Environ. Sci. Technol. 2005, 39, 4848-4856
Stable Isotope Evidence for Biodegradation of Chlorinated Ethenes at a Fractured Bedrock Site MICHELLE M. G. CHARTRAND, PENNY L. MORRILL,† GEORGES LACRAMPE-COULOUME, AND BARBARA SHERWOOD LOLLAR* Stable Isotope Laboratory, Department of Geology, University of Toronto, 22 Russell Street, Toronto, Ontario, Canada M5S 3B1
Stable carbon isotope analysis of chlorinated ethenes and ethene was performed at a site contaminated with trichloroethene (TCE), a dense non-aqueous phase liquid (DNAPL). The site is located in fractured bedrock and had variable groundwater hydraulic gradients during the study due to a local excavation project. Previous attempts to biostimulate a pilot treatment area at the site resulted in the production of cis-1,2-dichloroethene (cis-DCE), the first product of reductive dechlorination of TCE. Cis-DCE concentrations accumulated however, and there was no appreciable production of the breakdown products from further reductive dechlorination, vinyl chloride (VC) and ethene (ETH). Consequently, the pilot treatment area was bioaugmented with a culture of KB-1, a natural microbial consortium known to completely reduce TCE to nontoxic ETH. Due to ongoing dissolution of TCE from DNAPL in the fractured bedrock, and to variable hydraulic gradients, concentration profiles of dissolved TCE and its degradation products cis-DCE, VC, and ETH could not convincingly confirm biodegradation of the chlorinated ethenes. Isotopic analysis of cis-DCE and VC, however, demonstrated that biodegradation was occurring in the pilot treatment area. The isotope values of cis-DCE and VC became significantly more enriched in 13C over the last two sampling dates (in one well from -17.6‰ to -12.8‰ and from -22.5‰ to -18.2‰ for cis-DCE and VC, respectively). Quantification of the extent of biodegradation in the pilot treatment area using the Rayleigh model indicated that, depending on the well, between 21.3% and 40.7% of the decrease in cisDCE and between 15.2% and 36.7% of the decrease in VC concentrations can be attributed to the effects of biodegradation during this time period. Within each well, the isotope profile of TCE remained relatively constant due to the continuous input of undegraded TCE due to DNAPL dissolution.
Introduction The chlorinated solvent trichloroethene (TCE), a potential carcinogen, is commonly found in polluted groundwater in North America (1). TCE can be present in groundwater as a * Corresponding author phone: (416) 978-0770; fax: (416) 9783938; e-mail:
[email protected]. † Present address: Geophysical Laboratory, Carnegie Institution of Washington, 5251 Broad Branch Rd. NW, Washington, DC 20015. 4848
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dense non-aqueous phase liquid (DNAPL), which can provide a continuous source of dissolved phase TCE contamination over long time periods. In fractured bedrock, residual DNAPL settles in dead-end cracks and fissures, and due to slow dissolution of this product, remediation methods such as pump and treat techniques can take excessively long time periods (2). TCE may be degraded by anaerobic microorganisms via reductive dechlorination, which is the successive replacement of a chlorine atom with a hydrogen atom, to form cis-1,2dichloroethene (cis-DCE). Cis-DCE degrades to vinyl chloride (VC), and finally VC degrades to ethene (ETH). The isolate Dehalococcoides ethenogenes (DHE) strain 195 (3) is reported to completely transform TCE to nontoxic ETH. DHE-like phylotypes are present in the enrichment culture, KB-1, which was cultured from soil and groundwater from a southern Ontario site contaminated with TCE. KB-1 has been shown to completely dechlorinate TCE to ETH (4). TCE and cis-DCE serve as terminal electron acceptors in the respiratory pathway. However, the VC to ETH reductive dechlorination step is not energetically beneficial to DHE strain 195, and ETH is produced cometabolically from VC (3). DHE-like phylotypes are not present at many contaminated sites however, and bioremediation strategies reliant upon in situ reductive dechlorination can result in incomplete dechlorination and the accumulation of the degradation products cis-DCE and VC. At sites where TCE is not completely converted to ETH under in situ conditions, biostimulation (adding electron donors) and bioaugmentation (adding microorganisms capable of complete reductive dechlorination) (5-7) can be implemented to promote complete reductive dechlorination. The extent of biodegradation is determined by monitoring the concentrations of the primary contaminant and subsequent degradation products (e.g., TCE, cis-DCE, VC, and ETH), among other techniques. Ellis et al. (5) and Harkness et al. (6) studied soil and groundwater at a TCE contaminated site where, after biostimulation, the native