Biodegradation of a Mixture of Chlorinated Volatile Organic Compounds

L.J.W. Barnes1, S.R. Daniel1, and J.B. Warner2

1Department of Chemistry and Geochemistry, Colorado School of Mines, Golden, CO 80401, Phone: (303) 384-2053, FAX: (303) 273-3629; 2ERM-West, Inc., 1777 Botelho Drive, Suite 260, Walnut Creek, CA 94596


A mixture of vinyl chloride, cis- and trans-1,2-dichloroethene (DCE), and 1,1-dichloroethane (DCA) was biodegraded at 20 C in static microcosms by a consortium of indigenous microorganisms from a Superfund site contaminated with a variety of halogenated compounds. Microcosms were set up with sand and groundwater from the site to model biodegradation under aquifer conditions and biodegradation with various amendments in batch cultures. Under aerobic conditions, vinyl chloride and cis- and trans-1,2-DCE biodegraded slowly, although there was no change in the concentration of 1,1-DCA. The biodegradation rates for all three chlorinated ethenes were greatly increased by enriching for methanotrophs in an aerobic environment, but this had little effect on the concentration of 1,1-DCA. Under anaerobic conditions, vinyl chloride increased in concentration, while the concentrations of 1,1-DCA and the dichloroethene isomers decreased. The rate at which 1,1-DCA decreased from the VOC mixture correlated directly to the concentration of the chlorinated ethenes in that mixture. This relationship may be new in the literature and has important implications for the potential success for intrinsic bioremediation of sites contaminated with mixtures of chlorinated compounds.

Key words: biodegradation, vinyl chloride, 1,1-dichloroethane, dichloroethene, VOC


Vinyl chloride, cis- and trans-1,2-dichloroethene (DCE), and 1,1-dichloroethane (DCA), along with their parent compounds tetrachloroethene (PCE), trichloroethene (TCE), and 1,1,1-trichloroethane (TCA), are among the 5 halogenated aliphatic compounds, excluding trihalomethanes, most frequently found in finished water supplies that use groundwater throughout the United States (Westrick, Mello and Thomas, 1984). Their presence is of concern because of their known toxicity and suspected, or known, carcinogenicity (Barton, et al., 1995; Giri, 1995; Goldberg, et al., 1992; Harrison and Hathaway, 1990; Hurtt, Valentine and Alvarez, 1993; Jacobsen and Humayun, 1990; McCauley, et al., 1995; Mochida, Gomyoda and Fujita, 1995). Chlorinated aliphatics commonly have been used in industry as degreasers and solvents because they easily dissolve non-polar substances (Milde, Nerger and Mergler, 1988). They migrate into the groundwater system after being dumped, or accidentally spilled, and have become a problem because they do not easily degrade.

PCE and TCE are known to be biotransformed into several less-chlorinated compounds, including 1,1- and 1,2-dichloroethene, 1,2-dichloroethane, chloroethane, and vinyl chloride. If conditions are favorable, biodegradation may proceed to complete mineralization (Arvin, 1991; Barrio-Lage, Parsons and Nassar, 1987; Bouwer and McCarty, 1983; Broholm, Christensen and Jensen, 1993; DiStefano, Gossett and Zinder, 1991; Fathepure and Vogel, 1991; Fliermans, et al., 1988; Freedman and Gossett, 1989; Kastner, 1991; Lanzarone and McCarty, 1990; Nyer, Crossman and Boettcher, 1996; Vogel and McCarty, 1985). TCA degrades abiotically under anaerobic conditions, forming 1,1-DCE by an elimination reaction and acetic acid by hydrolysis (Klecka, Gonsior and Markham, 1990; Vogel and McCarty, 1987). It also readily biodegrades under both methanogenic and sulfate-reducing conditions, forming 1,1-DCA, chloroethane, 1,1-DCE, acetic acid, and carbon dioxide.

Sites contaminated by mixtures of chlorinated compounds often are difficult to clean up. Such is the case with the site that is the focus of this research. The results presented in this paper were obtained as part of a biofeasibility study investigating the potential for in situ bioremediation of a series of stacked aquifers in California's Central Valley. The study site originally was contaminated with PCE, TCE, and 1,1,1-TCA approximately 40 years ago. Since then, the parent compounds have been degrading naturally, by abiotic and biotic processes, but degradation has not been rapid enough to keep the plume from spreading. Today, the plume consists predominantly of a mixture of vinyl chloride, cis- and trans-1,2-DCE, and 1,1-DCA. The parent compounds occur only in hot spots and other halogenated compounds have been identified intermittently around the site.

