Daniel Hunkeler

Abstract: Three independent techniques were used to assess the biodegradation of monoaromatic hydrocarbons and low-molecular weight polyaromatic hydrocarbons in the alluvial aquifer at the site of a former cokery (Flemalle, Belgium). Firstly, a stable carbon isotope-based field method allowed quantifying biodegradation of monoaromatic compounds in situ and confirmed the degradation of naphthalene. No evidence could be deduced from stable isotope shifts for the intrinsic biodegradation of larger molecules such as methylnaphthalenes or acenaphthene. Secondly, using signature metabolite analysis, various intermediates of the anaerobic degradation of (poly-) aromatic and heterocyclic compounds were identified. The discovery of a novel metabolite of acenaphthene in groundwater samples permitted deeper insights into the anaerobic biodegradation of almost persistent environmental contaminants. A third method, microcosm incubations with (13)C-labeled compounds under in situ-like conditions, complemented techniques one and two by providing quantitative information on contaminant biodegradation independent of molecule size and sorption properties. Thanks to stable isotope labels, the sensitivity of this method was much higher compared to classical microcosm studies. The (13)C-microcosm approach allowed the determination of first-order rate constants for (13)C-labeled benzene, naphthalene, or acenaphthene even in cases when degradation activities were only small. The plausibility of the third method was checked by comparing (13)C-microcosm-derived rates to field-derived rates of the first approach. Further advantage of the use of (13)C-labels in microcosms is that novel metabolites can be linked more easily to specific mother compounds even in complex systems. This was achieved using alluvial sediments where (13)C-acenaphthyl methylsuccinate was identified as transformation product of the anaerobic degradation of acenaphthene. (C) 2011 Elsevier Ltd. All rights reserved.

Abstract: Crystalline aquifers of semi-arid southern India represent a vital water resource for farming communities. A field study is described that characterizes the hydrodynamic functioning of intensively exploited crystalline aquifers at local scale based on detailed well monitoring during one hydrological year. The main results show large water-table fluctuations caused by monsoon recharge and pumping, high spatial variability in well discharges, and a decrease of well yields as the water table decreases. Groundwater chemistry is also spatially variable with the existence of aquifer compartments within which mixing occurs. The observed variability and compartmentalization is explained by geological heterogeneities which play a major role in controlling groundwater flow and connectivity in the aquifer. The position of the water table within the fracture network will determine the degree of connectivity between aquifer compartments and well discharge. The presented aquifer conceptual model suggests several consequences: (1) over-exploitation leads to a drop in well discharge, (2) intensive pumping may contribute to the hydraulic containment of contaminants, (3) groundwater quality is highly variable even at local scale, (4) geological discontinuities may be used to assist in the location of drinking-supply wells, (5) modeling should integrate threshold effects due to water-table fluctuations.

Abstract: Analytical models were developed that simulate stable isotope ratios of volatile organic compounds (VOCs) near a point source contamination in the unsaturated zone. The models describe diffusive transport of VOCs, biodegradation and source ageing. The mass transport is governed by Fick's law for diffusion. The equation for reactive transport of VOCs in the soil gas phase was solved for different source geometries and for different boundary conditions. Model results were compared to experimental data from a one-dimensional laboratory column and a radial-symmetric field experiment. The comparison yielded a satisfying agreement. The model results clearly illustrate the significant isotope fractionation by gas phase diffusion under transient state conditions. This leads to an initial depletion of heavy isotopes with increasing distance from the source. The isotope evolution of the source is governed by the combined effects of isotope fractionation due to vaporisation, diffusion and biodegradation. The net effect can lead to an enrichment or depletion of the heavy isotope in the remaining organic phase, depending on the compound and element considered. Finally, the isotope evolution of molecules migrating away from the source and undergoing degradation is governed by a combined degradation and diffusion isotope effect. This suggests that, in the unsaturated zone, the interpretation of biodegradation of VOC based on isotopic data must always be based on a model combining gas phase diffusion and degradation. (C) 2010 Published by Elsevier B.V.

Abstract: Compound-specific isotope analysis (CSIA) can potentially be used to relate vapor phase contamination by volatile organic compounds (VOCs) to their subsurface sources. This field and modeling study investigated how isotope ratios evolve during migration of gaseous chlorinated ethenes across a 18 m thick unsaturated zone of a sandy coastal plain aquifer. At the site, high concentrations of tetrachloroethene (PCE up to 380 mu g/L), trichloroethene (TCE up to 31,600 mu g/L), and cis-1,2-dichloroethene (cDCE up to 680 mu g/L) were detected in groundwater. Chlorinated ethene concentrations were highest at the water table and steadily decreased upward toward the land surface and downward below the water table. Although isotopologues have different diffusion coefficients, constant carbon and chlorine isotope ratios were observed throughout the unsaturated zone, which corresponded to the isotope ratios measured at the water table. In the saturated zone, TCE became increasingly depleted along a concentration gradient, possibly due to isotope fractionation associated with aqueous phase diffusion. These results indicate that carbon and chlorine isotopes can be used to link vapor phase contamination to their source even if extensive migration of the vapors occurs. However, the numerical model revealed that constant isotope ratios are only expected for systems close to steady state.

Abstract: The fate of chlorinated ethenes in a large contaminant plume originating from a tetrachloroethene (PCE) source in a sandy aquifer in Denmark was investigated using novel methods including compound-specific carbon and chlorine isotope analysis and quantitative real-time polymerase chain reaction (qPCR) methods targeting Dehaloccocoides sp. and vcrA genes. Redox conditions were characterized as well based on concentrations of dissolved redox sensitive compounds and sulfur isotopes in SO(4)(2-). In the first 400 m downgradient of the source, the plume was confined to the upper 20 m of the aquifer. Further downgradient it widened in vertical direction due to diverging groundwater flow reaching a depth of up to 50 m. As the plume dipped downward and moved away from the source, O(2) and NO(3)(-) decreased to below detection levels, while dissolved Fe(2+) and SO(4)(2-) increased above detectable concentrations, likely due to pyrite oxidation as confirmed by the depleted sulfur isotope signature of SOY. In the same zone, PCE and trichloroethene (TCE) disappeared and cis-1,2-dichloroethene (cDCE) became the dominant chlorinated ethene. PCE and TCE were likely transformed by reductive dechlorination rather than abiotic reduction by pyrite as indicated by the formation of cDCE and stable carbon isotope data. TCE and cDCE showed carbon isotope trends typical for reductive dechlorination with an initial depletion of (13)C in the daughter products followed by an enrichment of (13)C as degradation proceeded. At 1000 m downgradient of the source, cDCE was the dominant chlorinated ethene and had reached the source delta(13)C value confirming that cDCE was not affected by abiotic or biotic degradation. Further downgradient (up to 1900 m), cDCE became enriched in (13)C by up to 8% demonstrating its further transformation while vinylchloride (VC) concentrations remained low (<1 mu g/L) and ethene was not observed. The correlated shift of carbon and chlorine isotope ratios of cDCE by 8 and 3.9 parts per thousand, respectively, the detection of Dehaloccocides sp genes, and strongly reducing conditions in this zone provide strong evidence for reductive dechlorination of cDCE. The significant enrichment of (13)C in VC indicates that VC was transformed further, although the mechanism could not be determined. The transformation of cDCE was the rate limiting step as no accumulation of VC occurred. In summary, the study demonstrates that carbon-chlorine isotope analysis and qPCR combined with traditional approaches can be used to gain detailed insight into the processes that control the fate of chlorinated ethenes in large scale plumes. (C) 2010 Elsevier B.V. All rights reserved.

