Environ. Sci. Technol. 2002, 36, 5139-5146
Use of Compound-Specific Stable Carbon Isotope Analyses To Demonstrate Anaerobic Biodegradation of MTBE in Groundwater at a Gasoline Release Site R A V I K O L H A T K A R , * ,† T O M A S Z K U D E R , ‡ PAUL PHILP,‡ JON ALLEN,‡ AND JOHN T. WILSON§ Group Environmental Management Company, 150 West Warrenville Road, Naperville, Illinois 60563, School of Geology and Geophysics, The University of Oklahoma, Norman, Oklahoma 73019, and ORD/NRMRL, EPA, Kerr Environmental Research Center, Ada, Oklahoma 74820
Currently it is unclear if natural attenuation is an appropriate remedial approach for groundwater impacted by methyl tertiary butyl ether (MTBE). Site-characterization data at most gasoline release sites are adequate to evaluate attenuation in MTBE concentrations over time or distance. But, demonstrating natural biodegradation of MTBE requires laboratory microcosm studies, which could be expensive and time-consuming. Recently, compound-specific carbon isotope ratio analyses (13C/12C expressed in δ13C notation) have been used to demonstrate aerobic biodegradation of MTBE in laboratory incubations. This study explored the potential of this approach to distinguish MTBE biodegradation from other abiotic processes in an anaerobic groundwater plume that showed extensive decrease in MTBE concentrations. To our knowledge, this is the first study to use δ13C of MTBE data in groundwater and laboratory microcosms to demonstrate anaerobic biodegradation of MTBE. The δ13C of MTBE in monitoring wells increased by up to 31‰ (-25.5‰ to +5.5‰) along with a 40-fold decrease in MTBE concentrations. Anaerobic incubations in laboratory microcosms indicated up to 20-fold reduction in MTBE concentrations with a corresponding increase in δ13C of MTBE of up to 33.4 ‰ (-28.7‰ to +4.7‰) in live microcosms. Little enrichment was observed in autoclaved controls. These results demonstrate that anaerobic biodegradation was the dominant natural attenuation mechanism for MTBE at this site. The estimated isotopic enrichment factors (field ) -8.10‰ and lab ) -9.16‰) were considerably larger than the range (-1.4‰ to -2.4‰) previously reported for aerobic biodegradation of MTBE in laboratory incubations. These observations strongly suggest that δ13C of MTBE
* Corresponding author phone: (630)420-3824; fax: (630)420-5016; e-mail:
[email protected]. † Group Environmental Management Company (a BP affiliated company). ‡ The University of Oklahoma. § ORD/NRMRL, EPA, Kerr Environmental Research Center. 10.1021/es025704i CCC: $22.00 Published on Web 10/29/2002
2002 American Chemical Society
could be potentially useful as an “indicator” of in-situ MTBE biodegradation.
Introduction Methyl tert-butyl ether (MTBE) has become a widespread contaminant in surface water and groundwater (1) following its increased use in gasoline as oxygenate to improve combustion efficiency. The New England Interstate Water Pollution Control Commission surveyed the fifty United States on the occurrence of MTBE in groundwater at gasoline spill sites (2). Nine states reported MTBE in groundwater at 80 to 100% of the spill sites. Fifteen states reported MTBE in 60 to 80% of spill sites, seven states reported MTBE in 40 to 60% of spill sites, four states reported MTBE in 20 to 40% of spill sites and eleven states reported MTBE in 0 to 20% of spill sites. Given the pervasiveness of MTBE and the potential for impacts to drinking water resources, a number of states are currently contemplating a ban or a phase-out of MTBE from gasoline (3). As MTBE is gradually eliminated from gasoline, new releases containing MTBE are likely to decrease over time; however, the existing MTBE plumes from the historical releases will need to be addressed. A risk-based decision making approach prioritizes plumes based on their potential to impact the health and the environment of the nearby receptors. Remediation by natural attenuation (RNA) or monitored natural attenuation (MNA) is a pragmatic approach in situations where the potential for impacts is moderate to low (4, 5). RNA and MNA both rely on the naturally occurring processes of dilution, dispersion, sorption, volatilization, and most importantly, biodegradation to reduce or control the mobility, toxicity and mass of contaminants in the subsurface over a reasonable time frame. It has long been recognized that natural biodegradation of dissolved constituents such as benzene, toluene, ethylbenzene and xylenes (BTEX) occurs rapidly and is relatively