Stable isotope evidence for biodegradation of chlorinated ethenes at a fractured bedrock site

Stable carbon isotope analysis of chlorinated ethenes and ethene was performed at a site contaminated with trichloroethene (TCE), a dense non-aqueous phase liquid (DNAPL). The site is located in fractured bedrock and had variable groundwater hydraulic gradients during the study due to a local excavation project. Previous attempts to biostimulate a pilot treatment area at the site resulted in the production of cis-1,2-dichloroethene (cis-DCE), the first product of reductive dechlorination of TCE. Cis-DCE concentrations accumulated however, and there was no appreciable production of the breakdown products from further reductive dechlorination, vinyl chloride (VC) and ethene (ETH). Consequently, the pilot treatment area was bioaugmented with a culture of KB-1, a natural microbial consortium known to completely reduce TCE to nontoxic ETH. Due to ongoing dissolution of TCE from DNAPL in the fractured bedrock, and to variable hydraulic gradients, concentration profiles of dissolved TCE and its degradation products cis-DCE, VC, and ETH could not convincingly confirm biodegradation of the chlorinated ethenes. Isotopic analysis of cis-DCE and VC, however, demonstrated that biodegradation was occurring in the pilot treatment area. The isotope values of cis-DCE and VC became significantly more enriched in 13C over the last two sampling dates (in one well from -17.6%o to -12.8%o and from -22.5%o to -18.2%o for cis-DCE and VC, respectively). Quantification of the extent of biodegradation in the pilot treatment area using the Rayleigh model indicated that, depending on the well, between 21.3% and 40.7% of the decrease in cis-DCE and between 15.2% and 36.7% of the decrease in VC concentrations can be attributed to the effects of biodegradation during this time period. Within each well, the isotope profile of TCE remained relatively constant due to the continuous input of undegraded TCE due to DNAPL dissolution.     

Stable carbon isotope fractionation during aerobic biodegradation of chlorinated ethenes

Stable isotope analysis is recognized as a powerful tool for monitoring, assessing, and validating in-situ bioremediation processes. In this study, kinetic carbon isotope fractionation factors (epsilon) associated with the aerobic biodegradation of vinyl chloride (VC), cis-1,2-dichloroethylene (cDCE), and trichloroethylene (TCE) were examined. Of the three solvents, the largest fractionation effects were observed for biodegradation of VC. Both metabolic and cometabolic VC degradation were studied using Mycobacterium aurum L1 (grown on VC), Methylosinus trichosporium OB3b (grown on methane), Mycobacterium vaccae JOB5 (grown on propane), and two VC enrichment cultures seeded from contaminated soils of Alameda Point and Travis Air Force Base, CA. M. aurum L1 caused the greatest fractionation (epsilon = -5.7) while for the cometabolic cultures, epsilon values ranged from -3.2 to -4.8. VC fractionation patterns for the enrichment cultures were within the range of those observed for the metabolic and cometabolic cultures (epsilon = -4.5 to -5.5). The fractionation for cometabolic degradation of TCE by Me. trichosporium OB3b was low (epsilon = -1.1), while no quantifiable carbon isotopic fractionation was observed during the cometabolic degradation of cDCE. For all three of the tested chlorinated ethenes, isotopic fractionation measured during aerobic degradation was significantly smaller than that reported for anaerobic reductive dechlorination. This study suggests that analysis of compound-specific isotopic fractionation could assist in determining whether aerobic or anaerobic degradation of VC and cDCE predominates in field applications of in-situ bioremediation. In contrast, isotopic fractionation effects associated with metabolic and cometabolic reactions are not sufficiently dissimilar to distinguish these processes in the field.     