One of the objectives of this study was to investigate how a mixed community of microorganisms from the aquifer biodegrades a mixture of vinyl chloride, cis- and trans-1,2-DCE and 1,1-DCA under conditions as they might exist in the aquifer, and with various amendments.


Sample Collection and Handling

Sand and groundwater were taken from the site to obtain a representative sample of the indigenous microbial community living attached to the sand grains or moving about in the water. Sand cores were taken from depths of 50 to 60 feet below the surface with a steam-cleaned, split-spoon coring device and sterile stainless steel liners. As soon as the sand cores were brought to the surface, the ends of the liners were tightly sealed with Teflon caps, and the sand cores were placed on ice in a cooler to be shipped "next day delivery" to the laboratory. Groundwater was collected by flowing water from an existing near-by monitoring well into sterile amber borosilicate glass jars with Teflon-lined caps. Sand cores and groundwater samples arrived at the laboratory in less than 24 hours and microcosms were immediately set up. Groundwater pH and dissolved oxygen were measured in the field when water samples were collected.

Microcosm Setup

Microcosms were set up to closely mimic physical and chemical conditions at the site, including temperature, pH, bioavailability of macro- and micronutrients, and the competitive or synergistic relationships between different factions within the microbial community. The microcosms were made by aseptically transferring approximately 60 grams of damp aquifer sand and 60 ml of groundwater into each sterile 125 ml borosilicate-glass serum bottle. Four different headspace atmospheres were studied, as shown in Table 1.

The more volatile organic compounds in the groundwater, including the compounds of interest, were lost during microcosm preparation, but some of the less volatile contaminants remained in measurable amounts. Microcosms were sealed with sterile, Teflon-lined gray butyl-rubber stoppers and aluminum crimp seals, then spiked either with a single compound-of-interest, or a mixture of all four compounds, to the initial concentrations summarized in Table 2. Vinyl chloride, cis- and trans-1,2-DCE, and 1,1-DCA, which were used to spike the microcosms, were purchased from Supelco Inc. (Bellefonte, PA) as certified single-component standards.

Sand and groundwater samples were subdivided to measure biodegradation rates in triplicate microcosms under four treatment conditions (Table 1). Each treatment was designed to favor a different subpopulation within the native microbial community. Unenhanced biodegradation effectively modeled unenhanced anaerobic biodegradation after the limited amount of bioavailable oxygen was consumed; dissolved oxygen within the plume usually is low, ranging between 0.5 and 1.5 mg/L. Oxygen gas, rather than air, was used in the aerobic treatment and in the enrichment for methanotrophs to ensure that oxygen would not become limiting in the batch microcosms. The numbers of methanotrophic bacteria in microcosms for that treatment were increased above the level at which they occurred in the aquifer by incubating the microcosms for approximately one week at 20 C with a headspace atmosphere of one part methane and two parts oxygen. Before the microcosms were spiked with the compounds of interest, the headspace atmosphere was changed to oxygen with 4% (v/v) methane.

Microcosms were incubated in the dark in a controlled environment chamber at 20 C, lying on their sides in trays on an orbital shaker operating at approximately 90 rpm. Microcosm control blanks were assembled according to the same protocol used for microcosms with live microbes, except they were autoclaved several times at 121 C, to kill the indigenous microorganisms, before the appropriate atmosphere and measured amounts of the compounds of interest were added. Two control blanks were set up for each microcosm with live microorganisms.

Analytical Procedures

Headspace analysis was conducted with a Perkin Elmer Autosystem gas chromatograph (GC) equipped with an electron capture detector (ECD) and a DB-VRX TM ( J & W Scientific, Folsom, CA) capillary column (.32 mm x 60 m) for volatiles. The sensitivity of the gas chromatograph was optimized for vinyl chloride by decreasing the split flow to two, lowering total flow through the ECD to 20 ml/min, and using a temperature-ramped program that remained isothermal at 35 C for 5 minutes, then ramped up to 150 C at 10 C/minute. Ultra high purity (UHP) helium was used for the carrier gas and UHP argon with 5% methane was used for the make-up gas (gases were supplied by General Air Service & Supply, Denver, CO). Method 8000B SW846 (USEPA, 1995) was followed to ensure quality control of the gas chromatography.