microbes failed to reductively dechlorinate cis-DCE. After bioaugmentation with the Pinellas culture, complete dechlorination to ETH was reported. Similarly, at Kelly Air Force Base (AFB), Major et al. (7) reported complete reductive dechlorination of PCE to ETH via bioaugmentation with KB-1, after previous attempts to biostimulate the native microbial population resulted only in the accumulation of cis-DCE. These studies indicate that the presence of microbes capable of complete reductive dechlorination of PCE or TCE to ETH is critical to the success of bioremediation strategies. Compound specific isotope analysis (CSIA) has been used in both laboratory and field studies to identify biodegradation of chlorinated ethenes (8-14). Isotopic fractionation is a shift in the ratio of heavy to light isotopes and is due to the different reaction rates of each isotopic species. Typically, heavier isotopes form stronger bonds than lighter isotopes, and molecules containing lighter isotopes have faster reaction rates than molecules containing heavier isotopes, particularly in biological systems. For instance, as TCE is reductively dechlorinated to cis-DCE, laboratory studies demonstrate that the isotope value of the remaining TCE will become less negative (i.e., more enriched in 13C) due to the preferential rate of incorporation of 12C molecules into the degradation product, cis-DCE (8). The cis-DCE will initially be more negative than the compound from which it was formed. Subsequently, as each degradation product is itself degraded, it too becomes increasingly enriched in 13C, reflected in a shift to less negative isotope values as biodegradation 10.1021/es048592z CCC: $30.25
2005 American Chemical Society Published on Web 05/26/2005
proceeds (9, 10, 12). Other processes of chlorinated ethene dispersal and mass transfer such as dissolution, volatilization, and sorption do not significantly alter the ratios of stable carbon isotopes at equilibrium (15-18). In contrast, the large fractionation due to biodegradation indicates that stable carbon isotope measurements are useful for determining whether a decrease in contaminant concentration is due to degradation or due to physical processes, and to confirm transformation of contaminants for both natural and enhanced biodegradation. Several recent studies have used CSIA to monitor in situ degradation of chlorinated ethenes at field sites. Hunkeler et al. (9) quantified isotope ratios for PCE, TCE, cis-DCE, VC, and ETH and determined that fractionation during the dechlorination step between cis-DCE and VC, and between VC and ETH, was much larger than that detected between TCE and cis-DCE, suggesting that CSIA was most useful for monitoring the last two products of reductive dechlorination. Sherwood Lollar et al. (11) used isotope enrichment trends of the primary pollutants PCE and TCE to verify in situ reductive dechlorination at Dover AFB. Morrill et al. (14) monitored the degradation of PCE to ETH at a site bioaugmented with KB-1, and reported significant isotopic fractionation patterns consistent with reductive dechlorination of the parent compound PCE, and subsequent degradation of the less chlorinated products TCE, cis-DCE, and VC. In contrast, in a field study of wells located close to a source zone, Song et al. (13) reported that the isotopic enrichment of TCE produced by reductive dechlorination to cis-DCE was masked due to continual dissolution of TCE from the source zone to the plume. Although substantial fractionation indicative of biodegradation was observed for the degradation products cis-DCE and VC, the constant influx of source TCE masked any isotopic shift associated with biodegradation of the dissolved TCE (13). To date, reported field applications of CSIA of chlorinated ethenes have focused on unconsolidated porous media and have largely relied on data collected from different parts of the plume, but at only one time point. In the current study, stable carbon isotope measurements were used to confirm biodegradation at a TCE contaminated site where the DNAPL source and dissolved plume are located in fractured bedrock. The objective of this study was to determine if stable carbon isotope measurements of the primary contaminant TCE, and its degradation products cis-DCE, VC, and ETH, could verify biodegradation in this complex fracture-controlled hydrogeologic environment with ongoing DNAPL dissolution contributing TCE to the dissolved plume. In addition, the study was carried out over a period of 6 months with four separate sampling intervals. The information on temporal variation in both concentrations and stable isotope values is important for assessing the effectiveness of biodegradation at a site where significant changes in these parameters were also produced due to fluctuating hydraulic gradients.
