditions and political context prevent extensive field surveys. The use of a three-dimensional (3D) hydrogeological conceptual model, integrating hydrochemical and multi-isotope data, is proposed for the Batna and Biskra area (NE Algeria). Geological data were assembled in 3D geological software, from which a 3D hydrogeological conceptual model was constructed, which included the delineation of groundwater flow directions. The isotopic characterization, including deuterium and oxygen isotopic composition of water (δ2H and δ18O), and tritium (3H), provided information regarding recharge sources, flow pathways and residence times of groundwaters. Hydrochemical parameters, measured on the same samples, supported the interpretation of isotope data. All data were processed in a geographic information system (GIS) environment. The effectiveness of this approach was tested on a complex system of aquifers with high hydrogeological heterogeneity. Results show the important role the tectonic setting of an area can play in the hydrogeology and hydrochemistry of its principal groundwater systems. The fault network in the study region connects different aquifers, resulting in the mixing of groundwaters. The region most influenced by geological structures is the southern part of the study area, close to Biskra city. In fact, besides a limited contribution of recharge from rain and surface water derived from flood events, the recharge of the Cenozoic aquifers seems to proceed from the ascension of deeper Cretaceous groundwaters through the fault network, as indicated by temperature, bulk chemistry and in particular δ2H, δ18O and 3H results. In contrast, results suggest that the recharge of the low mineralized Maastrichtian waters is primarily influenced by local precipitation and runoff in the mountainous northern part of the study area. Tritium content, low salinity, and bulk chemistry all suggest such waters to be a mix of pre-bomb (deeper flow-lines within the aquifer) and recent water, with no contribution from the deepest Continental Intercalaire groundwaters. The proposed approach reduces ambiguity about the studied aquifer systems, greatly improves the conceptual understanding of their behaviour, and could provide insights into the vulnerability of the aquifers to different anthropogenic pollution phenomena. The methodology used appears to be a valid tool that could be applied to other geographical areas, to inform the design and implementation of efficient management strategies aimed at improving the quality and availability of water resources. Moreover, three-dimensional modelling methods are becoming increasingly applied to different aspects of groundwater management, to obtain a detailed picture of subsurface conditions.

Dual-Element Isotope Analysis of Desphenylchloridazon to Investigate Its Environmental Fate in a Systematic Field Study: A Long-Term Lysimeter Experiment.

Desphenylchloridazon (DPC), the main metabolite of the herbicide chloridazon (CLZ), is more water soluble and persistent than CLZ and frequently detected in water bodies. When assessing DPC transformation in the environment, results can be nonconclusive if based on concentration analysis alone because estimates may be confounded by simultaneous DPC formation from CLZ. This study investigated the fate of DPC by combining concentration-based methods with compound-specific C and N stable isotope analysis (CSIA). Additionally, DPC formation and transformation processes were experimentally deconvolved in a dedicated lysimeter study considering three scenarios. First, surface application of DPC enabled studying its degradation in the absence of CLZ. Here, CSIA provided evidence of two distinct DPC transformation processes: one shows significant changes only in 13C/12C, whereas the other involves changes in both 13C/12C and 15N/14N isotope ratios. Second, surface application of CLZ mimicked a realistic field scenario, showing that during DPC formation, 13C/12C ratios of DPC were depleted in 13C relative to CLZ, while 15N/14N ratios remained constant. Finally, CLZ depth injection simulated preferential flow and demonstrated the importance of the topsoil for retaining DPC. The combination of the lysimeter study with CSIA enabled insights into DPC transformation in the field that are superior to those of studies of concentration trends.

Compound-specific chlorine isotope fractionation in biodegradation of atrazine.

Atrazine is a frequently detected groundwater contaminant. It can be microbially degraded by oxidative dealkylation or by hydrolytic dechlorination. Compound-specific isotope analysis is a powerful tool to assess its transformation. In previous work, carbon and nitrogen isotope effects were found to reflect these different transformation pathways. However, chlorine isotope fractionation could be a particularly sensitive indicator of natural transformation since chlorine isotope effects are fully represented in the molecular average while carbon and nitrogen isotope effects are diluted by non-reacting atoms. Therefore, this study explored chlorine isotope effects during atrazine hydrolysis with Arthrobacter aurescens TC1 and oxidative dealkylation with Rhodococcus sp. NI86/21. Dual element isotope slopes of chlorine vs. carbon isotope fractionation (Λ = 1.7 ± 0.9 vs. Λ = 0.6 ± 0.1) and chlorine vs. nitrogen isotope fractionation (Λ = -1.2 ± 0.7 vs. Λ = 0.4 ± 0.2) provided reliable indicators of different pathways. Observed chlorine isotope effects in oxidative dealkylation (εCl = -4.3 ± 1.8‰) were surprisingly large, whereas in hydrolysis (εCl = -1.4 ± 0.6‰) they were small, indicating that C-Cl bond cleavage was not the rate-determining step. This demonstrates the importance of constraining expected isotope effects of new elements before using the approach in the field. Overall, the triple element isotope information brought forward here enables a more reliable identification of atrazine sources and degradation pathways.