widespread under a variety of redox conditions (6). However, very few studies have demonstrated that MTBE will biodegrade naturally at environmentally significant rates under similar conditions (7-10). Landmeyer et al. (11) demonstrated natural biodegradation of MTBE under iron-reducing conditions in the bed sediments of a stream that received groundwater impacted by a gasoline spill. The removal was statistically significant, but the rate was very slow (first order rate constant of 0.06 per year or a half-life of 12 years). The rate of MTBE biodegradation under iron-reducing conditions may have been limited by the supply of biologically available iron in the aquifer sediment. When biologically available iron was added to aquifer sediments, the rate of biodegradation of MTBE was much faster (12). Bradley et al. (10) reported slow natural biodegradation of MTBE in the bed sediments of freshwater streams that received plumes from spills of gasoline. Under sulfate reducing conditions, from 9 to 20% of MTBE was degraded to carbon dioxide after 166 days of incubation. Under methanogenic conditions, approximately 8% of MTBE was degraded to TBA. These rates are also slow (first-order rate constant of 0.25 to 0.5 per year or a half-life of 2.8 to 1.4 years). Wilson et al. (7) demonstrated rapid and extensive MTBE biodegradation in methanogenic groundwater at a spill of JP-4 jet fuel. Anaerobic biodegradation of MTBE was also confirmed in laboratory microcosm studies using soil and groundwater from this site. Thirty-four states in the United States report that they use risk-based decision making to manage the risk associated VOL. 36, NO. 23, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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with releases of gasoline containing MTBE (2). To utilize RNA or MNA as a remedial strategy for MTBE-impacted sites, it is essential to demonstrate natural biodegradation of MTBE at a large number of sites under different redox conditions. BP and EPA undertook a groundwater survey at 74 BP retail sites in 1999 and 2000 to evaluate the potential for natural biodegradation of MTBE (8). The study site was chosen for further investigation based on that survey. Given the limited level of understanding of natural biodegradation of MTBE (13), the fact that a plume of dissolved MTBE is temporally or spatially stable or is shrinking is generally not adequate to justify using RNA or MNA as a remedial alternative. In addition, because tertiary butyl alcohol (TBA, a daughter product of MTBE bio-transformation) is also a contaminant of concern, it is important to distinguish between MTBE mineralization (complete conversion to carbon dioxide) and bio-transformation (partial conversion to daughter products, such as TBA). Detection of TBA in groundwater may be due to bio-transformation of MTBE; however, it is not an unequivocal indicator, as TBA is also likely to be a part of the original release (8, 14). TBA is completely miscible in water, and even trace levels of TBA in the gasoline release could result in significant TBA concentrations in groundwater (15). Traditional indicators such as depletion of electron acceptors (dissolved oxygen, nitrate, sulfate) or elevated concentrations of products of biodegradation (ferrous iron, dissolved methane and total alkalinity) are also not specific indicators of MTBE biodegradation, as degradation of other carbon sources (BTEX and other dissolved hydrocarbons) can produce similar changes in groundwater bio-geochemistry (13). An indicator parameter is needed to distinctly identify MTBE biodegradation in the subsurface. Such a parameter will also help evaluate the effectiveness of bioremediation technologies for MTBE cleanup. Compound specific stable carbon isotopic ratios (13C/12C) have a potential to demonstrate in-situ biodegradation of organic contaminants. Depending upon the source and release history, contaminant molecules have a particular 13C/12C ratio. This ratio is reported in δ13C (‰) notation as follows
δ13C )
(
)
Rsample - 1 × 1000 Rstandard
(1)