Methods Site Description. The site is an industrial facility located adjacent to an area undergoing major excavation activities which have significantly affected local groundwater hydraulic gradients and flow rates. The manufacturing building at the site sits on top of fill (to ∼3 m below ground surface (BGS)) (Figure 1). The fill is underlain by organic silt and peat (to ∼8 m BGS), clay (to ∼18 m BGS), glacial till (to ∼24-30 m BGS), and finally fractured bedrock, which dips northeasterly. The fractured bedrock is approximately 24-46 m BGS, and is underlain by competent bedrock. TCE was disposed of in a dry disposal well within the manufacturing building (Figures 1 and 2), where it migrated into the shallow overburden and down support piles, infiltrating the bedrock (shown in a
FIGURE 1. Geological cross section of site A-A′ (see also Figure 2). Monitoring wells and the disposal well (in the manufacturing building) in Figure 2 are projected onto A-A′. Bedrock wells 1B and 2B were cased to 34 m BGS in the fractured bedrock with open intervals between 34 and 40 m. Wells 3B, 4B, 5B, and 6B were cased to 37 m BGS in fractured rock with screened intervals from 37 to 40 m. The position of the support piles are not exact but illustrate the likely route by which DNAPL penetrated the fractured bedrock. schematic in Figure 1). TCE DNAPL remains in a shallow source area and likely a deep source area within the fractured bedrock. Hydraulic Gradients in the Pilot Treatment Area and Surrounding Area. The hydraulic gradient through the fractured bedrock in the vicinity of the pilot study area varied as much as 90° in response to excavation to the northeast of the manufacturing building (Figure 2). Under undisturbed conditions, the calculated flow rate in the shallow bedrock in the pilot treatment area was northwesterly from the manufacturing building at approximately 0.38 m d-1 (corresponding to a seepage velocity of 1.5 m d-1 if a porosity of 0.25 for highly fractured bedrock is used) (19). However, when the excavation at the neighboring site was dewatered, the hydraulic gradient shifted ∼90° to the west and groundwater flow rates were highly variable. To prevent subsidence of local building foundations, recharge wells were installed at the site and water was re-injected, resulting in a shift ∼90° back to northwesterly hydraulic gradients. Bioremediation Strategy. A bioremediation treatment system was installed in the pilot treatment area to remediate groundwater containing dissolved TCE in the fractured bedrock. The system consisted of a single extraction well (4B), a single injection well (1B), and two monitoring wells (2B and 3B) along the main groundwater flow path in the bioremediation treatment system (pilot treatment area) (Figure 2). Groundwater was extracted at 18.9 L min-1 (LPM) and amended using a system which was automated to control flow and mixing. Amended groundwater was pumped into well 1B at 18.9 LPM to create a recirculation loop between 1B and 4B. An iodide tracer study showed that shifts in hydraulic gradient due to localized excavation activities, in addition to the fracture-controlled hydraulic gradients at this site, prevented complete hydraulic gradient control in the pilot treatment area. Tracer recovery in the injection well was typically approximately 50%, indicating that the recirculation system within the pilot treatment area was not entirely closed. Up to 50% of the groundwater was being recirculated from zones outside the treatment area. While not ideal for the most effective remediation in the pilot treatment area, this does not affect the interpretations in this study. Molecular biological analysis of soil and groundwater samples obtained from various locations at the site indicated VOL. 39, NO. 13, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Map of the pilot treatment area located adjacent to the west side of the manufacturing building. The source of the spill was the disposal well located in the manufacturing building (see the text). In the pilot treatment area, water was injected into well 1B and extracted via well 4B. Wells 2B and 3B were within the groundwater flow path between the injection and extraction wells based on tracer tests. For a description of the other wells, see the text. A-A′ refers to the geologic cross-section in Figure 1.