Compound-Specific Chlorine Isotope Analysis of the Herbicides Atrazine, Acetochlor, and Metolachlor.

A gas chromatography-single quadrupole mass spectrometry method was developed and validated for compound-specific chlorine isotope analysis (Cl-CSIA) of three chlorinated herbicides, atrazine, acetochlor, and metolachlor, which are widespread contaminants in the environment. For each compound, the two most abundant ions containing chlorine (202/200 for atrazine, 225/223 for acetochlor, and 240/238 for metolachlor) and a dwell time of 30 ms were determined as optimized MS parameters. A limit of precise isotope analysis for ethyl acetate solutions of 10 mg/L atrazine, 10 mg/L acetochlor, and 5 mg/L metolachlor could be reached with an associated uncertainty between 0.5 and 1‰. To this end, samples were measured 10-fold and bracketed with two calibration standards that covered a wide range of δ37Cl values and for which amplitudes matched those of the samples within 20% tolerance. The method was applied to investigate chlorine isotope fractionation during alkaline hydrolysis of metolachlor, which showed a shift in δ37Cl of +46‰ after 98% degradation, demonstrating that chlorine isotope fractionation could be a sensitive indicator of transformation processes even when limited degradation occurs. This method, combined with large-volume solid-phase extraction (SPE), allowed application of Cl-CSIA to environmentally relevant concentrations of widespread herbicides (i.e., 0.5-5 µg/L in water before extraction). Therefore, the combination of large-volume SPE and Cl-CSIA is a promising tool for assessing the transformation processes of these pollutants in the environment.

Solid-phase extraction method for stable isotope analysis of pesticides from large volume environmental water samples.

Compound-specific isotope analysis (CSIA) is a valuable tool for assessing the fate of organic pollutants in the environment. However, the requirement of sufficient analyte mass for precise isotope ratio mass spectrometry combined with prevailing low environmental concentrations currently limits comprehensive applications to many micropollutants. Here, we evaluate the upscaling of solid-phase extraction (SPE) approaches for routine CSIA of herbicides. To cover a wide range of polarity, a SPE method with two sorbents (a hydrophobic hypercrosslinked sorbent and a hydrophilic sorbent) was developed. Extraction conditions, including the nature and volume of the elution solvent, the amount of sorbent and the solution pH, were optimized. Extractions of up to 10 L of agricultural drainage water (corresponding to up to 200 000-fold pre-concentration) were successfully performed for precise and sensitive carbon and nitrogen CSIA of the target herbicides atrazine, acetochlor, metolachlor and chloridazon, and metabolites desethylatrazine, desphenylchloridazon and 2,6-dichlorobenzamide in the sub-µg L-1-range. 13C/12C and 15N/14N ratios were measured by gas chromatography-isotope ratio mass spectrometry (GC/IRMS), except for desphenylchloridazon, for which liquid chromatography (LC/IRMS) and derivatization-GC/IRMS were used, respectively. The method validated in this study is an important step towards analyzing isotope ratios of pesticide mixtures in aquatic systems and holds great potential for multi-element CSIA applications to trace pesticide degradation in complex environments.

13C- and 15N-Isotope Analysis of Desphenylchloridazon by Liquid Chromatography-Isotope-Ratio Mass Spectrometry and Derivatization Gas Chromatography-Isotope-Ratio Mass Spectrometry.

The widespread application of herbicides impacts surface water and groundwater. Metabolites (e.g., desphenylchloridazon from chloridazon) may be persistent and even more polar than the parent herbicide, which increases the risk of groundwater contamination. When parent herbicides are still applied, metabolites are constantly formed and may also be degraded. Evaluating their degradation on the basis of concentration measurements is, therefore, difficult. This study presents compound-specific stable-isotope analysis (CSIA) of nitrogen- and carbon-isotope ratios at natural abundances as an alternative analytical approach to track the origin, formation, and degradation of desphenylchloridazon (DPC), the major degradation product of the herbicide chloridazon. Methods were developed and validated for carbon- and nitrogen-isotope analysis (δ13C and δ15N) of DPC by liquid chromatography-isotope-ratio mass spectrometry (LC-IRMS) and derivatization gas chromatography-IRMS (GC-IRMS), respectively. Injecting standards directly onto an Atlantis LC-column resulted in reproducible δ13C-isotope analysis (standard deviation <0.5‰) by LC-IRMS with a limit of precise analysis of 996 ng of DPC on-column. Accurate and reproducible δ15N analysis with a standard deviation of <0.4‰ was achieved by GC-IRMS after derivatization of >100 ng of DPC with 160-fold excess of (trimethylsilyl)diazomethane. Application of the method to environmental-seepage water indicated that newly formed DPC could be distinguished from "old" DPC by the different isotopic signatures of the two DPC sources.

