Dense Nonaqueous Phase Liquids (DNAPLs)

Isotope Analysis

A Guide for Assessing Biodegradation and Source Identification of Organic Ground Water Contaminants Using Compound Specific Isotope Analysis (CSIA)
Hunkeler, D., R.U. Meckenstock, B. Sherwood-Lollar, T.C. Schmidt, and J.T. Wilson.
EPA 600-R-08-148, 82 pp, 2008

When organic contaminants are degraded in the environment, the ratio of stable isotopes often will change, and the extent of degradation can be recognized and predicted from the change in the ratio of stable isotopes. Recent advances in analytical chemistry make it possible to perform compound-specific isotope analysis (CSIA) on dissolved organic contaminants, such as chlorinated solvents, aromatic petroleum hydrocarbons, and fuel oxygenates, at concentrations in water that are near their regulatory standards. Because CSIA is a new approach, no widely accepted standards have been established for accuracy, precision, and sensitivity, nor approaches to document accuracy, precision, sensitivity and representativeness. This text provides general recommendations on good practice for sampling, measurement, data evaluation and interpretation in CSIA. This guide is intended for managers of hazardous waste sites who must design sampling plans that will include CSIA and specify data quality objectives for CSIA analyses, for analytical chemists who must carry out the analyses, and for staff of regulatory agencies who must review the sampling plans, data quality objectives, and the data provided from the analyses.

Integrated Stable Isotope-Reactive Transport Model Approach for Assessment of Chlorinated Solvent Degradation: User's Guide
Kuder , T., P. Philp, B. van Breukelen, H. Thouement, M. Vanderford, and C. Newell.
ESTCP Project ER-201029, 245 pp, 2014

The objective of this document is to help site managers apply a reactive transport modeling approach for improved CSIA data interpretation and to estimate more accurate attenuation processes for chlorinated solvents. Quantification of destructive and transport processes and how they contribute to plume size and longevity may help extend MNA remedies to sites previously unable to use them. The report contains a description of standard CSIA laboratory methods, simple data interpretation, and a step-by-step guide to downloading and using software developed as part of this project. The approach presented has benefits over traditional data interpretation, i.e., (1) improvement of a conceptual site model by identification and quantification of prevalent attenuation pathways and identification of secondary inputs from DNAPL dissolution or nondegradative sinks, such as sorption or volatilization, diffusion, or dispersion; (2) a more accurate assessment of degradation of the parent contaminant; (3) and quantitative assessment of the net degradation/accumulation of the dechlorination intermediates. Additional information: Final report and model input files

Abstracts of Journal Articles

Application of Compound Specific 13C Isotope Investigations of Chlorinated Hydrocarbons in Contaminated Groundwaters
Osenbrueck, K.; M. Heidinger; A. Voropaev; S. Ertl; L. Eichinger, Hydroisotop GmbH, Schweitenkirchen, Germany. Proceedings of the Study of Environmental Change Using Isotope Techniques. International Atomic Energy Agency, Vienna (Austria). IAEA-CSP-13/P, 476-477, 2002 [Paper No: IAEA-CN-80/36P, OSTI: DE20328892]

The determination of the stable isotope ratio C-13/C-12 of chlorinated hydrocarbons in ground water may offer a promising tool to investigate the origin and the biodegradation characteristics of complex contamination situations with overlapping contaminant plumes. The application of the method is based on characteristic isotope fingerprints, which differ in chlorinated solvents. The isotope fingerprint derives from different production pathways and is not influenced by transport or by retardation processes in the underground. Due to the fact that two different contaminants can be distinguished by isotope ratios, an improved distinction of spatially and temporally different contamination plumes is possible. During biologically mediated degradation processes such as denitrification or sulfate reduction, a shift of the isotope ratios between the precursor and the product often can be observed. The isotope fractionation is due to a preferential reaction of the bonds formed by the lighter isotopes and leads to a progressive enrichment of the heavy isotopes in the precursor, while the product becomes depleted in the heavy isotopes. Biological degradation of highly chlorinated hydrocarbons results from co-metabolic dechlorination. Isotope ratios can be used to assess and quantify the degradation of the organic compounds at field sites. This application holds great interest for remediation strategies, including monitored natural attenuation. The author examined the results of 21 field studies where compound-specific C-13 isotope ratios had been applied. Isotope fractionation processes of chlorinated hydrocarbons due to biodegradation were not seen in all cases, and the author concludes that the occurrence and the degree of significant isotope fractionation of chlorinated hydrocarbons is still an open question whose answer may vary with the activity and type of the microbiological species, availability of cosubstrates, and hydrochemical and hydrogeological conditions. NOTE: The URL links to the entire proceedings, which can take quite some time to load.