Chlorine isotope fractionation during reductive dechlorination of chlorinated ethenes by anaerobic bacteria

Chlorine isotope fractionation during reductive dechlorination of trichloroethene (TCE) and tetrachloroethene (PCE) to cis-1,2-dichloroethene (cDCE) by anaerobic bacteria was investigated. The changes in the 37Cl/35Cl ratio observed during the one-step reaction (TCE to cDCE) can be explained by the regioselective elimination of chlorine accompanied by the Rayleigh fractionation. The fractionation factors (alpha) of the TCE dechlorination by three kinds of anaerobic cultures were approximately 0.994-0.995 at 30 degrees C. The enrichment of 37Cl in the organic chlorine during the two-step reaction (PCE to cDCE) can be explained by the random elimination of one chlorine atom in the PCE molecule followed by the regioselective elimination of one chlorine atom in the TCE molecule. The fractionation factors for the first step of the PCE dechlorination with three kinds of anaerobic cultures were estimated to be 0.987-0.991 at 30 degrees C using a mathematical model. Isotope fractionation during the first step would be the primary factor for the chlorine isotope fractionation during the PCE dechorination to cDCE. The developed models can be utilized to evaluate the fractionation factors of regioselective and multistep reactions.     

Monitoring biodegradation of ethene and bioremediation of chlorinated ethenes at a contaminated site using compound-specific isotope analysis (CSIA)

Chlorinated ethenes are commonly found in contaminated groundwater. Remediation strategies focus on transformation processes that will ultimately lead to nontoxic products. A major concern with these strategies is the possibility of incomplete dechlorination and accumulation of toxic daughter products (cis-1,2-dichloroethene (cDCE), vinyl chloride (VC)). Ethene mass balance can be used as a direct indicator to assess the effectiveness of dechlorination. However, the microbial processes that affect ethene are not well characterized and poor mass balance may reflect biotransformation of ethene rather than incomplete dechlorination. Microbial degradation of ethene is commonly observed in aerobic systems but fewer cases have been reported in anaerobic systems. Limited information is available on the isotope enrichment factors associated with these processes. Using compound-specific isotope analysis (CSIA) we determined the enrichment factors associated with microbial degradation of ethene in anaerobic microcosms (ε = -6.7‰ ± 0.4‰, and -4.0‰ ± 0.8‰) from cultures collected from the Twin Lakes wetland area at the Savannah River site in Georgia (United States), and in aerobic microcosms (ε = -3.0‰ ± 0.3‰) from Mycobacterium sp. strain JS60. Under anaerobic and aerobic conditions, CSIA can be used to determine whether biotransformation of ethene is occurring in addition to biodegradation of the chlorinated ethenes. Using δ(13)C values determined for ethene and for chlorinated ethenes at a contaminated field site undergoing bioremediation, this study demonstrates how CSIA of ethene can be used to reduce uncertainty and risk at a site by distinguishing between actual mass balance deficits during reductive dechlorination and apparent lack of mass balance that is related to biotransformation of ethene.     

Oxidation of chlorinated ethenes by heat-activated persulfate: kinetics and products

In situ chemical oxidation (ISCO) and in situ thermal remediation (ISTR) are applicable to treatment of groundwater contaminated with chlorinated ethenes. ISCO with persulfate (S2O8(2-)) requires activation, and this can be achieved with the heat from ISTR, so there may be advantages to combining these technologies. To explore this possibility, we determined the kinetics and products of chlorinated ethene oxidation with heat-activated persulfate and compared them to the temperature dependence of other degradation pathways. The kinetics of chlorinated ethene disappearance were pseudo-first-order for 1-2 half-lives, and the resulting rate constants-measured from 30 to 70 degrees C--fit the Arrhenius equation, yielding apparent activation energies of 101 +/- 4 kJ mol(-1) for tetrachloroethene (PCE), 108 +/- 3 kJ mol(-1) for trichloroethene (TCE), 144 +/- 5 kJ mol(-1) for cis-1,2-dichloroethene (cis-DCE), and 141 +/- 2 kJ mol(-1) for trans-1,2-dichloroethene (trans-DCE). Chlorinated byproducts were observed, but most of the parent material was completely dechlorinated. Arrhenius parameters for hydrolysis and oxidation by persulfate or permanganate were used to calculate rates of chlorinated ethene degradation by these processes over the range of temperatures relevant to ISTR and the range of oxidant concentrations and pH relevant to ISCO.     

