Environ. Sci. Technol. 2002, 36, 2464-2470
Hydrogen Isotopic Enrichment: An Indicator of Biodegradation at a Petroleum Hydrocarbon Contaminated Field Site SILVIA A. MANCINI,† GEORGES LACRAMPE-COULOUME,† HENDRIKUS JONKER,‡ BORIS M. VAN BREUKELEN,‡ JACOBUS GROEN,‡ FRANK VOLKERING,§ AND B A R B A R A S H E R W O O D L O L L A R * ,† Department of Geology, University of Toronto, 22 Russell Street, Toronto, Ontario, Canada M5S 3B1, Department of Hydrogeology, Faculty of Earth Sciences, Research School NSG, Free University, De Boelelaan 1085, NL-1081 HV Amsterdam, The Netherlands, and TAUW bv, Handelskade 11, P.O. Box 133, NL-7400 AC Deventer, The Netherlands
Compound-specific carbon and hydrogen isotope analysis was used to investigate biodegradation of benzene and ethylbenzene in contaminated groundwater at Dow Benelux BV industrial site. δ13C values for dissolved benzene and ethylbenzene in downgradient samples were enriched by up to 2 ( 0.5‰ in 13C, compared to the δ13C value of the source area samples. δ2H values for dissolved benzene and ethylbenzene in downgradient samples exhibited larger isotopic enrichments of up to 27 ( 5‰ for benzene and up to 50 ( 5‰ for ethylbenzene relative to the source area. The observed carbon and hydrogen isotopic fractionation in downgradient samples provides evidence of biodegradation of both benzene and ethylbenzene within the study area at Dow Benelux BV. The estimated extents of biodegradation of benzene derived from carbon and hydrogen isotopic compositions for each sample are in agreement, supporting the conclusion that biodegradation is the primary control on the observed differences in carbon and hydrogen isotope values. Combined carbon and hydrogen isotope analyses provides the ability to compare biodegradation in the field based on two different parameters, and hence provides a stronger basis for assessment of biodegradation of petroleum hydrocarbon contaminants.
Introduction Petroleum hydrocarbons such as benzene, toluene, ethylbenzene, and m-, o-, and p-xylenes (BTEX) are common groundwater pollutants that enter the subsurface by leaking underground storage tanks, improper disposal methods, or accidental spills. In addition to remediation strategies such as excavation, pump-and-treat, and physical containment by impermeable barriers, there is increasing interest in * Corresponding author phone: (416) 978-0770; fax: (416) 9783938; e-mail:
[email protected]. † University of Toronto. ‡ Free University. § TAUW. 2464
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 36, NO. 11, 2002
intrinsic bioremediation of BTEX-contaminated groundwater (1). This remediation strategy relies on the ability of indigenous microorganisms to degrade organic contaminants to nontoxic degradation products such as carbon dioxide and methane. Microbial degradation of BTEX compounds has been documented in the literature under aerobic (2, 3) and under anaerobic nitrate-reducing (4, 5), sulfate-reducing (5-9), iron-reducing (5, 10), and methanogenic (11, 12) conditions. Intrinsic bioremediation of organic contaminants is traditionally evaluated by measuring indicators of microbial activity such as depletion in contaminant and electronacceptor concentrations and increases in microbial biomass and biodegradation byproducts (13). However, these methods are limited in providing conclusive evidence of intrinsic bioremediation because physical processes such as volatilization, sorption, and plume dilution can also cause contaminant attenuation. In addition, accurate mass balances of contaminants, electron acceptors, and degradation byproducts can be difficult to obtain in the field. Recently, compound-specific isotope analysis (CSIA), or measurement of the 13C/12C or 2H/1H ratios of petroleum hydrocarbons, has been used to measure biodegradation of these contaminants (14-19). This is done by measuring the shift in isotopic composition (δ13C or δ2H values) of the residual contaminant compared to its original value. For certain compounds, as biodegradation proceeds, the residual contaminant becomes enriched in the heavier isotopes (13C, 2H) and depleted in the lighter isotopes (12C, 1H) due to faster reaction rates of bonds containing the light isotopes (20). The result is that the isotopic signature of the residual contaminant shifts to increasingly more enriched values as biodegradation proceeds. In contrast, recent laboratory studies have shown that processes such as volatilization (2123), dissolution (23, 24), and sorption (22, 25) do not cause carbon isotope fractionation outside of the analytical uncertainty typical for CSIA ((0.5‰). Similarly, laboratory experiments carried out using toluene have shown that under equilibrium conditions volatilization and dissolution do not cause significant hydrogen isotope