Assessing in Situ Mineralization of Recalcitrant Organic Compounds in Vadose Zone Sediments Using DELTA13C and 14C measurements
Kirtland, Brian C. and C. Marjorie Aeliona (Univ. of South Carolina, Columbia); Peter A. Stone (SC Department of Health and Environmental Control, Columbia). Journal of Contaminant Hydrology, Vol 76 Nos 1-2, p 1-18, Jan 2005

Predictions of biodegradation rates are needed for measuring the biodegradation of recalcitrant organic compounds in situ and thereby assessing intrinsic biodegradation, or natural attenuation. Traditional techniques measuring O2, CO2, or chemical concentrations (in situ respiration, metabolite and soil air monitoring) may not be sufficiently sensitive to estimate biodegradation rates for chlorinated hydrocarbons (CHC). Researchers combined isotopic measurements (14C and DELTA13C of CO2 and DELTA13C of CHCs) with traditional methods to assess in situ biodegradation of perchloroethene (PCE) and its metabolites in PCE-contaminated vadose zone sediments. This paper describes the conduct of the study over a period of 56 days. The researchers found 14C-CO2 analyses to be a sensitive measurement for quantifying in situ recalcitrant organic compound mineralization in vadose zone sediments.

Constraining Rates of Biodegradation of Chlorinated Ethenes at Steep Concentration Gradients Using Stable Carbon Isotopes
Morrill, P.L. (Carnegie Inst. of Washington); D.J. Seepersad, G. Lacrampe-Couloume, E.A. Edwards, and B.E. Sleep (Univ. of Toronto, Toronto, ON, Canada); M.L. McMaster and D.W. Major (Geosyntec Consultants, Guelph, ON Canada); B. Sherwood Lollar (Univ. of Toronto). Eos Trans. AGU, Vol 86 No 52, Fall Meet. Suppl., Abstract B31A-0983, 2005

Enhanced biodegradation of dense non-aqueous phase liquid (DNAPL) sources of tetrachloroethene (PCE) was monitored in a 2-D model aquifer and in a Dover Air Force Base pilot field study using the stable carbon isotope values of PCE and its biodegradation products to quantify first-order biodegradation rate constants during a study that assessed the potential for biological enhancement of PCE DNAPL dissolution. A maximum isotope fractionation of 2.3 per mil was observed in the dissolved PCE downgradient, while close to the source zone the carbon isotopic signature of the dissolved PCE remained largely unchanged, due to the continuing dissolution of unfractionated PCE DNAPL. Significant carbon isotopic fractionation was observed adjacent to and/or downstream from the source in the degradation products trichloroethene, 1,2-dichloroethene, and vinyl chloride. Close to the source zone, confirmation of PCE degradation is based primarily on the appearance of the lesser chlorinated ethene degradation products and isotopic signatures of those products consistent with biodegradation. This trend was observed on a small scale in the model aquifer and similar trends were observed in the field at a larger scale.