Carbon and chlorine isotope effects during abiotic reductive dechlorination of polychlorinated ethanes

We investigated the extent and variability of C and Cl isotope fractionation during the reduction of polychlorinated ethanes to evaluate the potential use of Cl isotope analysis for the assessment of contaminant transformation in subsurface environments. Kinetic isotope effects (KIE) for C and Cl for the reductive beta-elimination of 1,1,2,2-tetrachloroethane (1,1,2,2-TeCA), pentachloroethane (PCA), and hexachloroethane by Cr(II) used as model reductant in homogeneous solution were compared to KIEs measured for dehydrochlorination of 1,1,2,2-TeCA and PCA. Since isotopic reactions of polychlorinated compounds are complicated by the simultaneous presence of several Cl isotopologues and intramolecular isotopic competition, we present a procedure for the determination of KIEs for Cl from the initial reactant and final product Cl isotope ratios. Despite different reaction mechanisms, that is reduction via dissociative inner-sphere electron transfer by Cr(H2O)6(2+) and base-catalyzed, concerted elimination, respectively, apparent KIEs for C of both pathways fall within a similar range (1.021-1.031). In contrast, KIEs for Cl are significantly higher for reductive beta-elimination (1.013-1.021) than for dehydrochlorination (1.000-1.006). These results suggest that reductive transformations of polychlorinated contaminants might be identified on the basis of combined C and Cl isotope analysis.     

Seasonal variability and long term trends of chlorofluorocarbon mixing ratios in the unsaturated zone

To investigate processes that might affect chlorofluorocarbon (CFC) mixing ratios at the water table, a time series was obtained of unsaturated zone soil gas CFCs to depths of ca. 4 m at a site near New York City (NYC). Observed CFC 11, 12, and 113 mixing ratios were lower in winter than expected from either a local, high-resolution time series or remote atmospheric mixing ratios. A diffusion model, which includes seasonal changes in soil temperature, moisture, and CFC solubility, reproduces to first order the observed soil gas mixing ratios for CFC 11 and 12. Underestimation by the model of the seasonal cycle of CFC 11 points to changing levels of sorption to soils due to seasonal changes in temperature as an additional cause of the cycle seen in CFC 11 mixing ratios in soil air. In the case of spring recharge, low CFC mixing ratios in soil air caused by increased solubility may result in low CFC 11 concentrations in groundwater and, when dating groundwater recharged before the 1990s with CFCs, older apparent ages by up to 4 years. Attempts to observe average atmospheric CFC levels from soil gas are also significantly hindered by these seasonal fluctuations. Our results indicate the importance of considering seasonal changes in soil temperature when making precise observations of even very moderately soluble gases in the unsaturated zone and shallow groundwater.     

Assessment of degradation pathways in an aquifer with mixed chlorinated hydrocarbon contamination using stable isotope analysis

The demonstration of monitored natural attenuation (MNA) of chlorinated hydrocarbons in groundwater is typically conducted through the evaluation of concentration trends and parent-daughter product relationships along prevailing groundwater flow paths. Unfortunately, at sites contaminated by mixtures of chlorinated ethenes, ethanes, and methanes, the evaluation of MNA by using solely concentration data and parent-daughter relationships can result in erroneous conclusions regarding the degradation mechanisms that are truly active at the site, since many of the daughter products can be derived from multiple parent compounds. Stable carbon isotope analysis was used, in conjunction with concentration data, to clarify and confirm the active degradation pathways at a former waste solvent disposal site where at least 14 different chlorinated hydrocarbons have been detected in the groundwater. The isotope data indicate that TCE, initially believed to be present as a disposed product and/or a PCE dechlorination intermediate, is attributable to dehydrochlorination of 1,1,2,2-PCA. The isotope data further support that vinyl chloride and ethene in the site groundwater result from dichloroelimination of 1,1,2-trichlorethane and 1,2-dichloroethane, respectively, rather than from reductive dechlorination of the chlorinated ethenes PCE, TCE, or 1,2-DCE. The isotope data confirm that the chlorinated ethanes and chlorinated methanes are undergoing significant intrinsic degradation, whereas degradation of the chlorinated ethenes may be limited. In addition to the classical trend of enriched isotope values of the parent compounds with increasing distance associated to biodegradation, shifts of isotope ratios of degradation byproduct in the opposite direction due to mixing of isotopically light byproducts of biodegradation with compounds from the source are shown to be of high diagnostic value. These data underline the value of stable isotope analysis in confirming transformation processes at sites with complex mixtures of chlorinated compounds.     

