Environ. Sci. Technol. 2006, 40, 4245-4252
A Multitracer Test Proving the Reliability of Rayleigh Equation-Based Approach for Assessing Biodegradation in a BTEX Contaminated Aquifer ANKO FISCHER,† JANA BAUER,‡ RAINER U. MECKENSTOCK,§ WILLIBALD STICHLER,§ CHRISTIAN GRIEBLER,§ PIOTR MALOSZEWSKI,§ M A T T H I A S K A¨ S T N E R , | A N D H A N S H . R I C H N O W * ,† UFZ-Center for Environmental Research Leipzig-Halle, Department of Isotope Biogeochemistry, Permoserstrasse 15, D-04318 Leipzig, Germany, Research Centre Ju ¨ lich, Institute of Chemistry and Dynamics of the Geosphere: Agrosphere, D-52425 Ju ¨ lich, Germany, GSF-National Research Center for Environment and Health, Institute of Groundwater Ecology, Ingolsta¨dter Landstrasse 1, D-85764 Neuherberg, Germany, and UFZ-Center for Environmental Research Leipzig-Halle, Department of Bioremediation, Permoserstrasse 15, D-04318 Leipzig, Germany
Compound-specific stable isotope analysis (CSIA) is one of the most important methods for assessing biodegradation activities in contaminated aquifers. Although the concept is straightforward, the proof that the method cannot be only used for a qualitative analysis but also to quantify biodegradation in the subsurface was missing. We therefore performed a multitracer test in the field with ringdeuterated (d5) and completely (d8) deuterium-labeled toluene isotopologues (400 g) as reactive tracers as well as bromide as a conservative tracer. The compounds were injected into the anoxic zone of a BTEX plume located downgradient of the contaminant source. Over a period of 4.5 months the tracer concentrations were analyzed at two control planes located 24 and 35 m downgradient of the injection well. Deuterium-labeled benzylsuccinate was found in the aquifer, indicating the anaerobic biodegradation of deuterated toluene via the benzylsuccinate synthase pathway. Three independent methods were applied to quantify biodegradation of deuterated toluene. First, fractionation of toluene-d8 and toluene-d5 using the Rayleigh equation and an appropriate laboratory-derived isotope fractionation factor was used for the calculation of the microbial decomposition of deuterated toluene isotopologues (CSIAmethod). Second, the biodegradation was quantified by the changes of the concentrations of deuterated toluene * Corresponding author phone: ++49-341/235-2810; fax: ++49341/235-2492; e-mail:
[email protected]. † UFZ-Center for Environmental Research Leipzig-Halle, Department of Isotope Biogeochemistry. ‡ Research Centre Ju ¨ lich, Institute of Chemistry and Dynamics of the Geosphere: Agrosphere. § GSF-National Research Center for Environment and Health, Institute of Groundwater Ecology. | UFZ-Center for Environmental Research Leipzig-Halle, Department of Bioremediation. 10.1021/es052466t CCC: $33.50 Published on Web 06/03/2006
2006 American Chemical Society
relative to bromide. Both methods gave similar results, implying that the CSIA-method is a reliable tool to quantify biodegradation in contaminated aquifers. The results of both methods yielded a biodegradation of deuterated toluene isotopologues of approximately 23-29% for the first and 4451% for the second control plane. Third, the mineralization of deuterated toluene isotopologues was verified by determination of the enrichment of deuterium in the groundwater. This method indicated that parts of deuterium were assimilated into the biomass of toluene degrading microorganisms.