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 ±â€¯11% and 84 ±â€¯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.

Dual element (CCl) isotope approach to distinguish abiotic reactions of chlorinated methanes by Fe(0) and by Fe(II) on iron minerals at neutral and alkaline pH.

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 FeCl2(aq), and with Fe-bearing minerals (magnetite, Mag and pyrite, Py) amended with FeCl2(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 CS2 yield and is a likely reason for different Λ value (Δδ13C/Δδ37Cl) comparing with Fe(0) experiments at pH 7 (2.9 ±â€¯0.5 and 6.1 ±â€¯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 ±â€¯0.4) and Fe-bearing minerals (Mag, 2 ±â€¯1, and Py, 3.7 ±â€¯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 ±â€¯1) similar to that reported at pH 7 (8 ±â€¯2), suggesting CF hydrogenolysis as the main reaction and that CF alkaline hydrolysis (13.0 ±â€¯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).

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.

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.

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.

Feasibility of two low-cost organic substrates for inducing denitrification in artificial recharge ponds: Batch and flow-through experiments.

Anaerobic batch and flow-through experiments were performed to assess the capacity of two organic substrates to promote denitrification of nitrate-contaminated groundwater within managed artificial recharge systems (MAR) in arid or semi-arid regions. Denitrification in MAR systems can be achieved through artificial recharge ponds coupled with a permeable reactive barrier in the form of a reactive organic layer. In arid or semi-arid regions, short-term efficient organic substrates are required due to the short recharge periods. We examined the effectiveness of two low-cost, easily available and easily handled organic substrates, commercial plant-based compost and crushed palm tree leaves, to determine the feasibility of using them in these systems. Chemical and multi-isotopic monitoring (δ15NNO3, δ18ONO3, δ34SSO4, δ18OSO4) of the laboratory experiments confirmed that both organic substrates induced denitrification. Complete nitrate removal was achieved in all the experiments with a slight transient nitrite accumulation. In the flow-through experiments, ammonium release was observed at the beginning of both experiments and lasted longer for the experiment with palm tree leaves. Isotopic characterisation of the released ammonium suggested ammonium leaching from both organic substrates at the beginning of the experiments and pointed to ammonium production by DNRA for the palm tree leaves experiment, which would only account for a maximum of 15% of the nitrate attenuation. Sulphate reduction was achieved in both column experiments. The amount of organic carbon consumed during denitrification and sulphate reduction was 0.8‰ of the total organic carbon present in commercial compost and 4.4% for the palm tree leaves. The N and O isotopic fractionation values obtained (εN and εO) were -10.4‰ and -9.0‰ for the commercial compost (combining data from both batch and column experiments), and -9.9‰ and -8.6‰ for the palm tree column, respectively. Both materials showed a satisfactory capacity for denitrification, but the palm tree leaves gave a higher denitrification rate and yield (amount of nitrate consumed per amount of available C) than commercial compost.

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.

The use of alkaline hydrolysis as a novel strategy for chloroform remediation: the feasibility of using construction wastes and evaluation of carbon isotopic fractionation.

Laboratory and field-scale pilot experiments were performed to evaluate the feasibility of chloroform degradation by alkaline hydrolysis and the potential of δ(13)C values to assess this induced reaction process at contaminated sites. In batch experiments, alkaline conditions were induced by adding crushed concrete (pH 12.33 ± 0.07), a filtered concrete solution (pH 12.27 ± 0.04), a filtered cement solution (pH 12.66 ± 0.02) and a pH 12 buffer solution (pH 11.92 ± 0.11). The resulting chloroform degradation after 28 days was 94, 96, 99, and 72%, respectively. The experimental data were described using a pseudo-first-order kinetic model, resulting in pseudo-first-order rate constant values of 0.10, 0.12, 0.20, and 0.05 d(-1), respectively. Furthermore, the significant chloroform carbon isotopic fractionation associated with alkaline hydrolysis of chloroform (-53 ± 3‰) and its independence from pH in the admittedly limited tested pH range imply a great potential for the use of δ(13)C values for in situ monitoring of the efficacy of remediation approaches based on alkaline hydrolysis. The carbon isotopic fractionation obtained at the lab scale allowed the calculation of the percentage of chloroform degradation in field-scale pilot experiments where alkaline conditions were induced in two recharge water interception trenches filled with concrete-based construction wastes. A maximum of approximately 30-40% of chloroform degradation was achieved during the two studied recharge periods. Although further research is required, the treatment of chloroform in groundwater through the use of concrete-based construction wastes is proposed. This strategy would also imply the recycling of construction and demolition wastes for use in value-added applications to increase economic and environmental benefits.