Competition for sorption and degradation of chlorinated ethenes in batch zero-valent iron systems

The sorption and degradation of the chlorinated ethenes tetrachloroethene (PCE, 5 mg L(-1)) and trichloroethene (TCE, 10 mg L(-1)) were investigated in zero-valent iron systems (ZVI, 100 g L(-1)) in the presence of compounds common to contaminated groundwater with varying physicochemical properties. The potential competitors were chlorinated ethenes, monocyclic aromatic hydrocarbons, and humic acids. The effect of a complex matrix was tested with landfill contaminated groundwater. Nonlinear Freundlich isotherms adequately described chloroethene sorption to ZVI. In the presence of the more hydrophobic PCE (5 mg L(-1)), TCE sorption and degradation decreased by 33% and 30%, respectively, while TCE (10 mg L(-1)) decreased PCE degradation by 30%. In the presence of nonreactive hydrophobic hydrocarbons (i.e., benzene, toluene, and m-xylene at 100 mg L(-1)), TCE and PCE sorption decreased by 73% and 55%, respectively. The presence of the hydrocarbons had no effect on TCE degradation and increased PCE reduction rates by 50%, suggesting that the displacement of the chloroethenes from the sorption sites by the aromatic hydrocarbons enhanced the degradation rates. Humic acids did not interfere significantly with chloroethene sorption or with TCE degradation but lowered PCE degradation kinetics by 36% when present at high concentrations (100 mg L(-1)). The landfill groundwater with an organic carbon content of 109 mg L(-1) C had no effect on chloroethene sorption but inhibited TCE and PCE degradation by 60% and 70%, respectively.     

Groundwater acidification and the mobilization of trace metals in a sandy aquifer

The acidification of groundwater due to acid rain impact and the mobilization of the trace metals Ni, Be, Cd and Co was studied in a noncalcareous sandy aquifer. The groundwater is acidified down to pH 4.4 in the upper 3-4 m of the saturated zone. There is a sharp acidification front and below that the pH increases to 5.2-6.5. The acid zone groundwater contains an Al concentration of approximately 0.2 mM. These observations could be explained by a reactive transport model for downward groundwater movement based on ion exchange and equilibrium with Al(OH)3. At the acidification front, the Al3+ in groundwater exchanges for sorbed Ca2+ and Mg2+ and the coupled dissolution of Al(OH)3 causes the pH to increase. The downward migration rate of the acidification front is 3.5-5.0 cm/yr. Trace metals (Ni, Be, Cd and Co) are found to accumulate near the acidification front. Downward moving, low pH, and trace metal containing groundwater passes the acidification front, and the trace metals adsorb as the pH increases. The acidification front moves downward at a slower rate, and in this process the heavy metals are desorbed. Accordingly, the acidification front functions as a geochemical trap where trace metals accumulate, and their amount will increase with time. Different surface complexation models were explored to explain the behavior of Ni. Neither a simple iron oxide surface complexation model nor ion exchange could explain the field observations of the Ni distribution. The sediment appeared, even at low pH, to have a much stronger affinity toward Ni than predicted by the iron oxide model. The discrepancy can be accounted for in the model by increasing the Ni binding strength constant in combination with an increased number of reactive sites.     