A custom mixture of vinyl chloride (10000 ug/ml), cis-1,2-DCE (5000 ug/ml), trans-1,2-DCE (5000 ug/ml), and 1,1-DCA (5000 ug/ml) in methanol was purchased from AccuStandard, Inc. (New Haven, CT) as a certified standard. The batch standard was subdivided when it was fabricated into 52 amber-glass, 1-ml ampules so that fresh aliquots of the same standard could be used for GC calibration throughout the entire study. Chromatographic retention times were determined by injecting certified, single-component standards for each of the four compounds of interest, one at a time, into the chromatograph. Retention times for each of the peaks later were verified by GC/MS.

Changes in the headspace concentration of vinyl chloride, cis- and trans-1,2-DCE, and 1,1-DCA were measured periodically by withdrawing 200 ug/L of headspace gas from each microcosm into a GasTight TM syringe (Hamilton Company, Reno, Nevada), then injecting the sample directly into the GC.


Concentration versus time data were fit to both zero and first order kinetic models before graphing. First-order kinetics usually fit the data as well, or significantly better, than zero-order kinetics. Consequently, data were graphed as time (days) elapsed since the microcosms were spiked versus the natural logarithm of the ratio between concentration measured at any particular time (C) and the initial concentration (Co). Plotting (C/Co) makes it easier to see how concentration changes with time; the log transform fit the data to a first-order kinetic model. The lines on the graphs connect the mean values for each group of replicates. Error bars in these figures represent one standard deviation from the mean.

Even though vinyl chloride, cis- and trans-1,2-DCE, and 1,1-DCA were together as a mixture in the microcosms; unless otherwise noted, data for biodegradation of the chlorinated ethenes are presented separately from data for 1,1-DCA. This was done to highlight the results for 1,1-DCA.

Biodegradation of a Mixture of Chlorinated Ethenes

Under anaerobic or unenhanced conditions (Figures 1 and 2), cis- and trans-1,2-DCE both decreased in concentration, although the cis-isomer decreased more rapidly than the trans-isomer. The temporary increase in cis-1,2-DCE centered on 34 days in Figure 1 may reflect biodegradation of a more highly chlorinated compound in the groundwater from contamination at the site. During the first 22 - 34 days, the concentration of vinyl chloride decreased, as it did under aerobic conditions; thereafter, vinyl chloride concentrations continued to increase. Adding a small amount of methanol to the microcosms significantly reduced the rates at which these changes in concentration progressed and reduced the variability among replicates.

Under aerobic conditions (Figure 3), vinyl chloride concentrations decreased, instead of increasing as they did under anaerobic conditions. Cis- and trans-1,2-DCE concentrations also decreased, but much more slowly than they did in an anaerobic, or low oxygen, environment. The most dramatic decreases in the concentrations of all three chlorinated ethenes were achieved by enriching for methanotrophs (Figure 4)-a unique group of bacteria that use methane as a carbon and energy souce, but are able to co-metabolize low-molecular weight chlorinated molecules because they have a non-specific enzyme called methane monooxygenase. The variability among replicates also markedly increased in this treatment and the concentration of vinyl chloride temporarily increased after 69 days. This temporary increase may have occurred because vinyl chloride was forming by biodegradation of DCE at a rate greater than that at which the microbes could biodegrade newly-formed vinyl chloride plus the vinyl chloride still present from when the microcosm was spiked.

Biodegradation of 1,1-DCA

The concentration of 1,1-DCA decreased only in anaerobic microcosms or with low concentrations of oxygen. Under aerobic conditions with oxygen in the headspace, and in the enrichment for methanotrophs, 1,1-DCA concentrations remained unchanged over a period of 104 days.