Abstract: Chlorine isotope analysis of chlorinated hydrocarbons like trichloroethylene (TCE) is of emerging demand because these species are important environmental pollutants. Continuous flow analysis of noncombusted TCE molecules, either by gas chromatography/isotope ratio mass spectrometry (GC/IRMS) or by GC/quadrupole mass spectrometry (GC/qMS), was recently brought forward as innovative analytical solution. Despite early implementations, a benchmark for routine applications has been missing. This study systematically compared the performance of GC/qMS versus GC/IRMS in six laboratories involving eight different instruments (GC/IRMS, Isoprime and Thermo MAT-253; GC/qMS, Agilent 5973N, two Agilent 5975C, two Thermo DSQII, and one Thermo DSQI). Calibrations of (37)Cl/(35)Cl instrument data against the international SMOC scale (Standard Mean Ocean Chloride) deviated between instruments and over time. Therefore, at least two calibration standards are required to obtain true differences between samples. Amount dependency of delta(37)Cl was pronounced for some instruments, but could be eliminated by corrections, or by adjusting amplitudes of standards and samples. Precision decreased in the order GC/IRMS (1 sigma approximate to 0.1 parts per thousand), to GC/qMS (1 sigma approximate to 0.2-0.5 parts per thousand for Agilent GC/qMS and 1 sigma approximate to 0.2-0.9 parts per thousand for Thermo GC/qMS). Nonetheless, delta(37)Cl values between laboratories showed good agreement when the same external standards were used. These results lend confidence to the methods and may serve as a benchmark for future applications.

Abstract: Trichloroacetic acid (TCAA) is an important environmental contaminant present in soils, water and plants. A method for determining the carbon isotope signature of the trichloromethyl position in TCAA using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) was developed and tested with TCAA from different origins. Position-specific isotope analysis (PSIA) can provide direct information on the kinetic isotope effect for isotope substitution at a specific position in the molecule and/or help to distinguish different sources of a compound. The method is based on the degradation of TCAA into chloroform (CF) and CO(2) by thermal decarboxylation. Since thermal decarboxylation is associated with strong carbon isotope fractionation (epsilon = -34.6 +/- 0.2%) the reaction conditions were optimized to ensure full conversion. The combined isotope ratio of CF and CO(2) at the end of the reaction corresponded well to the isotope ratio of TCAA, confirming the reliability of the method. A method quantification limit (MQL) for TCAA of 18.6 mu g/L was determined. Samples of TCAA produced by enzymatic and non-enzymatic chlorination of natural organic matter (NOM) and some industrially produced TCAA were used as exemplary sources. Significant different PSIA isotope ratios were observed between industrial TCAA and TCAA samples produced by chlorination of NOM. This highlights the potential of the method to study the origin and the fate of TCAA in the environment. Copyright (C) 2011 John Wiley & Sons, Ltd.

Abstract: The anaerobic degradation potential at a chloroethene-contaminated site was investigated by operating two anoxic column aquifer microcosms enriched in iron(III). One column was fed with vinyl chloride (VC) only (column A) and one with VC and acetate (column B). In column A, after about 600 pore volume exchanges (PVEs), VC started to disappear and reached almost zero VC recovery in the effluent after 1000 PVEs. No formation of ethene was observed. In column B, effluent VC was almost always only a fraction of influent VC. Formation of ethene was observed after 800 PVEs and started to become an important degradation product after 1550 PVEs. However, ethene was never observed in stoichiometric amounts compared with disappeared VC. The average stable isotope enrichment factor for VC disappearance in column A was determined to be -4.3%. In column B, the isotope enrichment factor shifted from -10.7 to -18.5% concurrent with an increase in ethene production. Batch microcosms inoculated with column material showed similar isotope enrichment factors as the column microcosms. These results indicated that two degradation processes occurred, one in column A and two in parallel in column B with increasing importance of reductive dechlorination with time. This study suggests that in addition to reductive dechlorination, other degradation processes such as anaerobic oxidation should be taken into account when evaluating natural attenuation of VC and that isotope analysis can help to differentiate between different pathways of VC removal.

Abstract: This study investigated the use of radon ((222)Rn), Km a radioactive isotope with a half-life of 3.8 days, and CO(2) as natural tracers to evaluate the recharge dynamics of karst aquifer under varying hydrological conditions. Dissolved (222)Rn and carbon dioxide (CO(2)) were measured continuously in an underground stream of the Milandre test site, Switzerland. Estimated soil water (222)Rn activities were higher than baseflow (222)Rn activities, indicating elevated (222)Rn production in the soil zone compared to limestone, consistent with a (226)Ra enrichment in the soil zone compared to limestone. During small flood events, (222)Rn activities did not vary while an immediate increase of the CO(2) concentration was observed. During medium and large flood events, an immediate CO(2) increase and a delayed (222)Rn activity increase to up to 4.9 Bq/L and 11 Bq/L, respectively occurred. The detection of elevated (222)Rn activities during medium and large flood events indicate that soil water participates to the flood event. A soil origin of the (222)Rn is consistent with its delayed increase compared to discharge reflecting the travel time of (222)Rn from the soil to the saturated zone of the system via the epikarst. A three-component mixing model suggested that soil water may contribute 4-6% of the discharge during medium flood events and 25-43% during large flood events. For small flood events, the water must have resided at least 25 days below the soil zone to explain the background (222)Rn activities, taking into account the half-life of (222)Rn (3.8 days). In contrast to (222)Rn, the CO(2) increase occurred simultaneously with the discharge increase. This observation as well as the CO(2) increase during small flood events, suggests that the elevated CO(2) level is not due to the arrival of soil water as for (222)Rn. A possible explanation for the CO(2) trend is that baseflow water in the stream has lower CO(2) levels due to gas loss compared to water stored in low permeability zones. During flood event, the stored water is more rapidly mobilised than during baseflow with less time for gas loss. The study demonstrates that (222)Rn and CO(2) provides value information on the dynamics of groundwater recharge of karst aquifer, which can be of high interest when evaluating the vulnerability of such systems to contamination. (C) 2011 Published by Elsevier B.V.

Abstract: In this comment, we revisit equations concerning the analytical solutions presented by Gutierrez-Neri and co-workers for reactive transport for a pollutant undergoing core and fringe degradations. We state that a correction needs to be made in Eq. (9) of the work of Gutierrez-Neri et al. in order that the equation follows closely previous work published by). Bear (in 1-D) and P.A. Domenico (in 3-D). Furthermore we derive alternative solutions for Eqs. (13)-(16) which separate more clearly the first-order reaction and the instantaneous reaction. It is shown that the corrected solution agrees better with the results from the numerical model than the previous solution. An improvement is also made by giving a solution which avoids negative concentrations. Furthermore, the corresponding solution for the electron acceptor reacting with the pollutant is given. (c) 2010 Elsevier B.V. All rights reserved.

Abstract: With increasing awareness and concern for environmental quality, it is important to study the fate of pesticides in the subsurface. Laboratory studies were conducted to determine the behavior of atrazine and glyphosate within the root zone of an undisturbed sandy soil in Jianghan Plain, central China. Chloride as a tracer for water movement was applied to the soil as KCl for 26 hours before pesticide application for another 160 hours. Glyphosate, atrazine, and Cl concentrations (conc.) were determined as a function of time in breakthrough curves (BTCs). Atrazine BTC was fitted better in convection-dispersion equation equilibrium model. For glyphosate, however, a two-site non-equilibrium model was chosen. Leaching rate of atrazine from sandy soil was much higher than that of glyphosate and it took longer for glyphosate to leach through the column due to stronger sorption and degradation to its major metabolite, AMPA (aminomethylphosphonic acid, CH(6)NO(3)P), which was detected (up to 8890 ng/l) in the final leachate.

Abstract: Field and laboratory investigations have been conducted at a former coke plant, in order to assess pollutant attenuation in a contaminated alluvial aquifer, discharging to an adjacent river. Various organic (BTEX, PAHs, mineral oils) and inorganic (As, Zn, Cd) compounds were found in the aquifer in concentrations exceeding regulatory values. Due to redox conditions of the aquifer, heavy metals were almost immobile, thus not posing a major risk of dispersion off-site the brownfield. Field and laboratory investigations demonstrated that benzene, among organic pollutants, presented the major worry for off-site dispersion, mainly due to its mobility and high concentration, i.e. up to 750 mg L-1 in the source zone. However, benzene could never be detected near the river which is about 160 m downgradient the main source. Redox conditions together with benzene concentrations determined in the aquifer have suggested that degradation mainly occurred within 100 m distance from the contaminant source under anoxic conditions, and most probably with sulphate as main oxidant. A numerical groundwater flow and transport model, calibrated under transient conditions, was used to simulate benzene attenuation in the alluvial aquifer towards the Meuse River. The mean benzene degradation rate used in the model was quantified in situ along the groundwater flow path using compound-specific carbon isotope analysis (CSIA). The results of the solute transport simulations confirmed that benzene concentrations decreased almost five orders of magnitude 70 m downgradient the source. Simulated concentrations have been found to be below the detection limit in the zone adjacent to the river and consistent with the absence of benzene in downgradient piezometers located close to the river reported in groundwater sampling campaigns. In a transient model scenario including groundwater-surface water dynamics, benzene concentrations were observed to be inversely correlated to the river water levels, leading to the hypothesis that benzene dispersion is mainly controlled by natural attenuation. (C) 2009 Elsevier B.V. All rights reserved.