fractionation outside of typical analytical uncertainty for compound-specific hydrogen isotope analysis ((5‰) (18). During progressive vaporization of low molecular weight compounds (n-alkanes), Wang and Huang (26) report an isotopic depletion in 2H of up to 14‰, but this effect is in the opposite direction to the isotopic enrichment in residual contaminants associated with biodegradation. Laboratory studies investigating carbon isotope fractionation during biodegradation of chlorinated hydrocarbons have documented large and reproducible 13C enrichments on the order of tens of parts per mil (27-29). Fractionation or shifts in δ13C values of this magnitude have been demonstrated in the field for chlorinated ethenes such as PCE and TCE (30) and for cis-DCE, VC, and ethene (28). In both studies, shifts in the δ13C values were used to assess the extent of biodegradation of these contaminants. In contrast to chlorinated hydrocarbons, laboratory studies investigating carbon isotope fractionation during aerobic and anaerobic biodegradation of aromatic hydrocarbons have documented significantly smaller degrees of fractionation. Sherwood Lollar et al. (14) reported no resolvable carbon isotope fractionation during aerobic biodegradation of toluene using a mixed consortium. During aerobic biodegradation of benzene, Stehmeier et al. (15) reported a +2.0‰ carbon isotope fractionation using a mixed consortium. However, this shift was observed only at a late stage of biodegradation (>75% benzene degraded). Similarly, 10.1021/es011253a CCC: $22.00
2002 American Chemical Society Published on Web 04/27/2002
FIGURE 1. Cross-sectional view of the benzene plume at the site. Concentration contours represent benzene concentrations in units of µg/L. Area shaded with diagonal lines represents a clay layer. The aquifer is represented by the unshaded sand layer at 11-18 m and silt beds indicated by vertical lines. The area shaded with criss-cross lines represents a thick impermeable clay layer underlying the site. Shaded black vertical rectangle symbols represent well screens within the aromatic hydrocarbon plume for which samples were isotopically analyzed and results are listed in Table 1. DL3 is a groundwater sample containing pure-phase hydrocarbons collected at the water table (3) near well 1 (W1) at a depth of 1-2m. SA1 represents source spill that occurred in the vicinity of W1 in 1977. SA2 represents source spill that occurred in the vicinity of W2 and W3 in 1978. during anaerobic biodegradation of toluene by mixed consortia, a small carbon isotope fractionation of +2.4‰ and +2.0‰ was observed at >90% degradation for sulfate and methanogenic consortia, respectively (17). During anaerobic methanogenic biodegradation of benzene, a carbon isotope fractionation of +2.4‰ was observed at >60% degradation (31, 32). To date, there is no data available for isotope fractionation during biodegradation of ethylbenzene. Using pure cultures, Meckenstock et al. (16) reported carbon isotopic shifts of +60‰ fractionation (18), whereas the same consortia produced a carbon isotope fractionation of only +2.0‰ (17). Significantly, hydrogen isotope enrichment of the residual toluene became resolvable at ∼32% biodegradation, unlike carbon isotope fractionation which was only resolvable outside of error at >90% biodegradation. Hunkeler et al. (19) recently reported similar results of up to ∼+23‰ hydrogen isotopic fractionation at ∼70% biodegradation during aerobic biodegradation of benzene in laboratory experiments. On the basis of the large hydrogen isotope fractionation observed, Ward et al. (18) suggested that while carbon isotope analysis might be more appropriate for identifying different sources of BTEX at contaminated field sites (22, 23), hydrogen isotope analysis will provide more conclusive evidence of biodegradation of aromatic hydrocarbons in the field.
The objectives of the present study were (1) to test the applicability of using stable carbon and hydrogen isotopes to investigate biodegradation of aromatic hydrocarbons at a contaminated site and (2) to compare the effectiveness of carbon versus hydrogen isotope analysis as an indicator of biodegradation. This is one of the first studies to apply stable hydrogen isotope analysis in addition to carbon isotope analysis to assess intrinsic bioremediation of aromatic hydrocarbons in the field.
Site Background The field site is a Dow Benelux BV industrial site located in the southern Netherlands at the shore of the Westerschelde estuary. This study focuses on one area of groundwater contamination at the Dow Benelux BV site where benzene and ethylbenzene contaminant concentrations are up to 243 000 and 56 000 µg/L, respectively. Toluene, xylenes, and styrene are also present in the groundwater at low concentrations (