Under anaerobic or unenhanced conditions (Figure 5) in samples containing a mixture of chlorinated compounds, 1,1-DCA concentrations decreased to below, or near, the limit of detection in two of the three replicates within 70 days. The rate of decrease was relatively slow during the first 20 - 50 days, then markedly increased. Adding methanol under anaerobic conditions (Figure 6) increased the lag time before 1,1-DCA concentrations rapidly decreased in the mixture. However, when 1,1-DCA was the only chlorinated compound added to an anaerobic treatment amended with methanol (Figure7), its concentration rapidly decreased to the detection limit after a lag time of approximately 70 days. In a mixture, under the same conditions, 1,1-DCA decreased more slowly.

In anaerobic microcosms with no amendments (Figure 8), the rate of decrease for 1,1-DCA correlated directly with the relative concentrations of vinyl chloride and DCE. When 1,1-DCA was added, along with a mixture having the concentrations shown in Table 1, the concentration of 1,1-DCA rapidly decreased to the detection limit after a lag period of approximately 70 days. In a mixture spiked with only 50 ug/L of cis-1,2-DCE, 50 ug/L of trans-1,2-DCE, and 100 ug/L vinyl chloride, in addition to the 1,1-DCA, the concentration of 1,1-DCA decreased more slowly. When the microcosm was spiked with only 1,1-DCA, the concentration of 1,1-DCA remained relatively unchanged for almost 200 days.

Biodegradation of Unidentified Compounds in the Groundwater

Comparison of chromatograms produced by analysis of microcosm headspace soon after the microcosms were spiked with chromatograms produced by headspace analysis after the microcosms were incubated for three months readily shows that the microbes biodegraded other compounds in the groundwater, as well as the compounds of interest. The baseline cleaned up and flattened out best for samples from anaerobic microcosms. Several peaks increased in height, or new peaks appeared, in the headspace from aerobic microcosms or in microcosms enriched for methanotrophs.


Many of the results obtained in this study reproduce results previously reported in the literature. The literature on biodegradation of 1,1-DCA is not extensive, and in some cases, results seem to be contradictory. In this paper, we are reporting a direct relationship between the concentration of a mixture of chlorinated ethenes and the rate at which 1,1-DCA biodegrades in that mixture; this result has not, to our knowledge, previously been reported in the literature.

Enrichment for Methanotrophs

Chlorinated ethenes, like vinyl chloride and cis- and trans-1,2-DCE, have been shown to be readily biodegraded by methanotrophic bacteria in an aerobic environment (Arvis, 1991; Castro, Riebeth and Belser, 1992; Dolan and McCarty, 1995; Fogel, Taddeo and Fogel, 1986; Hensen, et al.., 1988; Nelson and Jewell, 1993; Phelps, et al., 1991; Strandberg, Donaldson and Farr, 1989); under similar conditions, 1,1-DCA has been reported to be recalcitrant to biodegradation (Roberts, et al.., 1989). The results obtained in this study, by enriching for methanotrophs, successfully reproduced those already in the literature. The wide variability in biodegradation rates among replicate samples demonstrated in our study also has been reported by Dolan and McCarty (1995).

Biodegradation Under Aerobic Conditions

Vinyl chloride has been shown to biodegrade aerobically as a primary substrate (Castro, Wade and Riebeth, 1992; Davis and Carpenter, 1990; Hartmans and deBont, 1992; Phelps, et al., 1991). Hartmans and deBont (1992) also demonstrated that dichloroethenes could be oxidized by cells grown on vinyl chloride. The results obtained in our study are in agreement with those reported in the literature. The slow rate at which vinyl chloride and the DCE isomers disappeared from microcosm headspace might be related to the high concentration of oxygen in the headspace atmosphere. Hauschild, et al..(1994) have shown that the rate at which vinyl chloride biodegrades is a function of oxygen concentration. High concentrations of oxygen may inhibit biodegradation due to oxygen toxicity, while low concentrations of oxygen may be limiting. Additional research is needed to determine the optimal range in oxygen concentration for vinyl chloride to biodegrade aerobicallly under aquifer conditions.

There is disagreement in the literature as to whether 1,1-DCA biodegrades aerobically (Parsons and Lage, 1985; Tabak, et al.., 1981; Wilson, et al.., 1983). In our study, 1,1-DCA appeared not to biodegrade, or to biodegrade very slowly, with an oxygen atmosphere in the headspace. This only means that 1,1-DCA does not readily biodegrade aerobically under the conditions as they existed in the aerobic microcosms. Perhaps the oxygen concentration was too high, or some essential nutrient was limiting. In any case, more research is needed.