Leaching of metribuzin metabolites and the associated contamination of a sandy Danish aquifer

As degradation products of metribuzin have received little attention as potential groundwater contaminants, we evaluated leaching of metribuzin and its primary metabolites desaminometribuzin (DA), desaminodiketometribuzin (DADK), and diketometribuzin (DK) at a sandy test site in Denmark. Soil water and groundwater were sampled monthly over a four-year period. Leaching of metribuzin and DA was negligible. DK and DADK leached from the root zone (1 meter below ground surface (mbgs)) in average concentrations considerably exceeding the EU limit value for drinking water (0.1 microg/L). Both metabolites appear to be relatively stable and persisted in soil water and groundwater several years after application. Past application of metribuzin at the site had contaminated the groundwater with both DK and DADK, which were detected in 99% and 48%, respectively, of the groundwater samples analyzed. Except for three of the groundwater samples, the DADK concentration never exceeded the EU limit value. In contrast, the annual concentration of DK exceeded 0.1 microg/L at 90% of the screens analyzed. The present findings suggest that as the degradation products of metribuzin can leach through sandy soil in high concentrations, they could potentially contaminate the groundwater. In view of this risk DK and DADK should both be included in monitoring programs and their ecotoxicological effects should be further investigated.     

Experimental evidence for a lack of thermodynamic control on hydrogen concentrations during anaerobic degradation of chlorinated ethenes

Hydrogen (H2) concentrations during reductive dechlorination of cis-dichloroethene (cDCE) and vinyl chloride (VC) were investigated with respectto the influence of parameters entering the Gibbs free energy expression of the reactions. A series of laboratory experiments was conducted employing a mixed, Dehalococcoides-containing enrichment culture capable of complete dechlorination of chlorinated ethenes. The objective was to investigate whether a constant energy gain controls H2 levels in dechlorinating systems, thereby evaluating the applicability of the partial equilibrium approach to microbial dechlorination at contaminated sites. Variations in the temperature between 10 and 30 degrees C did not affect the H2 concentration in a fashion that suggested thermodynamic control through a constant energy gain. In another set of experiments, H2 levels at constant ionic strength were independent of the chloride concentration between 10 and 110 mmol chloride per liter. These findings demonstrate that the partial equilibrium approach is not directly applicable to the interpretation of reductive degradation of chlorinated ethenes. We also present recalculated thermodynamic properties of aqueous chlorinated ethene species that allow for calculation of in-situ Gibbs free energy of dechlorination reactions at different temperatures.     

Dehalogenation of chlorinated ethenes and immobilization of nickel in anaerobic sediment columns under sulfidogenic conditions

A sediment column study was carried out to demonstrate the bioremediation of chloroethene- and nickel-contaminated sediment in a single anaerobic step under sulfate-reducing conditions. Four columns (one untreated control column and three experimental columns) with sediment from a chloroethene- and nickel-contaminated site were investigated for 1 year applying different treatments. By stimulating the activity of sulfate-reducing bacteria by the addition of sulfate as supplementary electron acceptor, complex anaerobic communities were maintained with lactate as electron donor (with or without methanol), which achieved complete dehalogenation of tetra- and trichloroethenes (PCE and TCE) to ethene and ethane. A few weeks after sulfate addition, production of sulfide increased, indicating an increasing activity of sulfate-reducing bacteria. The nickel concentration in the effluent of one nickel-spiked column was greatly reduced, likely due to the enhanced sulfide production, causing precipitation of nickel sulfide. At the end of the study, 94% of the initial amount of nickel added to that column was recovered in the sediment As compared to the untreated (nonspiked) control column, all chloroethene-spiked columns ladditions of PCE and TCE) showed a permanent release of small chloride ion quantities (approximately 0.5-0.7 mM chloride), which were detected in the effluents a few weeks after sulfide production was observed for the first time. The formation of ethene and ethane as final products after dechlorination of PCE and TCE was detected in some effluents and in some gas phases of the columns. Other metabolites or intermediates (such as DCE isomers) were only detected sporadically in negligible quantities. The results of this study demonstrated thatmicrobial activity stimulated under sulfate-reducing conditions can have a beneficial effect on both the precipitation of heavy metals and the complete dechlorination of organochlorines. The strongly negative redox potential created by the activity of sulfate-reducing bacteria may be one factor responsible for stimulating the activity of the dehalogenating bacteria in the test columns.     