Abstract: The effect of the number of carbon and chlorine atoms on carbon isotope fractionation during dechlorination of chlorinated alkanes by Xanthobacter autotrophicus GJ10 was studied using pure culture and cell-free extract experiments. The magnitude of carbon isotope fractionation decreased with increasing carbon number. The decrease can be explained by an increasing probability that the heavy isotope is located at a non-reacting position for increasing molecule size. The isotope data were corrected for the number of carbons as well as the number of reactive sites to obtain reacting-site-specific values denoted as apparent kinetic isotope effect (AKIE). Even after the correction, the obtained AKIE values varied (on average 1.0608, 1.0477, 1.0616, and 1.0555 for 1,2-dichloroethane, chloropentane, 1,3-dichloropentane and chlorobutane, respectively). Cell-free extract experiments were carried out to evaluate the effect of transport across the cell membrane on the observed variability in the AKIE values, which revealed that variability still persisted. The study demonstrates that even after differences related to the carbon number and structure of the molecule are taken into account, there still remain differences in AKIE values even for compounds that are degraded by the same pure culture and an identical reaction mechanism.

Abstract: The study investigated carbon and chlorine isotope fractionation during aerobic oxidation and reductive dechlorination of vinyl chloride (VC) and cis-1,2-dichloroethene (cDCE). The experimental data followed a Rayleigh trend. For aerobic oxidation, the average carbon isotope enrichment factors were -7.2 parts per thousand and -8.5 parts per thousand for VC and cDCE, respectively, while average chlorine isotope enrichment factors were only -0.3 parts per thousand for both compounds. These values are consistent with an initial transformation by epoxidation for which a significant primary carbon isotope effect and only a small secondary chlorine isotope effect is expected. For reductive dechlorination, larger carbon isotope enrichment factors of -25.2 parts per thousand for VC and -18.5 parts per thousand for cDCE were observed consistent with previous studies. Although the average chlorine isotope enrichment factors were larger than those of aerobic oxidation (-1.8 parts per thousand for VC, -1.5 parts per thousand for cDCE), they were not as large as typically expected for a primary chlorine isotope effect suggesting that no cleavage of C-Cl bonds takes place during the initial rate-limiting step. The ratio of isotope enrichment factors for chlorine and carbon were substantially different for the two reaction mechanisms suggesting that the reaction mechanisms can be differentiated at the field scale using a dual isotope approach.

Abstract: There is increasing interest in combined carbon-chlorine compound-specific isotope analysis (CSIA) to differentiate between contaminant sources and to assess transformation processes, However, the significant abundance of polychlorinated molecules with several heavy chlorine isotopes complicates the evaluation of chlorine isotope trends. Therefore, the goal of this study was to develop a conceptual and mathematical framework that describes the expected chlorine isotope fractionation patterns during multistep transformation of chlorinated compounds. Reductive dechlorination of chlorinated ethenes served as an example. The study demonstrates that chlorine isotope trends can be simulated by reproducing the average behavior of light and heavy isotopes or by explicitly simulating molecules with different numbers of heavy isotopes (isotopologues). The calculations reveal that initial chlorine isotope ratios of products equal the isotope ratios of their parent compounds in the absence of secondary isotope effects, while steadily increasing during transformation. The slopes in dual isotope plots are linear for reactant and product during a one-step reaction. They become nonlinear for products that are degraded further but converge to characteristic slopes. Consideration of different scenarios reveals that combined carbon-chlorine isotope analysis bears high potential to differentiate between contaminant sources, to elucidate reaction mechanisms in laboratory studies, and to identify transformation processes in the field.

Abstract: At a former wood preservation plant severely contaminated with coal tar oil, in situ bulk attenuation and biodegradation rate constants for several monoaromatic (BTEX) and polyaromatic hydrocarbons (PAH) were determined using (1) classical first order decay models, (2) Michaelis-Menten degradation kinetics (MM), and (3) stable carbon isotopes, for o-xylene and naphthalene. The first order bulk attenuation rate constant for o-xylene was calculated to be 0.0025 d(-1) and a novel stable isotope-based first order model, which also accounted for the respective redox conditions, resulted in a slightly smaller biodegradation rate constant of 0.0019 d(-1). Based on MM-kinetics, the o-xylene concentration decreased with a maximum rate of k(max)=0.1 mu g/L/d. The bulk attenuation rate constant of naphthalene retrieved from the classical first order decay model was 0.0038 d(-1). The stable isotope-based biodegradation rate constant of 0.0027 d(-1) was smaller in the reduced zone, while residual naphthalene in the oxic part of the plume further downgradient was degraded at a higher rate of 0.0038 d(-1). With MM-kinetics a maximum degradation rate of k(max)=12 mu g/L/d was determined. Although best fits were obtained by MM-kinetics, we consider the carbon stable isotope-based approach more appropriate as it is specific for biodegradation (not overall attenuation) and at the same time accounts for the dominant electron-accepting process. For o-xylene a field based isotope enrichment factor epsilon(field) of - 1.4 could be determined using the Rayleigh model, which closely matched values from laboratory studies of o-xylene degradation under sulfate-reducing conditions. (C) 2008 Elsevier B.V. All rights reserved.

Abstract: The occurrence of chlorinated ethene transformation in a streambed was investigated using concentration and carbon isotope data from water samples taken at different locations and depths within a 15 x 25 m study area across which a tetrachloroethene (PCE) plume discharges. Furthermore, it was evaluated how the degree of transformation is related to groundwater discharge rates, redox conditions, solid organic matter content (SOM) and microbial factors. Groundwater discharge rates were quantified based on streambed temperatures, and redox conditions using concentrations of dissolved redox-sensitive species. The degree of chlorinated ethene transformation was highly variable in space from no transformation to transformation beyond ethene. Complete reductive dechlorination to ethane and ethene occurred at locations with at least sulfate-reducing conditions and with a residence time in the samples streambed zone (80 cm depth) of at least 10 days. Among these locations, Dehalococcoides was detected using a PCR method where SOM contents were > 2% w/w and where transformation proceeded beyond ethene. However, it was not detected at locations with low SOM, which may cause an insufficient H-2 supply to sustain a detectably dense Dehalococcoides population. Additionally, it is possible that other organisms are responsible for the biodegradation. A microcosm study with streambed sediments demonstrated the potential of VC oxidation throughout the site even at locations without a pre-exposure to VC, consistent with the detection of the epoxyalkane:coenzyme M transferase (EaCoMT) gene involved in the degradation of chlorinated ethenes via epoxiclation. In contrast, no aerobic transformation of cDCE in microcosms over a period of 1.5 years was observed. In summary, the study demonstrated that carbon isotope analysis is a sensitive tool to identify the degree of chlorinated ethene transformation even in hydrologically and geochemically complex streambed systems. In addition, it was observed that the degree of transformation is related to redox conditions, which in turn depend on groundwater discharge rates. (C) 2009 Elsevier B.V. All rights reserved.

Abstract: The study focuses on the effect of volatilization, diffusion, and biodegradation on the isotope evolution of volatile organic compounds (VOCs) in a 1.06 m long column filled with alluvial sand. A liquid mixture of 10 VOCs was placed at one end of the column, and measurements of VOC vapor concentrations and compound-specific isotope ratios (delta C-13) were performed at the source and along the column. Initially, the compounds became depleted in C-13 by up to -4.8 parts per thousand along the column axis, until at 26 h, uniform isotope profiles were observed for most compounds, which is expected for steady-state diffusion. Subsequently, several compounds (n-pentane, benzene, n-hexane) became enriched in C-13 throughout the column. For the same compounds, a significant decrease in the source vapor concentration and a gradual enrichment of C-13 by up to 5.3 parts per thousand at the source over a period of 336 h was observed. This trend can be explained by a larger diffusive mass flux for molecules with light isotopes compared to those with a heavy isotope, which leads to a depletion of light isotopes in the source. The isotope evolution of the source followed closely a Rayleigh trend and the obtained isotope enrichment factor corresponded well to the ratio between the diffusion coefficients for heavy and light molecules as expected based on theory. In contrast to diffusion, biodegradation had generally only a small effect on the isotope profiles, which is expected because in a diffusion-controlled system the isotope shift per decrease of mass flux is smaller than in an advection-controlled system. These findings open interesting perspectives for monitoring source depletion with isotope and have implications for assessing biodegradation and source variability in the unsaturated zone based on isotopes.