Introduction Benzene, toluene, ethylbenzene, and xylene isomers (BTEX) are among the most frequent pollutants in contaminated aquifers. In many cases, natural attenuation (NA) of these compounds has emerged as an appropriate remediation strategy (1). Abiotic NA-processes such as advection, dispersion, sorption, and volatilization cause a displacement of BTEX compounds from contaminated groundwater into other compartments. Only microbial degradation leads to a sustainable reduction of BTEX concentrations associated with an effective mass reduction in groundwater. Since most BTEX-contaminated sites are anoxic, anaerobic biodegradation is of special interest in the evaluation of contaminated aquifers. The application of NA as a remediation strategy requires methods to prove and quantify in situ biodegradation (2). Often spatial or temporal changes in concentrations of BTEX, electron acceptors, or mineralization products are used to assess BTEX-biodegradation. However, this approach involves a significant uncertainty as it is extremely difficult to distinguish between biotic and abiotic sinks of organic pollutants. In recent years, compound-specific stable isotope analysis (CSIA) for assessing in situ biodegradation of organic pollutants in contaminated aquifers has gained more and more attention (3-6). The concept relies on the observation of the shift in stable isotope ratios of carbon, hydrogen, or other elements that are involved in the breakage or generation of chemical bonds during biodegradation of organic contaminants. The detection of a significant enrichment of the heavier stable isotope in the residual fraction of a pollutant gives indications for the biodegradation of this substance. The changes in stable isotope ratios of pollutants are used to calculate the extent of biodegradation in contaminated aquifers using the Rayleigh equation associated with laboratory-derived isotope fractionation factors. CSIA is commonly used for pollutants consisting of stable carbon or hydrogen isotopes in natural abundance. An alternative technique for investigating the microbial BTEX-decomposition by CSIA is to determine fractionation of labeled BTEX isotopologues. The fractionation of toluene isotopologues with and without a deuterated methyl group during anaerobic biodegradation is caused by the first enzymatic attack on the toluene molecule (7). The reaction is performed by benzylsuccinate synthase which catalyzes the addition of fumarate to the methyl group of toluene (8, 9). The principle of fractionation during anaerobic biodegradation of toluene with and without a deuterated methyl group is that more energy is required to break a bond between carbon and deuterium than to break a bond between carbon and hydrogen (7). Hence, toluene without a labeled methyl group is preferentially biodegraded VOL. 40, NO. 13, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Map of the Zeitz field site showing the BTEX plume within the upper aquifer with the detailed location and sampling point arrangement of the tracer test transect. during anaerobic decomposition in comparison to toluene with a deuterated methyl group. The consequence is an enrichment of toluene with a deuterium-labeled methyl group in the fraction that has not yet been biodegraded. In batch experiments, laboratory-derived fractionation factors for the biodegradation of mixtures of differently labeled toluene by several anaerobic bacteria were obtained (7, 10). The applicability of deuterium-labeled compounds for the assessment of BTEX-biodegradation in tracer tests has been shown in several studies (11, 12). The advantage of deuterium-labeled compounds is that they can be easily detected even though the nonlabeled toluene is present in high concentrations in an aquifer. The disadvantage of fully deuterium-labeled BTEX-compounds is a slower transformation relative to nondeuterated analogues (7, 13). Hence, the use of fully deuterated tracer species can lead to an underestimation of the occurred BTEX-biodegradation in the field. To evaluate the applicability of the CSIA-method for the assessment of in situ biodegradation of different labeled isotopologues we designed a multitracer test with a halfand-half mixture of ring-deuterated (d5) and completely (d8) deuterium-labeled toluene (i.e., toluene without and with a deuterated methyl group) as reactive tracers and bromide as conservative tracer. With this approach, we were able to monitor and assess the biotransformation and mineralization of deuterated toluene by means of several techniques: (1) CSIA-method based on the fractionation of toluene-d5 and toluene-d8, (2) the changes of labeled toluene isotopologues relative to bromide, and (3) the enrichment of deuterium in groundwater. Although biodegradation of pollutants was often demonstrated by CSIA in contaminated aquifers, it is questioned whether the concept can be practically used to quantify microbial decomposition. As recently has been shown by an analytical modeling approach, the quantification of biodegradation by the CSIA-method gives reliable results in terms of aquifer heterogeneities such as various dispersive conditions, degrees of biodegradation, and plume geometries, as well as travel times (14). However, there is only one study 4246
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showing the reliability of the CSIA-method for the quantification of in situ biodegradation using independent methods (15). The process of isotope fractionation of a nonlabeled pollutant as well as the fractionation of labeled isotopologues upon microbial degradation is similar and can be described by the Rayleigh equation (5, 16, 17). Thus, we applied a multitracer test to validate the Rayleigh approach with two independent methods to demonstrate the applicability of the CISA method for the assessment of biodegradation in contaminated aquifers.
Materials and Methods Field Site. The test site is located in the area of a former benzene plant close to the city of Zeitz (Saxony-Anhalt, Germany). The dominant contaminants are benzene and toluene with concentrations up to 850 mg L-1 and 50 mg L-1, respectively. At the test site, the biodegradation of benzene and toluene was indicated by means of CSIA (18, 19). The predominant electron acceptor used for biodegradation was sulfate (18-20). Additional information about the hydrogeology and the hydrochemistry of the contaminated aquifer in Zeitz are given in the Supporting Information. The multitracer test was performed within the upper aquifer downstream of the source of the BTEX plume (Figure 1). The aquifer thickness in the area where the test took place varied from 4 to 6 m and the groundwater table was located approximately 9.20 m below ground level. The effective porosity ranged between 0.19 and 0.24 and the organic carbon content of the aquifer sediment (Corg) was 137 m above sea level), the middle was referred to as P2 (