Biodegradation Under Anaerobic and Low Oxygen Conditions

Under anaerobic conditions, or with very low concentrations of dissolved oxygen, vinyl chloride has been shown to have little potential for biotransformation (Barrio-Lage, et al., 1990; Freedman and Gossett, 1989; Hauschild, et al., 1994; Vogel and McCarty, 1985). However, cis- and trans-1,2-DCE biodegrade well in an anaerobic environment (Barrio-Lage, et al.., 1986; Vogel and McCarty, 1985). The results obtained in our study are in good agreement with those in the literature.

Barrio-Lage, et al.. (1990), using a flow-through packed column with soil from the study site, reported that the addition of methanol enhanced anaerobic biodegradation of vinyl chloride. Those results are not in agreement with the ones obtained in our study, nor do they agree with results Barrio-Lage, et al.., (1990) obtained using static microcosms. Microorganisms can have a strong affinity for methanol as a primary substrate (Harrison, 1973; Wilkinson and Harrison, 1973). The noticeably slower rates at which vinyl chloride accumulated and the dichloroethene isomers disappeared in our study are consistent with a scenario in which methanol competitively inhibited vinyl chloride biodegradation.

1,1-DCA has been reported to biodegrade slowly under anaerobic conditions (Klecka, Gonsior and Markham, 1990; Vogel and McCarty, 1987). In our study, 1,1-DCA began to biodegrade anaerobically in some microcosms in less than one month, although the lag time varied widely from one replicate to another. The addition of methanol to anaerobic microcosms quadrupled the lag time before biodegradation of 1,1-DCA began, suggesting that the microbes responsible for biodegrading 1,1-DCA also have a high affinity for methanol as a carbon source. In anaerobic microcosms amended with methanol, 1,1-DCA biodegraded more rapidly when it was the only chlorinated compound in the microcosms, rather than occurring in a mixture of VOCs. In both cases, there was a lag time of almost 80 days before 1,1-DCA began to be biodegraded more rapidly. The direct correlation between the rate at which 1,1-DCA biodegraded and the concentration of chlorinated ethenes suggests that the chlorinated ethenes may be necessary to induce the enzyme needed for 1,1-DCA biodegradation. Additional research is needed to more clearly define this system.

Biodegradation of Unidentified Compounds

Along with the compounds of interest, the indigenous microbial community from this site biodegraded several other unidentified compounds that also were in the groundwater. This is important for several reasons; first, it means that the demand for electron acceptors will be higher than would be calculated from just the concentrations of the compounds of interest. Second, microbes already present in the aquifer can clean up more than just the compounds that currently are regulated. Third, biodegradation by-products from the unidentified compounds also may need to be considered when evaluating the outcome from in situ bioremediation.


Microorganisms from the indigenous microbial community living 50 to 60 feet below the surface were able to biodegrade all four of the compounds of interest: vinyl chloride, cis- and trans-1,2-dichloroethene, and 1,1-dichloroethane. However, all four compounds did not biodegrade equally well under the same treatment conditions. Many of the results obtained in this study reproduce those obtained previously by other researchers working with less complex, better-defined systems. These include the reluctance of vinyl chloride to biodegrade under anaerobic conditions, the biodegradability of cis- and trans-1,2-dichloroethene under anaerobic conditions, and the ability of methanotrophs to rapidly biodegrade vinyl chloride and cis- and trans-1,2-dichloroethene using oxygen as an electron acceptor.

Results from this study suggest that the rates at which mixtures of chlorinated compounds biodegrade in situ will be affected by all of the bioavailable organic compounds present in the aqueous environment. In some cases, the effect may be synergistic, as shown by the increased anaerobic biodegradation rate of 1,1-dichloroethane in a higher concentration mixture of chlorinated ethenes. In other cases, the effect may be antagonistic, as when small amounts of methanol were added to anaerobic microcosms with the same higher concentration mixture. Further research currently is in progress to more clearly define the mechanism(s) operating during biodegradation of 1,1-dichloroethane in a mixture of chlorinated ethenes and the conditions required to biodegrade 1,1-dichloroethane.