Abstract: Compound-specific chlorine isotope analysis receives much interest to assess the fate of chlorinated hydrocarbons in contaminated environments. This paper provides a theoretical basis to calculate isotope ratios and quantify isotope fractionation from ion-current ratios of molecular- and fragment-ion multiplets. Because both Cl-35 and Cl-37 are of high abundance, polychlorinated hydrocarbons consist of molecules containing different numbers of Cl-37 denoted as isotopologues. We show that, during reactions, the changes in isotopologue ratios are proportional to changes in the isotope ratio assuming a nonselective isotope distribution in the initial compound. This proportionality extents even to fragments formed in the ion source of a mass spectrometer such as C2Cl2 (double dechlorinated fragment of perchloroethylene, PCE). Fractionation factors and kinetic isotope effects (KIE) may, therefore, be evaluated from isotope, isotopologue or even fragment ratios according to conventional simple equations. The proportionality is exact with symmetric molecules such as dichloroethylene (DCE) and PCE, whereas it is approximately true with molecules containing non-reactive positions such as trichloroethylene (TCE). If in the latter case isotope ratios are derived from dechlorinated fragments, e.g., C2HCl2, it is important that fragmentation in the ion source affect all molecular positions alike, as otherwise isotopic changes in reactive positions may be underrepresented.

Abstract: A field experiment was conducted in Denmark in order to evaluate the fate of 13 volatile organic compounds (VOCs) that were buried as an artificial fuel source in the unsaturated zone. Compound-specific isotope analysis showed distinct phases in the C-13/C-12 ratio evolution in VOC vapors within 3 m from the source over 114 days. At day 3 and to a lesser extent at day 6, the compounds were depleted in C-13 by up to -5.7%o with increasing distance from the source compared to the initial source values. This trend can be explained by faster outward diffusion of the molecules with C-12 only compared to molecules with a C-13. Then, the isotope profile leveled out, and several compounds started to become enriched in C-13 by up to 9.5 parts per thousand with increasing distance from the source, due to preferential removal of the molecules with C-12 only, through biodegradation. Finally, as the amount of a compound diminished in the source, a C-13 enrichment was also observed close to the source. The magnitude of isotope fractionation tended to be larger the smaller the mass of the molecule was. This study demonstrates that, in the unsaturated zone, carbon isotope ratios of hydrocarbons are affected by gas-phase diffusion in addition to biodegradation,which was confirmed using a numerical model. Gas-phase diffusion led to shifts in delta C-13 >1 parts per thousand during the initial days after the spill, and again during the final stages of source volatilization after >75% of a compound had been removed. In between, diffusion has less of an effect, and thus isotope data can be used as an indicator for hydrocarbon biodegradation.

Abstract: Microcosm experiments were conducted to quantify carbon isotope fractionation during aerobic biodegradation of n-alkanes (from C-3 to C-10) and monoaromatic hydrocarbons in unsaturated alluvial sand. In single compound experiments with n-alkanes, the largest enrichment factor was obtained for propane (- 10.8 +/- 0.7 parts per thousand). The magnitude of the enrichment factor decreased with increasing number of carbon atoms from propane to n-decane (-0.2 +/- 0.1 parts per thousand). This trend can partly be explained by the decreasing probability that a C-13 is located at the reacting site in the molecule with increasing chain length. After correcting for the presence of non-reacting positions, a chain length dependence of the calculated apparent isotope effect persisted. This observation suggests that transport and binding steps before the actual reaction step become increasingly rate limiting with increasing chain length. For aromatic compounds tested individually, the enrichment factor was the largest (-1.4 +/- 0.1 parts per thousand) for benzene (B), followed by toluene (T) (-0.8 7 +/- 0.1 parts per thousand) and m-xylene (X) (-0.6 +/- 0.1%o). Enrichment factors for BTX were systematically smaller than for n-alkanes with equivalent number of carbons, which is likely related to different biodegradation mechanisms. The study demonstrates that significant carbon isotope fractionation occurs during aerobic biodegradation of n-alkanes and aromatic compounds under unsaturated conditions and that the magnitude of isotope enrichment is linked to molecule size and molecule structure. (c) 2007 Elsevier Ltd. All rights reserved.

Abstract: A microcosm study was conducted to investigate the degradation of mono- and polyaromatic hydrocarbons under in situ-like conditions using alluvial sediments from the site of a former cokery. Benzene, naphthalene, or acenaphthene were added to the sediments as C-13-labeled substrates. Based on the evolution of C-13-CO2 determined by gas chromatography isotope-ratio mass spectrometry (GC-IRMS) it was possible to prove mineralization of the compound of interest in the presence of other unknown organic substances of the sediment material. This new approach was suitable to give evidence for the intrinsic biodegradation of benzene, naphthalene, and acenaphthene under oxic and also under anoxic conditions, due to the high sensitivity and reproducibility of C-13/C-12 stable isotope analysis. This semi-quantitative method can be used to screen for biodegradation of any slowly degrading, strongly sorbing compound in long-term experiments. (c) 2007 Elsevier Ltd. All rights reserved.

Abstract: Deposition, turnover and movement of persistent organic pollutants (POP) were investigated in the EU integrated project "AquaTerra", which is among the first funded environmental projects within the 6th Framework Program by the European Commission. Project work integrates across various disciplines that range from biogeochemistry, environmental engineering, computer modelling and chemistry to socio-economic sciences. Field study areas are the river basins of the Ebro, the Meuse, the Elbe and the Danube as well as the 3-km(2) French catchment of the Brevilles Spring. Within the first 2 years of the project more than 1700 samples of atmospherically deposited particles, sediments, and water have been collected in the above-mentioned systems. Results show clear spatial patterns of deposition of polyaromatic hydrocarbons (PAHs) with the highest rates in the Meuse Basin. For local inputs, in the Brevilles sandy aquifer, the contamination of the groundwater by the pesticides atrazine (AT) and deethylatrazine did not decrease even 5 years after their agricultural inputs were stopped. On the other hand, herbicides such as mecroprop (MCPP), and PAHs, were at least partially degraded microbiologically in laboratory studies with soils and aquifer material from selected sites. For sediment transport of contaminants, new flood sampling techniques revealed highest deposition rates of beta-hexachlorocyclohexane (beta-HCH) in river sediments at hotspot areas on the Mulde River in the Bitterfeld region (Elbe Basin, Germany). These selected preliminary results of AquaTerra help to improve fundamental understanding of persistent organic pollutants (POP) in the environment. (c) 2007 Elsevier B.V. All rights reserved.

Abstract: A field investigation of a TCE plume in a surficial sand aquifer shows that groundwater-surface water interactions strongly influence apparent plume attenuation. At the site, a former industrial facility in Connecticut, depth-discrete monitoring along three cross-sections (transects) perpendicular to groundwater flow shows a persistent VOC plume extending 700 in from the DNAPL source zone to a mid-size river. Maximum TCE concentrations along a transect 280 in from the source were in the 1000s of mu g/L with minimal degradation products. Beyond this, the land surface drops abruptly to a lower terrace where a shallow pond and small streams occur. Two transects along the lower ten-ace, one midway between the facility and river just downgradient of the pond and one along the edge of the river, give the appearance that the plume has strongly attenuated. At the river, maximum TCE concentrations in the 10s of mu g/L and similar levels of its degradation product cis-DCE show direct plume discharge from groundwater to the river is negligible. Although degradation plays a role in the strong plume attenuation, the major attenuation factor is partial groundwater plume discharge to surface water (i.e. the pond and small streams), where some mass loss occurs via water-air exchange. Groundwater and stream mass discharge estimates show that more than half of the plume mass discharge crossing the first transect, before surface water interactions occur, reaches the river directly via streamflow, although river concentrations were below detection due to dilution. This study shows that groundwater and surface water concentration measurements together provide greater confidence in identifying and quantifying natural attenuation processes at this site, rather than groundwater measurements alone. (c) 2006 Elsevier B.V. All rights reserved.