FIGURE 3. Concentrations of TCE (squares), cis-DCE (tilted squares), VC (triangles), and ETH (circles) at well 2B. that microorganisms closely related to DHE were present at several site locations both inside and outside the pilot treatment area (20). Despite this evidence of the presence of DHE, there was no quantifiable evidence of activity of these microorganisms under in situ conditions. Hence, the pilot treatment area was biostimulated in March 2001 to induce reductive dechlorination of TCE using the native DHE-like microorganisms. Acetate was added at a time-weighted average concentration of 100 mg L-1 (1.7 mM), based on groundwater flux through the pilot treatment area. In the 3 months following biostimulation, the concentrations of TCE decreased to below the detection limit at well 2B (Figure 3). Increasing cis-DCE concentrations to between 30 and 40 mg L-1 confirmed that TCE was being degraded (Figure 3). However, during the biostimulation phase there was no appreciable production of VC or ETH, suggesting that reductive dechlorination had stalled at cis-DCE degradation to VC. To facilitate complete conversion of TCE to ETH, the 4850
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pilot treatment area was then bioaugmented with a culture of KB-1 and biostimulated with methanol at a time-weighted average concentration of 250 mg L-1 (7.8 mM). One 40 L pulsed injection of a culture of KB-1 was added to the recirculation loop in June 2001. In the subsequent 8 months after bioaugmentation, the concentration of cis-DCE at well 2B decreased to approximately 30 mg L-1, increased to approximately 55 mg L-1 in January 2002, and subsequently decreased to 8 mg L-1 in February 2002 (Figure 3). VC and ETH concentrations increased from less than 0.1 mg L-1 each to 20 and 0.25 mg L-1, respectively (Figure 3). However, beginning in March 2002, concentrations of cis-DCE and VC showed substantial fluctuations, alternately increasing and decreasing between sampling dates (Figure 3). The substantial fluctuations in concentrations of chlorinated ethenes made confirmation of biodegradation difficult. Therefore, in June 2002, an isotope study was initiated to measure the isotope values of TCE, cis-DCE, VC, and ETH to assess biodegradation
and in particular to confirm, if possible, whether complete reductive dechlorination to ETH was occurring. Samples were collected in June, July, September, and November 2002 to monitor the course of degradation. Sample Collection and Groundwater Chemical Analyses. Groundwater samples for quantification of chlorinated ethenes and their isotope values were periodically collected from wells in and outside the pilot treatment area in 40 mL amber volatile organics analysis (VOA) vials containing 1 mL of 12 N HCl. The vials were completely filled, and sealed with no headspace using a PTFE/silicone septum fitted screw cap. Samples were shipped on ice to Alpha Analytical Laboratories (Westborough, MA) for volatile organic compound (VOC) analysis using EPA method 8260 and to the SiREM laboratory (Guelph, ON, Canada) for ethene analysis using EPA method Modified RSK 175. Uncertainty in these analyses is 30%. Samples for isotope analyses were shipped to the Stable Isotope Laboratory at the University of Toronto and were kept at 4 °C in the dark until analyzed. Well Selection for Isotope Analyses. Seven wells (four located in the pilot treatment area) were sampled during the course of the isotope study (Figure 2). Sampling wells 1B and 2B had a diameter of 0.15 m, and the diameter of wells 3B, 4B, 5B, 5T, 6B, and 7B was 0.10 m. Wells 1B and 2B were cased to approximately 34 m BGS in the fractured bedrock, with open intervals between 34 and 40 m. Wells 3B, 4B, 5B, and 6B were cased to approximately 37 m BGS in the fractured rock with screened intervals between 37 and 40 m (Figure 1). Well 5T was cased to approximately 28 m BGS and screened in the till overlying the fractured bedrock. Samples were collected from well 4B (the extraction well) and two wells in the main groundwater flow path in the pilot treatment area (wells 2B and 3B) where tracer was detected. Well 5 was chosen because it provided the opportunity to compare isotope values of the chlorinated ethenes in a bedrock well (5B) to the overlying till (5T). The detection of electron donor amendments from the pilot treatment area (i.e., acetate and methanol) in well 5T indicated some degree of hydraulic connectivity between the till layer and underlying shallow bedrock. Well 7B, located approximately 93 m southwest of the pilot treatment area (well 1B), was selected as a background well because the absence of electron donor amendments from the pilot treatment area indicated no hydraulic connection to the pilot treatment area. In addition, well 6B, located approximately 93 m to the southeast and upgradient from the pilot treatment area (well 1B), and at which no electron donor amendments from the pilot treatment area were detected, was selected as a second background well. Isotope