Degradation under Different Redox Conditions in the Unsaturated Zone
Crowley, O.A., G. Boshoff, K. Redeker, and R.M. Kalin, Queens University, Belfast. Geophysical Research Abstracts, Vol. 5, 12099, 2003

Chlorinated solvents, in particular trichloroethene (TCE), are very mobile in the subsurface and often exist as volatilized constituents within the vadose zone due to their low water solubility. Limited information is available regarding the fate of TCE in unsaturated conditions. Compound-specific stable carbon isotope measurements for TCE at contaminated sites are useful indicators for the assessment of natural attenuation. Sorption, volatilization, chemical reactions, dispersion, dilution, and biodegradation processes all contribute to natural attenuation and in turn will have a resultant isotope signature. Contaminated site soil and ground water were used in this investigation to set up representative unsaturated microcosms. The microcosms were used to assess the isotopic signature as a result of the natural attenuation processes, under nitrate, sulfate, and methanogenic reducing conditions. The research determined the degradation efficiencies at each of the identified redox conditions. Accurate evaluation of in situ bioremediation of TCE is dependant on accurate identification of degradation occurring. The investigations of carbon isotope signatures as indicators of degradation may provide a novel environmental monitoring tool, allowing for the identification of redox-specific degradation at contaminated sites.

Development and Evaluation of Stable Isotope and Fluorescent Labeling and Detection Methodologies for Tracking Injected Bacteria During In Situ Bioremediation
Fuller, M. E. (Shaw Environmental Inc., Lawrenceville, NJ); T.C. Onstott (Princeton Univ., NJ). Report No: DOE/ER/62712-1, 11 pp, Nov 2003

This report summarizes the results of a research project conducted to develop new methods to label bacterial cells so that they could be tracked and enumerated as they move in the subsurface after they are introduced into the groundwater (i.e., during bioaugmentation). Labeling methods based on stable isotopes of carbon (13C) and vital fluorescent stains were developed. Both approaches proved successful for effectively labeling bacterial cells. Several methods for enumeration of fluorescently-labeled cells were developed and validated, including near-real time microplate spectrofluorometry that could be performed in the field; however, the development of a novel enumeration method for the 13C-enriched cells, chemical reaction interface/mass spectrometry (CRIMS), was not successful due to difficulties with the proposed instrumentation. Both labeling methodologies were successfully evaluated and validated during laboratory- and field-scale bacterial transport experiments. The methods developed during this research should be useful for future bacterial transport work, as well as other microbial ecology research in a variety of environments.

Distinguishing Abiotic and Biotic Reductive Dechlorination of Tetrachloroethylene by Stable Carbon Isotope Fractionation
Butler, E.C. (Univ. of Oklahoma, Norman); X. Liang; Y. Dong; T. Kuder; L. Krumholz; R.P. Philp. Partners in Environmental Technology Technical Symposium & Workshop, 29 November - 1 December 2005, Washington, DC: Abstracts. Poster presentation, p G-19, 2005

Stable carbon isotope fractionation is a promising tool for monitored natural attenuation (MNA) assessments to evaluate the fate of chlorinated organic compounds. Researchers assessed whether stable carbon isotope fractionation could be used to distinguish abiotic and biotic reductive dechlorination of tetrachloroethylene (PCE). Abiotic microcosms were set up using different iron minerals, including iron sulfide, green rust, pyrite, and magnetite simultaneously with biotic microcosms containing two isolated pure cultures (Dehalospirillum multivorans and Desulfuromonas michiganensis strain BB1) and one bacteria consortium (BioDechlor INOCULUM, or BDI [TM])). Significant fractionation of carbon isotopes was observed in the abiotic reductive dechlorination of PCE by iron sulfide. The study results show that stable carbon isotope analysis can distinguish abiotic (beta-elimination) and biotic (hydrogenolysis) reductive dechlorination of PCE, at least under certain conditions, and may be useful as a tool in MNA assessments. This project (ER-1368) receives SERDP funding.