where Rsample and Rstandard represent 13C/12C ratios of the sample and the international standard, respectively. It is normally observed that physical, chemical and biological reactions proceed faster and/or have lower bond activation energies for the lighter isotope, 12C compared to the heavier isotope, 13C (16). The result is that reaction products are enriched in 12C, while the residual substrate is enriched in 13C. The observed difference between the δ13C values of the original and the residual substrate is referred to as isotopic fractionation. If the isotopic fractionation due to biodegradation is significantly greater than that due to nonbiological processes (dissolution, sorption and volatilization), an increase in δ13C of the parent contaminant could be a useful indicator of its ongoing biodegradation. It is important to note that biodegradation of parent substrate does not imply complete mineralization to carbon dioxide and water. Increase in δ13C of a substrate associated with decrease in substrate concentrations are indicative of substrate biotransformation. Large changes in δ13C have been reported during reductive dechlorination of chlorinated ethenes (1720), aerobic oxidation of dichloromethane (21) and aerobic oxidation of 1,2-dicholoroethane (22). Studies of biodegradation of BTEX compounds and higher alkylbenzenes showed typically smaller changes in δ13C than was the case with chlorinated ethenes (23-29). It is important to note that the 5140
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extent of fractionation due to microbial biodegradation in these experiments was significantly (at least an order of magnitude) greater than fractionations due to abiotic processes, such as dissolution, sorption and volatilization (22, 30-33). The utility of using δ13C of MTBE as an indicator of MTBE biodegradation in aerobic microcosm enrichments was reported recently (34). Laboratory studies were carried out to evaluate the effect of relevant physical and biological processes on δ13C of MTBE. They reported δ13C enrichments from 5.1‰ to 6.9‰ after biodegradation of 95 to 97% of MTBE in experiments with MTBE as a substrate and cometabolic experiments with 3-methylpentane as the primary substrate. In comparison, none to negligible 13C enrichments were observed in sterile controls. Isotopic fractionation observed during various interphase partitioning experiments were an order of magnitude smaller than due to biodegradation. More recently Gray et al. (35) demonstrated isotopic fractionation of carbon and hydrogen during laboratory experiments on aerobic biodegradation of MTBE using a pure bacterial culture and mixed consortia from an MTBEimpacted site. They reported 13C enrichment of up to 8.1‰ at 99.7% biodegradation of MTBE. In addition, 2H enrichment in excess of 80‰ was observed at 90% MTBE biodegradation. This study was initiated to demonstrate the utility of stable carbon isotopic analyses as an indicator of in-situ MTBE biodegradation. This technique was applied to a field setting where the long-term monitoring data indicated that the plume of MTBE was stable or shrinking and where an empirical evaluation of the field data indicated that natural anaerobic biodegradation of MTBE was an important process (8). The first objective of this work was to compare changes in the δ13C of MTBE in groundwater along a flow path where the concentration of MTBE was attenuated by a factor of 40 and to correlate any increase in δ13C of MTBE to the reduction in the concentrations of MTBE. The second objective was to compare the δ13C enrichment of MTBE with attenuation in concentration as seen at field scale to δ13C enrichment in laboratory microcosms that were constructed using aquifer sediments from the midpoint of the MTBE plume. The sediment was acquired from an anaerobic region of the plume, and the microcosms were constructed and incubated under anaerobic conditions. Site Background. The study site is a retail gasoline station in New Jersey. Figure 1 is a site map showing the locations of monitoring wells studied during this investigation. These are 10.2 cm outer diameter, PVC wells screened from 0.6 to 4.6 m. There are additional monitoring wells located on either sides of the plume (not shown). The gasoline release was first detected in 1990. The site is underlain by alternating layers of medium to coarse sand (up to 0.9 m below grade), silty clay with traces of coarse sand (0.9 to 2.1 m), coarse sand (2.1 to 3.3 m) and a clay layer that extends from 3.3 to 4.6 m. The depth to groundwater varies from 0.3 to 1.0 m. Slug tests conducted at the site indicated hydraulic conductivity values from 0.37 to 0.91 m/d with an average of 0.57 m/d. Historically, the hydraulic gradient has varied from 0.004 m/m to 0.015 m/m with an average of 0.009 m/m. The estimates of groundwater velocity range from 3 m/year to 26 m/year with an average velocity of 9.5 m/year (assuming a porosity of 0.2).