The authors appreciate the assistance of David Slayter and Greg Murphy, who collected sand and groundwater samples from the site for this study. They also thank Dr. David M. Updegraff for technical assistance setting up anaerobic microcosms. Support for this research was provided by ERM-West, Inc., Walnut Creek, CA.


Arvin, E., 1991. Biodegradation Kinetics of Chlorinated Aliphatic Hydrocarbons with Methane-Oxidizing Bacteria in an Aerobic Fixed Biofilm Reactor, Water Res., 25, pp. 873-881.

Barrio-Lage, G., F. Z. Parsons, R. M. Narbaitz, P. A. Lorenzo and H. E. Archer, 1990. Enhanced Anaerobic Biodegradation of Vinyl Chloride in Groundwater, Environ. Chem., 9, pp. 403-415.

Barrio-Lage, G., F. Z. Parsons and R. S. Nassar, 1987. Kinetics of the Depletion of Trichloroethene, Environ. Sci. Technol., 21, pp. 366-370.

Barrio-Lage, G., F. Z. Parsons, R. S. and P.A. Lorenzo, 1986. Sequential Dehalogenation of Chlorinated Ethenes, Environ. Sci. Technol., 20, pp. 96-99.

Barton, H. A., J. R. Creech, C. S. Godin, G. M. Randall and C. S. Seckel, 1995. Chloroethylene Mixtures: Pharmacokinetic Modeling and In Vitro Metabolism of Vinyl Chloride, Trichloroethylene, and Trans-1,2-Dichloroethylene in Rat, Toxicol. Appl. Pharmacol., 130, pp. 237-247.

Bouwer, E. J. and P. L. McCarty, 1983. Transformation of 1- and 2- Carbon Halogenated Aliphatic Organic Compounds Under Methanogenic Conditions, Appl. Environ. Microbiol., 45, pp. 1286-1294.

Broholm, K., T. H. Christensen and B. K. Jensen, 1993. Different Abilities of Eight Mixed Cultures of Methane-Oxidizing Bacteria to Degrade TCE, Water Res., 27, pp. 215-224.

Castro, C. E., D. M. Riebeth and N. O. Belser, 1992. Biodehalogenation: The Metabolism of Vinyl Chloride by Methylosinus trichosporium OB-3b: A Sequential Oxidative and Reductive Pathway Through Chloroethylene Oxide, Environ. Toxicol. Chem., 11, pp. 749-755.

Castro, C. E., R. S. Wade, D. M. Riebeth, E. W. Bartnicki and N. O. Belser, 1992. Biodehalogenation: Rapid Metabolism of Vinyl Chloride by a Soil Pseudomonas sp.: Direct Hydrolysis of a Vinyl C - Cl Bond, Environ. Toxicol. Chem., 11, pp. 757-764.

Davis, J. W. and C. L. Carpenter, 1990. Aerobic Biodegradation of Vinyl Chloride in Groundwater Samples, Appl. Environ. Microbiol., 56, pp. 3878-3880.

DiStefano, T. D., J. M. Gossett and S. H. Zinder, 1991. Reductive Dechlorination of High Concentrations of Tetrachloroethene to Ethene by an Anaerobic Enrichment Culture in the Absence of Methanogenesis, Appl. Environ. Microbiol., 57, pp. 2287-2292.

Dolan, M. E. and P. L. McCarty, 1995. Small-Column Microcosm for Assessing Methane-Stimulated Vinyl Chloride Transformation in Aquifer Samples, Environ. Sci. Technol., 29, pp. 1892-1897.

Fathepure, B. Z. and T. M. Vogel, 1991. Complete Degradation of Polychlorinated Hydrocarbons by a Two-Stage Biofilm Reactor, Appl. Environ. Microbiol., 57, pp. 3418-3422.

Fliermans, C. B., T. J. Phelps, D. Ringelberg, A. T. Mikell and D. C. White, 1988. Mineralization of Trichloroethylene by Heterotrophic Enrichment Cultures, Appl. Environ. Microbiol., 54, pp. 1709-1714.

Fogel, M. M., A. R. Taddeo and S. Fogel, 1986. Biodegradation of Chlorinated Ethenes by a Methane-Utilizing Mixed Culture, Appl. Environ. Microbiol., 51, pp. 720-724.