Abstract: Stable isotope data have been increasingly used to assess in situ biodegradation of organic contaminants in groundwater. The data are usually evaluated using the Rayleigh equation to evaluate whether isotope data follow a Rayleigh trend, to calculate the extent of contaminant biodegradation, or to estimate first-order rate constants. However, the Rayleigh equation was developed for homogeneous systems while in the subsurface, contaminants can migrate at different velocities due to physical heterogeneity. This paper presents a method to quantify the systematic effect that is introduced by applying the Rayleigh equation to field isotope data. For this purpose the travel time distribution between source and sampling point is characterized by an analytical solution to the advection-dispersion equation. The systematic effect was evaluated as a function of the magnitude of physical heterogeneity, geometry of the contaminant plume, and degree of biodegradation. Results revealed that the systematic effect always leads to an underestimation of the actual values of isotope enrichment factors, the extent of biodegradation, or first-order rate constants, especially in the dispersion-dominant region representing a higher degree of physical heterogeneity. A substantial systematic effect occurs especially for the quantification of first-order rate constants (up to 50% underestimation of actual rate) while it is relatively small for quantification of the extent of biodegradation (< 5% underestimation of actual degree of biodegradation). The magnitude of the systematic effect is in the same range as the uncertainty due to uncertainty of the analytical data, of the isotope enrichment factor, and the average travel time.

Abstract: The current knowledge of microbial biocenoses (communities) in pristine aquifers is presented in a review, which also discusses their relevance for questions of groundwater protection. Aquifers are heterogeneous on all scales and structured in a variety of habitats. The void spaces in many aquifers are small. The biocenoses are thus predominantly composed of microorganisms and, often, microinvertebrates. Larger voids and macroorganisms occur in karst cavities. Due to the absence of light, the biocenoses depend on chemical energy resources, which are, however, scarce in non-contaminated groundwater. The microorganisms thus show small cell sizes, low population densities and reduced activity; they developed specific strategies to survive oligotrophic conditions. The review also discusses the impact of contamination on the biocenoses, and the potential use of the biocenoses or specific organisms as indicators for groundwater quality, and the limits of this approach. Bacteria are either planktonic or attached to aquifer material, which requires both fluid and solid phase sampling. Most groundwater bacteria are viable but non-culturable. Consequently, cultivation techniques give an incomplete picture of the biocenoses, while methods from molecular microbiology provide genetic fingerprints of the entire community. Different analytical methods are available to count microorganisms, identify species, characterise microbial diversity, and measure activity.

Abstract: 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 MA 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.

Abstract: Compound-specific isotope analysis (CSIA) enables quantification of biodegradation by use of the Rayleigh equation. The Rayleigh equation fails, however, to describe the sequential degradation of chlorinated aliphatic hydrocarbons (CAHs) involving various intermediates that are controlled by simultaneous degradation and production. This paper shows how isotope fractionation during sequential degradation can be simulated in a 1D reactive transport code (PHREEGC-2). C-12 and C-13 isotopes of each CAH were simulated as separate species, and the ratio of the rate constants of the heavy to light isotope equaled the kinetic isotope fractionation factor for each degradation step. The developed multistep isotope fractionation reactive transport model (IF-RTM) adequately simulated reductive dechlorination of tetra chloroethene (PCE) to ethene in a microcosm experiment. Transport scenarios were performed to evaluate the effect of sorption and of different degradation rate constant ratios among CAH species on the downgradient isotope evolution. The power of the model to quantify degradation is illustrated for situations where mixed sources degrade and for situations where daughter products are removed by oxidative processes. Finally, the model was used to interpret the occurrence of reductive dechlorination at a field site. The developed methodology can easily be incorporated in 3D solute transport models to enable quantification of sequential CAH degradation in the field by CSIA.

Abstract: Measuring stable isotope fractionation of carbon, hydrogen, and other elements by Compound Specific Isotope Analysis (CSIA) is a new, innovative approach to assess organic pollutant degradation in the environment. Central to this concept is the Rayleigh equation which relates degradation-induced decreases in concentrations directly to concomitant changes in bulk (= average over the whole compound) isotope ratios. The extent of in situ transformation may therefore be inferred from measured isotope ratios in field samples, provided that an appropriate enrichment factor (epsilon(bulk)) is known. This epsilon(bulk) value, however, is usually only valid for a specific compound and for specific degradation conditions. Therefore, a direct comparison of epsilon(bulk) values for different compounds and for different types of reactions has in general not been feasible. In addition, it is often uncertain how robust and reproducible epsilon(bulk) values are and how confidently they can be used to quantify contaminant degradation in the field. To improve this situation and to achieve a more in-depth understanding, this critical review aims to relate fundamental insight about kinetic isotope effects (KIE) found in the physico(bio)chemical literature to apparent kinetic isotope effects (AKIE) derived from epsilon(bulk) values reported in environmentally oriented studies. Starting from basic rate laws, a quite general derivation of the Rayleigh equation is given, resulting in a novel set of simple equations that take into account the effects of (1) nonreacting positions and (2) intramolecular competition and that lead to position-specific AKIE values rather than bulk enrichment factors. Reevaluation of existing epsilon(bulk) literature values result in consistent ranges of AKIE values that generally are in good agreement with previously published data in the (bio)chemical literature and are typical of certain degradation reactions (subscripts C and H indicate values for carbon and hydrogen): AKIE(C) = 1.01-1.03 and AKIE(H) = 2-23 for oxidation of C-H bonds; AKIE(C) = 1.03-1.07 for S(N)2-reactions; AKIE(C) = 1.02-1.03 for reductive cleavage of C-Cl bonds; AKIE(C) = 1.00-1.01 for C=C bond epoxidation; AKIEC = 1.02-1.03 for C=C bond oxidation by permanganate. Hence, the evaluation scheme presented bridges a gap between basic and environmental (bio)chemistry and provides insight into factors that control the magnitude of bulk isotope fractionation factors. It also serves as a basis to identify degradation pathways using isotope data. It is shown how such an analysis may be even possible in complex field situations and/or in cases where AKIE values are smaller than intrinsic KIE values, provided that isotope fractionation is measured for two elements simultaneously ("two-dimensional isotope analysis"). Finally, the procedure is used (1) to point out the possibility of estimating approximate epsilon(bulk) values for new compounds and (2) to discuss the moderate, but non-negligible variability that may quite generally be associated with epsilon(bulk) values. Future research is suggested to better understand and take into account the various factors that may cause such variability.

Abstract: Geochemical heterogeneities may cause spatial variations in virus inactivation rates resulting from interactions with minerals leading to differences in natural disinfection capacity within an aquifer. Column studies investigating the interaction of the bacteriophage H40/1 with natural sands sampled from the Kappelen test site (Kappelen), Bern, Switzerland indicated that inactivation rates are higher for adsorbed bacteriophages than for those suspended in groundwater. Moreover, breakthrough curves obtained from field-based tracer tests at Kappelen indicated that the adsorbed H40/1 is inactivated in-situ at comparable rates. Statistical analyses of mineralogical data failed to demonstrate significant spatial variations in aquifer composition either across the site or with depth. In contrast hydrochemical analyses of groundwater samples collected at Kappelen demonstrated that iron-reducing groundwater occurs below aerobic waters. Tracer breakthrough curves indicate that H40/1 survival is not affected by variable redox conditions. Investigation results suggest that spatial geochemical variability does not significantly affect H40/1s inactivation rate at Kappelen.