Materials and Methods Groundwater Sampling. The potential for natural biodegradation of MTBE at this site was first evaluated in 1999 during a groundwater survey conducted jointly by BP and EPA at BP’s 74 retail sites in the Eastern United States (8). After measuring the depth to groundwater, three well casing
FIGURE 1. Site map showing the locations of monitoring wells and direction of groundwater flow at the study site. The plume footprint is identified by the shaded area and includes wells with dissolved methane concentration exceeding 0.5 mg/L. volumes were purged. Groundwater samples were collected using disposable bailers. Groundwater samples for VOC analyses were collected in duplicate 40-mL VOA vials containing 0.4 g tri-sodium phosphate preservative (36). Samples for dissolved methane were collected in 60-mL serum bottles containing 1% w/w tri-sodium phosphate preservative, whereas samples for the geochemical parameters (nitrate, nitrite, sulfate, total alkalinity, pH and total organic carbon) were collected in 60-mL serum bottles without any preservative. Laboratory Analytical Procedures. The concentrations of MTBE, TBA, and the BTEX compounds were determined by headspace gas chromatography/mass spectrometry (GC/ MS) using a modification of EPA Method 524.2. Samples preserved with 1% w/w tri-sodium phosphate were loaded on an automated static headspace sampler. Analytes were determined by gas chromatography/mass spectrometry using a Finnigan Ion Trap Detector. In groundwater samples, the lower limit of quantitation was 1 µg/L for MTBE, 10 µg/L for TBA, and 0.5 µg/L for individual BTEX compounds. The effective quantitation limits and method detection limits in the pore water samples of the microcosms were fifteen times higher than the above analytical limits due to 15-fold dilution during sub-sample collection. The lowest calibration standard was 1.0 µg/L for MTBE and 10 µg/L for TBA; the method detection limit was 0.28 µg/L for MTBE and 2.4 µg/L for TBA. The lowest calibration standard for the BTEX compounds was 0.5 µg/L; the method detection limit varied from 0.16 to 0.30 µg/L. The lowest calibration standard for methane was 1 µg/L in groundwater. The fuel oxygenates and BTEX compounds were identified and determined by mass spectrometry. There was little possibility of misidentification of an analyte due to a coeluting peak. The instruments were recalibrated if the calibration check standards diverged from the expected concentration by more than twenty percent of the expected value. The concentration of dissolved methane was determined by headspace analysis and gas chromatography using a flame ionization detector (37). The limit of quantitation was 1 µg/L in groundwater. Stable Carbon Isotope Ratio Analyses. MTBE and TBA were extracted from the water samples using OI 4560 purge and trap apparatus (P&T) interfaced to Varian 3410 gas
chromatograph (GC) and Finnigan MAT 252 isotope ratio mass spectrometer (IRMS). An on-line combustion reactor was installed as an interface between GC and IRMS to convert organic analytes to carbon dioxide and water without affecting chromatographic resolution (38, 39). A Nafion membrane installed prior to the IRMS removed excess water that could be detrimental to the IRMS detector. P&T conditions were optimized for recovery of TBA and MTBE and to minimize water transfer onto GC-IRMS. A Tenax-silica-charcoal trap was used with the following program: purge time 8 min at 40 °C; sample temperature 60 °C; desorb 4 min at 180 °C; bake 20 min at 200 °C. Analyte was transferred onto GC without cryofocusing via heated transfer line to injector with a 1:2.5 split ratio (total flow of helium carrier gas ca. 7 mL/ min). GC column was DB-MTBE, 60 m × 0.32 mm i.d., film thickness 1.8 µm. Flow rate was 2 mL/min (constant pressure mode); temperature program: isothermal at 33 °C for 10 min., then programmed to 80 °C at 30 °C/min, to 100 °C at 4 °C/min. The combustion reactor was a ceramic tube with oxidized nickel wires and platinum catalyst, with auxiliary oxygen trickle. The reactor was held at 980 °C. The isotopic composition of the samples was measured relative to a carbon dioxide standard that was introduced directly into the ion source as a series of pulses. Pulses of carbon dioxide were introduced directly before and after the analyte peaks, to minimize detector precision fluctuations