Carbon isotope fractionation during permanganate oxidation of chlorinated ethylenes (cDCE,TCE, PCE)

Permanganate oxidation of chlorinated ethylenes is an attractive technique to effect remediation of these important groundwater contaminants. Stable carbon isotope fractionation associated with permanganate oxidation of trichloroethylene (TCE), tetrachloroethylene (PCE), and cis-1,2-dichloroethylene (cDCE) has been measured, to study the possibility of applying stable carbon isotope analysis as a technique to assess the efficacy of remediation implemented by permanganate oxidation. Average carbon isotope fractionation factors of alphaTCE = 0.9786, alphaPCE = 0.9830, and alphacDCE = 0.9789 were obtained, although the fractionation factor for PCE may be interpreted to change from a value of 0.9779-0.9871 during the course of the reaction. The fractionation factors for all three compounds are quite similar, in contrast to the variation of fractionation factors vs degree of chlorination observed for other degradative processes, such as microbial dechlorination. This may be due to a common rate-determining step for permanganate oxidation of all three compounds studied. The large fractionation factors and the relative lack of dependence of the fractionation factors upon other environmental factors (e.g. oxidation rate, presence of multiple contaminants, incomplete oxidation, presence of chloride in solution) indicate that monitoring delta13C values of chlorinated ethylenes during oxidation with permanganate may be a sensitive, and potentially quantitative, technique to investigate the extent of degradation.     

Changes in interfacial tension of chlorinated solvents following flow through U.K. soils and shallow aquifer material

The interfacial tension (IFT) that arises at the interface between water and an immiscible organic liquid is a key parameter affecting the transport and subsequent fate of the organic liquid in water-saturated porous media. In this paper, data are presented that show how contact between a range of soil types and chlorinated hydrocarbon solvent (CHS) dense nonaqueous phase liquids (DNAPLs) can affect DNAPL/water IFT values. The soils examined are indicative of U.K. soil types and shallow aquifer materials. The solvents investigated were tetrachloroethylene (PCE) and trichloroethylene (TCE). Lab grade, recovered field DNAPL and industrial waste chlorinated solvent mixtures were used. The data from batch and column experiments invariably revealed that water/DNAPL IFT values change following contact with unsaturated soils. In the majority of cases, the IFT values increase following soil exposure. However, after contact with an organic-rich soil, the IFT of the lab grade solvents decreased. The experimental evidence suggests that these reductions are linked to the removal of organic material from the soil and its subsequent incorporation into the solvent IFT increases in the case of lab solvents are shown to be linked to the removal of stabilizers (added by the manufacturers to obviate degradation) that are removed by adsorption to soil mineral surfaces. Similarly, it is conjectured that adsorption of surface-active compounds from the industrial waste samples to soil surfaces is responsible for increases in the IFT in these samples. Finally, it was observed that invading CHSs are capable of dissolving and subsequently mobilizing in-situ soil contaminants. GC/MS analysis revealed these mobilized soil contaminants to be polyaromatic hydrocarbons and phthalate esters.     

Monitoring oxidation of chlorinated ethenes by permanganate in groundwater using stable isotopes: laboratory and field studies

Permanganate injection is increasingly applied for in situ destruction of chlorinated ethenes in groundwater. This laboratory and field study demonstrates the roles that carbon isotope analysis can play in the assessment of oxidation of trichloroethene (TCE) by permanganate. In laboratory experiments a strong carbon isotope fractionation was observed during oxidation of TCE with similar isotopic enrichment factors (-25.1 to -26.8 per thousand) for initial KMnO4 concentrations between 67 and 1,250 mg/L. At the field site, a single permanganate injection episode was conducted in a sandy aquifer contaminated with TCE as dense nonaqueous liquid (DNAPL). After injection, enriched delta13C values of up to +204% and elevated Cl- concentrations were observed at distances of up to 4 m from the injection point. Farther away, the Cl- increased without any change in delta13C of TCE suggesting that Cl- was not produced locally but migrated to the sampling point Except for the closest sampling location to the injection point, the delta13C rebounded to the initial 613C again, likely due to dissolution of DNAPL Isotope mass balance calculations made it possible to identify zones where TCE oxidation continued to occur during the rebound phase. The study indicates that delta13C values can be used to assess the dynamics between TCE oxidation and dissolution and to locate zones of oxidation of chlorinated ethenes that cannot be identified from the Cl- distribution alone.