Abstract: A column containing four concentric layers of progressively finer-grained glass beads (graded column) was used to study the transport of the bacteriophage T7 in water flowing parallel to layering through a fining-upwards (FU) sedimentary structure. By passing a pulse of T7, and a conservative solute tracer upwards through a column packed with a single bead size (uniform column), the capacity of each bead type to attenuate the bacteriophage was determined. Solute and bacteriophage responses were modelled using an analytical solution to the advection-dispersion equation, with first-order kinetic deposition simulating bacteriophage attenuation. Resulting deposition constants for different flow velocities indicated that filtration theory-determined values differed from experimentally determined values by less than 10%. In contrast, the responses of solute and bacteriophage tracers passing upwards through graded columns could not be reproduced with a single analytical solution. However, a flux-weighted summation of four one-dimensional advective-dispersive analytical terms approximated solute breakthrough curves. The prolonged tailing observed in the resulting curve resembled that typically generated from field-based tracer test data, reflecting the potential importance of textural heterogeneity in the transport of dissolved substances in groundwater. Moreover, bacteriophage deposition terms, determined from filtration theory, reproduced the T7 breakthrough curve once desorption and inactivation on grain surfaces were incorporated. To evaluate the effect of FU sequences on mass transport processes in more detail, bacteriophage passage through sequences resembling those sampled from a FU bed in a fluvioglacial gravel pit were carried out using an analogous approach to that employed in the laboratory. Both solute and bacteriophage breakthrough responses resembled those generated from field-based test data and in the graded column experiments.. Comparisons with the results of simulations using averaged hydraulic conductivities show that simulations employing averaged parameters overestimate bacteriophage travel times and underestimate masses recovered and peak concentrations. (C) 2004 Elsevier B.V. All rights reserved.

Abstract: Chlorinated ethenes often migrate over extended distances in aquifers and may originate from different sources. The aim of this study was to determine whether stable carbon isotope ratios remain constant during dissolution and transport of chlorinated ethenes and whether the ratios can be used to link plumes to their sources. Detailed depth-discrete delineation of the carbon isotope ratio in a tetrachloroethene (PCE) plume and in a trichloroethene (TCE) plume was done along cross-sections orthogonal to groundwater flow in two sandy aquifers in the Province of Ontario, Canada. At the TCE site, TCE concentrations up to solubility were measured in one high concentration zone close to the bottom of the aquifer from where dense non-aqueous phase liquid (DNAPL) was collected. A laboratory experiment using the DNAPL indicated that only very small carbon isotope fractionation occurs during dissolution of TCE (0.26parts per thousand), which is consistent with field observations. At most sampling points, the delta(13)C of dissolved TCE was similar to that of the DNAPL except for a few sampling points at the bottom of the aquifer close to the underlying aquitard. At these points, a C-13 enrichment of up to 2.4parts per thousand was observed, which was likely due to biodegradation and possibly preferential diffusion of TCE with C-12 into the aquitard. In contrast to the TCE site, several distinct zones of high concentration were observed at the PCE site and from zones to zone, the delta(13)C values varied substantially from -24.3parts per thousand to -33.6parts per thousand. Comparison of the delta(13)C values in the high concentration zones made it possible to divide the plume in the three different domains, each probably representing a different episode and location of DNAPL release. The three different zones could still be distinguished 220 m from the DNAPL sources. This demonstrates that carbon isotope ratios can be used to differentiate between different zones in chlorinated ethene plumes and to link plume zones to their sources. In addition, subtle variations in delta(13)C at plume fringes provided insight into mechanisms of plume spreading in transverse vertical direction. These variations were identified because of the high-resolution provided by the monitoring network. (C) 2004 Elsevier B.V. All rights reserved.

Abstract: Virus inactivation and virus adsorption, resulting from interactions with minerals, constitute important aspects of an aquifers disinfection capacity. Investigations using a 20 cm. column tilled with medium-grained natural sands demonstrated that the sands can attenuate up to 62% of a pulse of viruses injected. Experiments using repeatedly washed sands had significantly lower attenuation capacity than fresh sands, due to removal of fine-grained (silt and clay-sized) coatings on grain surfaces. X-ray diffraction analyses of the sand, and the associated fine-grained coating indicated that no significant mineralogical differences existed between these two materials. The experimental data suggested that rougher surfaces/crystal edges in the grain coatings reduced repulsive forces between viruses and the sands permitting greater virus adsorption to the column matrix. Soaking all sands with Tryptone solution after testing released adsorbed viruses indicated that short-term viral inactivation due to interactions with the column matrix was a negligible part of the attenuation process. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Abstract: Currently there is no in situ method to detect and quantify complete mineralization of chlorinated hydrocarbons (CHCs) to CO2. Combined isotopic measurements in conjunction with traditional chemical techniques were used to assess in situ biodegradation of trichloroethylene (TCE) and carbon tetrachloride (CT). Vadose zone CHC, ethene, ethane, methane, O-2, and CO2 concentrations were analyzed using gas chromatography over 114 days at the Savannah River Site. delta(13)C of CHC and delta(13) C and C-14 of vadose zone CO2, sediment organic matter, and groundwater dissolved inorganic carbon (DIC) were measured. Intermediate metabolites of TCE and CT accounted for less than or equal to10% of total CHCs. delta(13)C of cis-1,2-dichloroethylene (DCE) was always heavier than TCE indicating substantial DCE biodegradation. C-14-CO2 values ranged from 84 to 128 percent modern carbon (pMC), suggesting that plant root-respired CO2 was dominant. C-14-CO2 values decreased over time (up to 12 pMC), and contaminated groundwater C-14-DIC (76 pMC) was substantially depleted relative to the control (121 pMC). C-14 provided a direct measure of complete CHC mineralization in vadose zone and groundwater in situ and may improve remediation strategies.

Abstract: 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 parts per thousand) for initial KMnO4 concentrations between 67 and 1250 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 delta(13)C 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 delta(13)C 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 delta(13)C rebounded to the initial delta(13)C 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 delta(13)C 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.

Abstract:

Abstract: A diesel fuel contaminated aquifer in Menziken, Switzerland was treated for 4.5 years by injecting aerated groundwater, supplemented with KNO3 and NH4H2PO4 to stimulate indigenous populations of petroleum hydrocarbon (PHC) degrading microorganisms. After dissolved PHC concentrations had stabilized at a low level, engineered in situ bioremediation was terminated. The main objective of this study was to evaluate the efficacy of intrinsic in situ bioremediation as a follow-up measure to remove PHC remaining in the aquifer after terminating engineered in situ bioremediation. In the first 7 months of intrinsic in situ bioremediation, redox conditions in the source area became more reducing as indicated by lower concentrations of SO42- and higher concentrations of Fe(II) and CH4. In the core of the source area, strongly reducing conditions prevailed during the remaining study period (3 years) and dissolved PHC concentrations were higher than during engineered in situ bioremediation. This suggests that biodegradation in the core zone was limited by the availability of oxidants. In lateral zones of the source area, however, gradually more oxidized conditions were reestablished again, suggesting that PHC availability increasingly limited biodegradation. The total DIC production rate in the aquifer decreased within 2 years to about 25% of that during engineered in situ bioremediation and remained at that level. Stable carbon isotope analysis confirmed that the produced DIC mainly originated from PHC mineralization. The total rate of DIC and CH4 production in the source area was more than 300 times larger than the rate of PHC elution. This indicates that biodegradation coupled to consumption of naturally occurring oxidants was an important process for removal of PHC which remained in the aquifer after terminating engineered measures. (C) 2002 Elsevier Science B.V All rights reserved.

Abstract: Accumulation of vinyl chloride (VC) is often a main concern at sites contaminated with chlorinated ethenes and ethanes due to its high toxicity. Since there can be several possible sources of VC and ethene at such sites, assessing the origin and fate of VC can be complicated. Aim of this study was to evaluate carbon isotope fractionation associated with various anaerobic processes that lead to the production of VC and ethene in view of using isotopes to evaluate the origin and fate of these compounds in groundwater. Microcosms were constructed using sediments and groundwater from a contaminated site and amended with potential precursors for VC and ethene production. In the microcosms with dichloroethene isomers, sequential reductive dechlorination was observed, and isotopic enrichment factors of -19.9+/-1.5%,for cis- 1,2-dichloroethene -30.3+/-1.9%o for trans-1,2-dichloroethene, and -7.3+/-0.4%o for 1,1,1-dichloroethene were obtained. In microcosms with chlorinated ethanes, 1,2-dichloroethane (1,2-DCA) and 1,1,2-trichloroethane (1,1,2-TCA) were predominantly transformed by dichloroelimination to ethene and VC, respectively, and enrichment factors of -32.1+/-1.1%o for 1,2-DCA and -2.0+/-0.2%o for 1,1,2-TCA were observed. Except for 1,1,2-TCA, a strong C-13 enrichment in each of the potential precursor of VC was observed, which opens the possibility to trace the origin of VC based on the isotope ratio of potential precursors, Furthermore, it was possible to model the isotope evolution of VC present as substrate or intermediate product as a function of time. The study demonstrates that carbon isotope ratios can potentially be used for qualitative and possibly quantitative evaluation of the origin and fate of VC at sites with complex contaminant mixtures.