Freedman, D. L. and J. M. Gossett, 1989. Biological Reductive Dechlorination of Tetrachloroethylene and Trichloroethylene Under Methanogenic Conditions, Appl. Environ. Microbiol., 55, pp. 2144-2151.

Giri, A. K., 1995. Genetic Toxicology of Vinyl Chloride-A Review, Mutat. Res., 339, pp. 1-14.

Goldberg, S. J., B. V. Dawson, P. D. Johnson, H. E. Hoyme and J. B. Ulreich, 1992. Cardiac Teratogenicity of Dichloroethylene in a Chick Model, Pediatr. Res., 32, pp. 23-26.

Harrison, D. E. F., 1973. Studies on the Affinity of Methanol- and Methane-Utilizing Bacteria for Their Carbon Substrates, J. Appl. Bacteriol., 36, pp. 301-308.

Harrison, R. and G. Hathaway, 1990. Case Studies in Environmental Medicine: Vinyl Chloride Toxicity, Clin. Toxicol., 28, pp. 267-286.

Hartmans, S. and J. A. deBont, 1992. Aerobic Vinyl Chloride Metabolism in Mycobacterium aurum L1, Appl. Environ. Microbiol., 58, pp. 1220-1226.

Hauschild, I., A. Schroer, M. Siedersleben and J. Starnick, 1994. The Microbial Growth of Mycobacterium aurum L1 on Vinyl Chloride with Respect to Inhibitory and Limiting Influence of Substrate and Oxygen, Water Sci. Technol., 30, pp. 125-132.

Henson, J. M., M. V. Yates, J. W. Cochran and D. L. Shackleford, 1988. Microbial Removal of Halogenated Methanes, Ethanes and Ethylenes in an Aerobic Soil Exposed to Methane, FEMS Microbial. Ecol., 53, pp. 193-201.

Hurtt, M. E., R. Valentine and L. Alvarez, 1993. Developmental Toxicity of Inhaled Trans-1,2-Dichloroethylene in the Rat, Fundam. Appl. Toxicol., 20, pp. 225-230.

Jacobsen, J. S. and M. Z. Humayun, 1990. Mechanisms of Mutagenesis by Vinyl Chloride Metabolite Chloroacetaldehyde: Effect of Gene-Targeted In Vitro Adduction of M13 DNA on DNA Template Activity In Vivo and In Vitro, Biochem., 29, pp. 496-504.

Kastner, M., 1991. Reductive Dechlorination of Tri- and Tetrachloroethylene Depends on Transition from Aerobic to Anaerobic Conditions. Appl. Environ. Microbiol., 57, pp. 2039-2046.

Klecka, G. M., S. J. Gonsior and D. A. Markham, 1990. Biological Transformations of 1,1,1-Trichloroethane in Subsurface Soils and Groundwater, Environ. Toxicol. Chem., 9, pp. 1437-1451.

Lanzarone, N. A. and P. L. McCarty, 1990. Column Studies on Methanotrophic Degradation of Trichloroethylene and 1,2-Dichloroethane, Groundwater, 28, pp. 910-919.

McCauley, P. T., M. Robinson, F. B. Daniel and G. R. Olson, 1995. The Effects of Subacute and Subchronic Oral Exposure to Cis-1,2-Dichloroethylene in Sprague-Dawley Rats, Drug Chem. Toxicol., 18, pp. 171-184.

Milde, G., M. Nerger and R. Mergler, 1988. Biological Degradation of Volatile Chlorinated Hydrocarbons in Groundwater, Water Sci. Technol., 20. pp. 67-73.

Mochida, K., M. Gomyoda and T. Fujita, 1995. Toxicity of 1,1-Dichloroethane and 1,2-Dichloroethylene Determined Using Cultured Human KB Cells, Bull. Environ. Contam. Toxicol., 55, pp. 316-319.

Nyer, E. K., T. L. Crossman and G. Boettcher, 1996. In Situ Bioremediation, In: Nyer, E. K., S. Fam, D. F. Kidd, F. J. Johns II, P. L. Palmer, G. Boettcher, T. L. Crossman and S. S. Suthersan (eds), In Situ Treatment Technology, Lewis Publishers, Boca Raton, pp. 61-100.