Evidence of Biodegradation at a DNAPL Contaminated Fractured Bedrock Field Site Using Stable Carbon Isotopes
Chartrand, M.M.G., P.L. Morrill, and G. Lacrampe-Couloume (Univ. of Toronto); K.T. Finneran, P. Chang, and P. Zeeb (Geosyntec Inc.); B. Sherwood Lollar (Univ. of Toronto). Proceedings: 2004 U.S. EPA/NGWA Fractured Rock Conference: State of the Science and Measuring Success in Remediation, Portland, Maine, 13-15 September 2004. p 385-387 [abstract only], 2004

Stable carbon isotope analysis of chlorinated ethenes and ethene was performed at a site where the TCE DNAPL source and dissolved plume are located in fractured bedrock. Previous attempts to biostimulate the pilot test area (PTA) at the site resulted in the accumulation of cis-1,2-dichloroethene (cis-DCE). Since there was no appreciable production of vinyl chloride (VC) or ethene (ETH), there was no evidence for further reductive dechlorination beyond cis-DCE. Subsequently, the PTA was bioaugmented with KB-1, a natural microbial consortium shown in laboratory experiments to completely reduce TCE to non-toxic ETH. While the appearance of breakdown products (VC, ETH) suggested that bioaugmentation was successful to some extent, due to the continuous source of TCE from the DNAPL in the fractured bedrock and variability in the hydraulic gradient, concentration profiles of TCE and degradation products cis-DCE, VC and ETH were unable to unambiguously verify bioaugmentation. Compound specific carbon isotope analysis of the chlorinated ethenes was able to confirm biodegradation. At any given sampling well, the isotopic signatures of the breakdown products were more depleted than that of their parent compound. The isotopic signatures of cis-DCE and VC became increasingly enriched over the four sampling events consistent with the effects of biodegradation. The isotopic profile of TCE remained relatively consistent due to the continuous input of undegraded TCE from DNAPL dissolution. Stable carbon isotope measurements can provide an important line of evidence for biodegradation in complex hydrogeologic systems.

Laboratory and In Situ Evaluation of Enzyme Activity-Dependent Fluorescent Probes Specific for Chlorinated Solvent Degrading Bacteria
Watwood, M.E., Idaho State Univ., Pocatello. NTIS: DE2007-895979, 7 pp, Apr 2006

Several bacterial groups commonly found in soil and ground water are capable of oxidative, cometabolic degradation of trichloroethene (TCE). This research effort has developed enzyme activity-dependent probes for most of the toluene oxygenase pathways that cometabolize TCE. In conjunction with Frank Roberto and Amber Miller of Idaho National Engineering and Environmental Laboratory (INEEL), an enzyme activity-dependent probe for soluble methane monooxygenase has been developed that also cometabolizes TCE. These probes are surrogate substrates that are transformed by the enzyme to yield fluorescent and/or colored products. They give a clear, quantifiable signal only when the enzyme of interest is actively functioning. Their pathway specificities have been demonstrated, and response of the probes has been correlated with TCE degradation in microcosm studies. PCR primer sets specific for the genes encoding the initial hydrolase for each of the toluene oxidizing pathways have also been developed. The primers were located within the open reading frame of the gene so that they could be used with extracted DNA to detect the presence of the gene and with extracted mRNA to detect active transcription. Additional approaches to analyzing bacteria containing the toluene oxygenase pathways include fluorescent in situ hybridization (FISH) and 5-cyano,2,3-ditotyl tetrazolium chloride (CTC) reduction. FISH of whole cells using 16s rRNA oligonucleotide probes is a powerful technique for evaluating the identity, abundance, and relative activity of microbial cells. CTC is a monotetrazolium redox dye that produces a fluorescent formazan crystal when biologically or chemically reduced. CTC provides a direct visualization of actively respiring bacterial cells. These techniques allow examination of relationships between microbial community metabolic activity, single-cell metabolic activity, and specific enzyme activity in laboratory test cultures. The status of microbes containing the desired degradative enzymes has been assessed by applying the entire suite of these techniques. The techniques were applied to ground-water samples from the TCE-contaminated aquifer at the Test Area North (TAN) site at INEEL. Cells were harvested from the samples by sterile filtration of the ground water. The results from two full field trials of the enzyme activity probes at the TAN site, along with molecular analysis and other approaches, indicate substantial levels of toluene oxygenase and soluble methane monooxygenase activity. This type of information can play a pivotal role in verification of monitored natural attenuation, as well as active bioremediation monitoring.