Abstract: Methyl tert-butyl ether (MTBE), the most common gasoline oxygenate, is frequently detected in surface water and groundwater. The aim of this study was to evaluate the potential of Compound-specific isotope analysis to assess in situ biodegradation of MTBE in groundwater. For that purpose, the effect of relevant physical and biological processes on carbon isotope ratios of MTBE was evaluated in laboratory studies. Carbon isotope fractionation during organic phase/gas-phase partitioning (0.50 +/- 0.15 parts per thousand), aqueous phase/gas-phase partitioning (0.17 +/- 0.05 parts per thousand), and organic phase/aqueous-phase partitioning (0.18 +/- 0.24 parts per thousand) was small in comparison to carbon isotope fractionation measured during biodegradation of MTBE in microcosms based on aquifer sediments of the Borden site. In experiments with MTBE as the only substrate and a cometabolic experiment with 3-methypentane as primary substrate, MTBE became enriched in C-13 by 5.1 to 6.9 parts per thousand after 95 to 97% degradation. For both experiments, similar isotopic enrichment factors were obtained (-1.52 +/- 0.06 to -1.97 +/- 0.05 parts per thousand). Biodegradation of TBA, which accumulated transiently in the cometabolic microcosms, was also accompanied by carbon isotope fractionation, with an isotopic enrichment factor of -4.21 +/- 0.07 parts per thousand. This study suggests that carbon isotope analysis is a potential tool to trace in situ biodegradation of MTBE and TBA and thus to better understand the fate of these contaminants in the environment.

Abstract: The main aim of the study was to evaluate hydrogen and carbon isotope fractionation during biodegradation of benzene as a possible tool to trace the process in contaminated environments. Aerobic biodegradation of benzene by two bacterial isolates, Acinetobacter sp. and Burkholderia sp., was accompanied by significant hydrogen and carbon isotope fractionation with hydrogen isotope enrichment factors of -12.8 0.7 parts per thousand and -11.2 1.8 parts per thousand respectively, and average carbon isotope enrichment factors of -1.46 +/- 0.06 parts per thousand and -3.53 +/- 0.26 parts per thousand, respectively. Inorganic carbon produced by Acinetobacter sp. was depleted in C-13 by 3.6-6.2 parts per thousand as compared to the initial delta C-13 of benzene, while the produced biomass was enriched in C-13 by 3.8 parts per thousand. The secondary aim was to determine isotope ratios of benzenes from different manufacturers with regard to the use of isotopes for source differentiation. While two of the four analyzed benzenes had similar delta C-13 values, each of them had a distinct,delta H-2-delta C-13 pair and delta H-2 values spread over a range of 66.5 parts per thousand. Thus, combined analyses of hydrogen and carbon isotopes may be a more promising approach to trace sources and/or biodegradation of benzene than measuring carbon isotopes only.

Abstract: Compound-specific carbon isotope ratio analysis is a promising tool to assess the, origin and fate of organic contaminants in groundwater. The aim of this study was to develop and evaluate a reliable,fast method to determine carbon isotope ratios of chlorinated methanes, ethanes, and ethenes in aqueous samples. Direct solid-phase microextraction (dSPME) and headspace solid-phase microextraction (hSPME) were selected as extraction method and compared to headspace equilibration. For dSPME and hSPME, deviations between carbon isotope ratios in the aqueous phase and an the SPME fiber were less than or equal to 0.40%. For headspace equilibration, molecules in the gas phase were enriched in C-13 compared to molecules in the aqueous phase by up to 1.46% in particular for chlorinated methanes. The absence of significant carbon isotope fractionation during dSPME and hSPME could be explained by the fact that both the aqueous phase and the SPME fiber coating discriminate against molecules with C-13 to a similar degree, and thus no net carbon isotope fractionation occurs. If aqueous phase/gas-phase carbon isotope fractionation during headspace equilibration is taken into account, all methods, dSPME, hSPME, and headspace equilibration, provide accurate delta(13)C values with a similar precision. Direct SPME was the most sensitive method with detection limits as low as 130 ppb.

Abstract: Carbon isotope fractionation during dechlorination of chlorinated ethenes was investigated using a methanogenic microbial enrichment culture. Subcultures were amended with trichloroethene (TCE), cis-1,2-dichloroethene ( cis-DCE), and vinyl chloride (VC), respectively. Carbon isotope ratios and concentrations of reactants and of all dechlorination products were monitored during two experiments. All dechlorination steps were accompanied by significant isotope fractionation. The isotope ratios of the reactants were described with a Rayleigh type model, and the following enrichment factors (epsilon(P/R)) were obtained: -6.6 and -2.5% for dechlorinationof TCE, -14.1 and -16.1% for dechlorination of cis-DCE, and -26.6 and -21.5% for dechlorination of VC. isotope and mass balances suggested that ethene (ETH) was degraded. In additional experiments with ETH as reactant, ETH became enriched in C-13 as its concentration decreased indicating the cultures were capable of degrading ETH. The average value for the enrichment factor obtained for the degradation of ETH was -3.0%. The large carbon isotope fractionation observed in this study confirms that carbon isotope ratios are a sensitive tool for monitoring dechlorination of chlorinated ethenes to nontoxic end products.

Abstract: Carbon isotope fractionation during aerobic mineralization of 1,2-dichloroethane (1,2-DCA) by Xanthobacter autotrophicus GJ10 was investigated. A strong enrichment of C-13 in residual 1,2-DCA was observed, with a mean fractionation factor ex a standard deviation of 0.968 +/- 0.0013 to 0.973 +/- 0.0015. In addition, a large carbon isotope fractionation between biomass and inorganic carbon occurred. A mechanistic model that links the fractionation factor or to the rate constants of the first catabolic enzyme was developed. Based on the model, it was concluded that the strong enrichment of C-13 in 1,2-DCA arises because the first irreversible step of the initial enzymatic transformation of 1,2-DCA consists of an S(N)2 nucleophilic substitution. S(N)2 reactions are accompanied by a large kinetic isotope effect. The substantial carbon isotope fractionation between biomass and inorganic carbon could be explained by the kinetic isotope effect associated with the initial 1,2-DCA transformation and by the metabolic pathway of 1,2-DCA degradation. Carbon isotope fractionation during 1,2-DCA mineralization leads to 1,2-DCA, inorganic carbon, and biomass with characteristic carbon isotope compositions, which may be used to trace the process in contaminated environments.

Abstract: A concept is proposed to assess in situ petroleum hydrocarbon mineralization by combining data on oxidant consumption, production of reduced species, CH4, alkalinity and dissolved inorganic carbon (DIC) with measurements of stable isotope ratios, The concept was applied to a diesel fuel contaminated aquifer in Menziken, Switzerland, which was treated by engineered in situ bioremediation. In the contaminated aquifer, added oxidants (O-2 and NO3-) were consumed, elevated concentrations of Fe(II), Mn(II), CH4, alkalinity and DIC were detected and the DIC was generally depleted in C-13 compared to the background. The DIC production was larger than expected based on the consumption of dissolved oxidants and the production of reduced species. Stable carbon isotope balances revealed that the DIC production in the aquifer originated mainly from microbial petroleum hydrocarbon mineralization, and that geochemical reactions such as carbonate dissolution produced little DIC. This suggests that petroleum ydrocarbon mineralization can be underestimated if it is determined based on concentrations of dissolved oxidants and reduced species. (C) 1999 Elsevier Science B.V. All rights reserved.