Analyses. Samples were prepared for isotopic analysis using two methods. For groundwater samples containing >1 mg L-1 dissolved VC, the contents of two 40 mL VOA vials were poured into a 160 mL glass bottle, which was then capped with a PTFE/silica septum and crimp sealed after the technique of Slater et al. (15). The bottles were equilibrated overnight at 4 °C. Two duplicate 160 mL bottles were prepared for each sampling well. For groundwater samples containing 1 mg L-1 ETH, the 40 mL VOA vials were inverted to create a gaseous headspace by loosening the cap and allowing approximately 10 mL of the sample to drain from the vial while retaining the headspace. This second method allowed the sample to be analyzed using direct headspace analysis without the possibility of VC or ETH volatilizing during the transfer from 40 mL VOA bottles to 160 mL bottles. Laboratory protocol tests showed neither approach affected the isotope value of VC or ETH. Headspace samples (200-1000 µL) were obtained using Supelco Pressure-Lok series A-2 syringes. The stable isotope ratio for an individual compound is measured and compared to an international standard. For
carbon, the isotope ratio is expressed by the following equation:
δ13Ccompound ) ((Rcompound/Rstandard) - 1) × 1000 (1) where R ) 13C/12C ratio of the compound or standard (for carbon, the international standard used is Vienna Peedee Belemnite). Throughout the paper where the terms enrichment and depletion are used, these consistently refer to enrichment or depletion in the heavy isotope (13C). The δ13C value is expressed in permil (‰) units, and the analytical error is (0.5 ‰ (21, 22). This error incorporates both the accuracy of the measurement with respect to international standards and the reproducibility on replicate measurements of the sample. The first sampling date for the isotope study was June 2002. TCE and cis-DCE were analyzed using a temperature program of 35 °C held for 4 min, increased to 90 °C at 15 °C min-1, and increased to 180 °C at 25 °C min-1 on a Varian 3400 gas chromatograph (GC) (DB-624 column, Chrompack, 30 m × 0.25 mm i.d., flow 2.0 mL min-1) interfaced with a combustion oven and a Finnigan MAT 252 mass spectrometer. Cis-DCE and VC were analyzed using a temperature program commencing at 40 °C, which was increased at a rate of 10 °C min-1 to 200 °C and held for 5 min, on an HP 6890 GC (Poraplot Q column, Chrompack, 30 m × 0.25 mm i.d., flow 1.6 mL min-1) on line with a combustion oven and a Finnigan Mat Delta plus XL mass spectrometer. Each reported value is the average of measurements from at least duplicate samples. The standard deviation on replicates for each sample ranged from 0.01‰ to 0.46‰, always less than the reported analytical error (incorporating both reproducibility and accuracy) of (0.5‰ (21, 22). The second sampling date was July 2002. TCE, cis-DCE, and VC were analyzed using a Poraplot U column (Varian, 30 m × 0.25 mm i.d., flow 1.5 mL min-1) with a temperature program of 40 °C held for 5 min, increased to 135 °C at 5 °C min-1, increased to 185 °C at 15 °C min-1, and held for 5 min on the Finnigan Mat Delta plus XL mass spectrometer, and this method was used for the subsequent sampling dates (September and November 2002). ETH was analyzed using the same instrument and column but with a temperature program of 20 °C held for 6 min, increased to 135 °C at 5 °C min-1, increased to 185 °C at 15 °C min-1, and held for 5 min. Isotope standards run throughout confirmed that, regardless of the specific temperature program, mass spectrometer, or column used, all isotope standards were accurate within (0.5‰.
Results and Discussion Wells Outside the Pilot Treatment Area. The concentrations and isotope values of TCE, cis-DCE, VC, and ETH measured on each sampling day for all wells sampled are presented in Tables 1 and 2, respectively. Well 7B was chosen as a background well because electron donor amendments from the pilot treatment area were not detected at this location. The presence of cis-DCE (2.6-0.64 mg L-1), the reductive dechlorination product of TCE, and on one date (November 2002) the low but significant detection of VC (0.092 mg L-1;Table 1), a metabolite of cis-DCE dehalogenation, nonetheless suggest that some in situ biodegradation has occurred at this well. Previous studies confirmed the presence of DHElike microorganisms at this well, supporting the possibility of in situ biodegradation (20). However, the δ13C values for TCE and cis-DCE measured at well 7B were among the most depleted values measured at the site (Table 2) and remained constant within analytical error throughout the four sampling dates. These isotopic data and the absence of significant VC and ETH in well 7B suggest the degree of biodegradation of the chlorinated ethenes at this location is limited. VOL. 39, NO. 13, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. TCE, cis-DCE, VC, and ETH Concentrations (mg L-1) for All Wellsa cis-DCE
TCE well
July
Sept
Nov
June
July
VC
Sept
Nov
June
July
Sept
ETH Nov