due to variable water transfer during the run. The performance of purge and trap and GCIRMS was monitored at least once a day with an external MTBE/TBA standard. Standard deviations of δ13C of the external standard were calculated for each data subset when samples were analyzed (footnotes to Tables 1 and 3). Quantitation limits with 5 mL sample size were ca. 5 µg/L MTBE and ca. 60 µg/L TBA. It is noteworthy that these quantitation limits are lower than the most stringent cleanup standards for MTBE (10 µg/L in New York) and TBA (100 µg/L in New Jersey). Construction, Sampling and Analysis of Microcosms. Microcosms were constructed with aquifer material that was collected near monitoring well MW-6 (40). This represented a location where the apparent natural attenuation of MTBE was extensive, but not complete. The highest concentration of MTBE in any permanent monitoring well at any sampling event at the site was 1500 µg/L. The highest concentration of MTBE in Monitoring Well 6 at any sampling event was 270 µg/L. At the sample location, the concentration of MTBE was reduced approximately 10 fold from the concentration in the presumed source area. Another 10-fold reduction would approach concentrations that would meet regulatory standards for MTBE. Microcosms were prepared in 25-mL glass serum bottles. Groundwater from MW-6 was added to the sediment to make thick slurry. This slurry was transferred to the serum bottles with a scoop. Each microcosm received 45 g of slurry and 1.0 mL of a dosing solution containing various amendments. The microcosms were sealed with a gray butyl rubber septum and a crimp cap. The microcosms were prepared and stored in a glovebox, under an atmosphere containing 2 to 5% v/v hydrogen and less than 1 ppmv oxygen and incubated at room temperature (20 to 22 °C). A total of 21 microcosms was set up for each treatment and triplicates were sacrificed during each sampling event. Data are presented from three “living” treatments in the microcosm study. The sediment already contained measurable concentrations of MTBE, TBA, and BTEX compounds. One treatment was amended with MTBE and BTEX, one was amended with BTEX and ethanol, and one was amended with MTBE, BTEX, and ethanol. To prepare abiotic controls, a portion of the sediment was autoclaved overnight. The controls were amended with MTBE, BTEX, and TBA. VOL. 36, NO. 23, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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the plume had not reached MW-12, or the plume passed to one side or the other. Because MW-12 was outside the geochemical footprint of the plume of gasoline, the absence of MTBE in water from this well was not be attributed to natural biodegradation. Only the wells that were within the footprint of the gasoline plume were evaluated for natural anaerobic biodegradation of MTBE. Quantification of Isotopic Fractionation in the Field. MTBE. The following simplified form of Rayleigh equation can be used to describe carbon isotopic fractionation due to MTBE biodegradation (41) FIGURE 2. MTBE (solid symbols) and dissolved methane (hollow symbols) concentrations (µg/L) in groundwater as a function of distance away from the presumed source in 1999 (triangle), 2000 (circle) and 2001 (square). MTBE concentrations show up to 40-fold attenuation, whereas methane concentrations remain stable along the flow path indicating that these wells are within the methanogenic plume footprint. The microcosms were sampled as follows. The contents were vigorously stirred with a vortex mixer while they were still sealed in the microcosm. The solids were allowed to settle, then the crimp cap and septa were removed, and approximately 1 mL supernatant water was removed and diluted with 14 mL distilled water containing 0.15 gm trisodium phosphate preservative. These samples were analyzed as described above.
Results and Discussion Attenuation of MTBE in Groundwater. Figure 1 shows the relationship between the source area of MTBE in groundwater, the predominant direction of groundwater flow, and the location of the monitoring wells that were sampled for this study. Wells MW-5, MW-6, MW-7, MW-10 and MW-11 had high concentrations of dissolved methane (> 0.5 mg/L). They are within the footprint of the plume of gasoline contaminants in groundwater. Water from these wells was devoid of nitrate or nitrite nitrogen (