Abstract: The determination of compound-specific stable isotope ratios is a promising new tool to assess biodegradation of organic compounds in groundwater. In this study, the occurrence of carbon isotope fractionation during dechlorination of tetrachloroethene (PCE) to ethene was evaluated in a PCE-contaminated aquifer and in a microcosm that was based on aquifer material from the site. In the microcosm, all dechlorination steps were accompanied by carbon isotope fractionation. The largest fractionation occurred during dechlorination of cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC), resulting in a large enrichment of C-13 in the remaining cDCE and VC. Stable carbon isotope ratios (delta(13)C) of cDCE and VC increased from -25.7 to -1.5 parts per thousand and -37.0 to -2.5 parts per thousand, respectively. The delta(13)C of ethene was initially -60.2 parts per thousand and approached the delta(13)C of the added PCE (-27.3 parts per thousand) as dechlorination came to completion. A similar carbon isotope pattern was observed for PCE dechlorination at the field site. Strong enrichment of C-13 in cDCE and VC during microbial dechlorination may serve as a powerful tool to monitor the last two dechlorination steps, which frequently determine the rate of complete dechlorination of chlorinated ethenes at field sites undergoing intrinsic bioremediation.

Abstract: This study presents a stepwise concept to assess the in situ microbial mineralization of petroleum hydrocarbons (PHC) in aquifers. A new graphical method based on stable carbon isotope ratios (delta(13)C) was developed to verify the origin of dissolved inorganic carbon (DIC). The concept and the isotope method were applied to an aquifer in Studen, Switzerland, in which more than 34,000 liters of heating oil were accidentally released. Chemical analyses of ground water revealed that in this aquifer locally, anaerobic conditions prevailed, and that PHC mineralization was linked to the consumption of oxidants such as O-2, NO3-, and SO42- and the production of reduced species such as Fe2+, Mn2+, H2S and CH4. However, alkalinity and DIC balances showed a quantitative disagreement in the link between oxidant consumption and DIC production, indicating that chemical data alone may not be a reliable assessment tool. delta(13)C ratios in DIC have been used before for bioremediation assessment, but results were reported to be negatively influenced by methanogenesis. Using the new graphical method to display delta(13)C data, it was possible to identify anomalies found in methanogenic monitoring wells. It could be shown that 88% of the DIC produced in the contaminated aquifer originated from microbial PHC mineralization. Thus, the new graphical method to display delta(13)C ratios appears to be a useful tool for the assessment of microbial hydrocarbon mineralization in a complex environment.

Abstract: Engineered in situ bioremediation is an economically and ecologically sound technology for the clean-up of contaminated soils and aquifers. However, a successful bioremediation requires solid evidence for the detoxification of the contaminants, preferably proven by complete mineralization. This paper discusses a stepwise evaluation leading to the demonstration of successful engineered in situ bioremediation. Five major evaluation steps assess whether: (1) the contaminants can be mineralized by the indigenous microbial population (2) the mineralization rates can be increased (3) the remediation concept can be simulated under continuous flow conditions (4) the increase of mineralization rates can be achieved at the field site (scale-up), and (5) complete mineralization to harmless end products is achieved at the field site. For these evaluations, the applicability of four experimental approaches (field investigations, laboratory aquifer columns, microcosms and microbial cultures) and the relevance of various microbiological or chemical monitoring parameters are discussed. The evaluations are illustrated using a specific engineered in situ bioremediation of a diesel fuel-contaminated aquifer in Menziken, Switzerland. The case study demonstrates that microbiological and chemical monitoring parameters as well as field tracer studies and stable carbon isotopes should be combined for the unequivocal evaluation of engineered in situ bioremediation. (C) 1998 Elsevier Science B.V.

Abstract: The anaerobic biodegradation of hydrocarbons at mineral oil contaminated sites has gathered increasing interest as a naturally occurring remediation process, The aim of this study was to investigate biodegradation of hydrocarbons in laboratory aquifer columns in the absence of O-2 and NO3- and to calculate a mass balance of the anaerobic biodegradation processes. The laboratory columns contained aquifer material from a diesel fuel contaminated aquifer. They were operated at 25 degrees C for 65 days with artificial groundwater that contained only SO42- and CO2 as externally supplied oxidants. After 31 days of column operation, stable concentration profiles were found for most of the measured dissolved species. Within 14 h residence time, about 0.24 mM SO42- were consumed and dissolved Fe(II) (up to 0.012 mM), Mn(II) (up to 0.06 mM), and CH4 (up to 0.38 mM) were produced. The alkalinity and the dissolved inorganic carbon (DIC) concentration increased and the DIC became enriched in C-13. In the column, n-alkanes were selectively removed while branched alkanes persisted, suggesting a biological degradation. Furthermore, based on changes of concentrations of aromatic compounds with similar physical-chemical properties in the effluent, it was concluded that toluene, p-xylene and naphthalene were degraded. A carbon mass balance revealed that 65% of the hydrocarbons removed from the column were recovered as DIG, 20% were recovered as CH4, and 15% were eluted from the column. The calculations indicated that hydrocarbon mineralization coupled to SO42- reduction and methanogenesis contributed in equal proportions to the hydrocarbon removal. Hydrocarbon mineralization coupled to Fe(III) and Mn(IV) reduction was of minor importance. DIG, alkalinity, and stable carbon isotope balances were shown to be a useful tool to verify hydrocarbon mineralization. (C) 1998 Elsevier Science B.V. All rights reserved.

Abstract: The use of Rn-222, a naturally occurring radioactive isotope, was investigated as a partitioning tracer to detect and quantify the amount of non-aqueous-phase liquids (NAPLs) in contaminated aquifers. Diesel fuel was chosen as a model NAPL. The diesel fuel-water partition coefficient for Rn-222 was 40 +/- 2.3, in bottles containing diesel fuel and water at 12 degrees C. In water-saturated quartz sand contaminated with diesel fuel, the Rn-222 emanating from the sand partitioned between diesel fuel and water as expected based on this partition coefficient. In a column containing uncontaminated quartz sand, the Rn-222 activity in infiltrated water increased from (0.2 to 4.9 kBq m(-3), and in a subsequent column containing diesel fuel-contaminated quartz sand, the Rn-222 activity in the water phase decreased to 3.3 kBq m(-3). This decrease corresponds to what has been predicted using a mathematical model. At a contaminated field site, the Rn-222 activity of groundwater decreased by about 40% between monitoring wells upgradient of the contaminated zone and monitoring wells within the contaminated zone. On the basis of this decrease, the average diesel fuel saturation was estimated using the mathematical model. The calculated diesel fuel saturation was in the range of that found in excavated aquifer material.

Abstract: The in situ bioremediation of aquifers contaminated with petroleum hydrocarbons is commonly based on the infiltration of groundwater supplemented with oxidants (e.g., O-2, NO3-) and nutrients (e.g., NH4+, PO43-). These additions stimulate the microbial activity in the aquifer and several field studies describing the resulting processes have been published. However, due to the heterogeneity of the subsurface and due to the limited number of observation wells usually available, these field data do not offer a sufficient spatial and temporal resolution. In this study, flow-through columns of 47-cm length equipped with 17 sampling ports were filled with homogeneously contaminated aquifer material from a diesel fuel contaminated in situ bioremediation site. The columns were operated over 96 days at 12 degrees C with artificial groundwater supple mented with O-2, NO3- and PO43-. Concentration profiles of O-2, NO3-, NO2-, dissolved inorganic and organic carbon (DIC and DOC, respectively), protein, microbial cells and total residual hydrocarbons were measured. Within the first 12 cm, corresponding to a mean groundwater residence time of < 3.6 h, a steep O-2 decrease from 4.6 to < 0.3 mg l(-1), denitrification, a production of DIC and DOC, high microbial cell numbers and a high removal of hydrocarbons were observed. Within a distance of 24 to 40.5 cm from the infiltration, O-2 was below 0.1 mg l(-1) and a denitrifying activity was found. In the presence and in the absence of O-2, n-alkanes were preferentially degraded compared to branched alkanes. The results demonstrate that: (1) infiltration of aerobic groundwater into columns filled with aquifer material contaminated with hydrocarbons leads to a rapid depletion of O-2; (2) O-2 and NO3- can serve as oxidants for the mineralization of hydrocarbons; and (3) the modelling of redox processes in aquifers has to consider denitrifying activity in presence of O-2.