Parsons, F. and G. B. Lage, 1985. Chlorinated Organics in Simulated Groundwater Environments, Res. Technol., 5, pp. 52-59.

Phelps, T. J., K. Malachowsky, R. M. Schram and D. C. White, 1991. Aerobic Mineralization of Vinyl Chloride by a Bacterium of the Order Actinomycetales, Appl. Environ. Microbiol., 57, pp. 1252-1254.

Phelps, T. J., J. J. Niedzielski, K. J. Malachowsky, R. M. Schram, S. E. Herbes and D. C. While, 1991. Biodegradation of Mixed Organic Wastes by Microbial Consortia in Continuous-Recycle Expanded-Bed Bioreactors, Environ. Sci. Technol., 25, pp. 1461-1465.

Roberts, P. V., L. Semprini, G. D. Hopkins, D. Grbic-Galic, P. L. McCarty and M. Reinhard, 1989. In Situ Aquifer Restoration of Chlorinated Aliphatics by Methanotrophic Bacteria, EPA Report EPA/600/2-89/033.

Strandberg, G. W., T. L. Donaldson and L. L. Farr, 1989. Degradation of Trichloroethylene and trans-1,2-Dichloroethylene by a Methanotrophic Consortium in a Fixed-Film, Packed-Bed Bioreactor, Environ. Sci. Technol., 23, pp. 1422-1425.

Tabak, H. H., S. A. Quave, C. I. Mashni and E. F. Barth, 1981. Biodegradability Studies with Organic Priority Pollutant Compounds, J Water Pollut Control Fed., 53, pp. 1503-1518.

U. S. Environmental Protection Agency, 1995. EPA Test Methods for Evaluating Solid Waste: Laboratory Manual Physical/Chemical Methods, SW846, Revision 2.

Vogel, T. M. and P. L. McCarty, 1987. Abiotic and Biotic Transformations of 1,1,1-Trichloroethane Under Methanogenic Conditions, Environ. Sci. Technol., 21, pp. 1208-1213.

Vogel, T. M. and P. L. McCarty, 1985. Biotransformation of Tetrachloroethylene to Trichloroethylene, Dichloroethylene, Vinyl Chloride and Carbon Dioxide Under Methanogenic Conditions, Appl. Environ. Microbiol., 49, pp. 1080-1083.

Westrick, J. J., J. W. Mello and R. F. Thomas, 1984. The Groundwater Supply Survey, J. Am. Water Works Assoc., 76, pp. 52-59.

Wilkinson, T. G. and D. E. F. Harrison, 1973. The Affinity for Methane and Methanol of Mixed Cultures Grown on Methane in Continuous Culture, J. Appl. Bacteriol., 36, pp. 309-313.

Wilson, J. T., J. F. McNabb, B. H. Wilson and M. J. Noonan, 1983. Biotransformation of Selected Organic Pollutants in Groundwater, Devel. Indust. Microbiol., 24, pp. 225-233.

Table 1. Microcosm treatments.
Biodegradation Type Amendment Headspace Atmosphere

Unenhanced none argon, plus a measured amount of

oxygen to approximate oxygen

dissolved in groundwater

Anaerobic amended .08 % (v/v) nitrogen

with methanol methanol

Aerobic oxygen oxygen

Enrichment for oxygen + methane 4% (v/v) methane in oxygen


Table 2. Initial concentrations of the compounds of interest used to spike microcosms.
Compound Concentration (ug/L)

Vinyl chloride 1500

Trans-1,2-DCE 500

Cis-1,2-DCE 500

1,1-DCA 1000

Figure 1. Unenhanced anaerobic biodegradation of a mixture of chlorinated ethenes.

Figure 2. Anaerobic biodegradation of a mixture of chlorinated ethenes amended with methanol.

Figure 3. Aerobic bidegradation of a mixture of chlorinated ethenes.

Figure 4. Biodegradation of a mixture of chlorinated ethenes in an enrichment for methanotrophs.

Figure 5. Unenhanced biodegradation of 1,1-DCA.

Figure 6. Anaerobic biodegradation of 1,1-DCA amended with methanol.

Figure 7. Anaerobic biodegradation of 1,1-DCA amended with methanol.

Figure 8. Anaerobic biodegradation of 1,1-DCA with no amendments added.