Monitoring Oxidation of Chlorinated Ethenes by Permanganate in Groundwater Using Stable Isotopes: Laboratory and Field Studies
Hunkeler, D. (Univ. of Waterloo, Waterloo, ON, Canada), R. Aravena, B. L. Parker, J. A. Cherry, and X. Diao. Environmental Science & Technology, Vol 37 (4), 798 -804, 2003

Permanganate injection is increasingly applied for in situ destruction of chlorinated ethenes in groundwater. This laboratory and field study demonstrates the roles that carbon isotope analysis can play in the assessment of oxidation of trichloroethene (TCE) by permanganate. In laboratory experiments a strong carbon isotope fractionation was observed during oxidation of TCE with similar isotopic enrichment factors (-25.1 to -26.8 ") for initial KMnO4 concentrations between 67 and 1250 mg/L. At the field site, a single permanganate injection episode was conducted in a sandy aquifer contaminated with TCE as dense nonaqueous liquid (DNAPL). After injection, enriched 13C values of up to +204% and elevated Cl- concentrations were observed at distances of up to 4 m from the injection point. Farther away, the Cl- increased without any change in 13C of TCE suggesting that Cl- was not produced locally but migrated to the sampling point. Except for the closest sampling location to the injection point, the 13C rebounded to the initial 13C again, likely due to dissolution of DNAPL. Isotope mass balance calculations made it possible to identify zones where TCE oxidation continued to occur during the rebound phase. The study indicates that 13C values can be used to assess the dynamics between TCE oxidation and dissolution and to locate zones of oxidation of chlorinated ethenes that cannot be identified from the Cl- distribution alone.

Quantifying Chlorinated Ethene Degradation During Reductive Dechlorination at Kelly AFB Using Stable Carbon Isotopes
Morrill, P.L. (Univ. of Toronto, Toronto ON, Canada); G. Lacrampe-Couloume; G.F. Slater; B.E. Sleep; E.A. Edwards; M.L. McMaster; D.W. Major; B.S. Lollar. Journal of Contaminant Hydrology, Vol 76 Nos 3-4, p 279-293, Feb 2005

Stable isotope analysis of chlorinated ethene contaminants, primarily cis-1,2-dichloroethene (cDCE), was carried out during a bioaugmentation pilot test at Kelly Air Force Base, San Antonio, TX. Following bioaugmentation with a mixed microbial enrichment culture, KB-1(TM), perchloroethene (PCE), trichloroethene (TCE) and cDCE concentrations declined, while vinyl chloride (VC) concentrations increased and subsequently decreased as ethene became the dominant transformation product. Shifts in carbon isotopic values up to 2.7, 6.4, 10.9 and 10.6 per thousand were observed for PCE, TCE, cDCE and VC, respectively, after bioaugmentation, consistent with the effects of biodegradation. Seventy-two days post-bioaugmentation, a rising trend of VC concentrations and the first appearance of ethene were indicative of biodegradation, but the most compelling evidence of biodegradation was the substantial carbon isotope enrichment (2.0 to 5.0 per thousand) in 13C-cDCE.

Stable Isotope Evidence for Biodegradation of Chlorinated Ethenes at a Fractured Bedrock Site
Chartrand, M.M.G. (Univ. of Toronto, Toronto, ON, Canada); P.L. Morrill; G. Lacrampe-Couloume; B.S. Lollar. Environmental Science and Technology, Vol 39 No 13, p 4848-4856, 1 July 2005

At a fractured bedrock site contaminated with TCE, a DNAPL, investigators performed stable carbon isotope analysis of chlorinated ethenes and ethene to examine the progress of a bioaugmentation treatment. A pilot treatment area was bioaugmented with a culture of KB-1™, a natural microbial consortium known to completely reduce TCE to nontoxic ETH. Ongoing dissolution of TCE from DNAPL in the fractured bedrock and variable hydraulic gradients interfered with the concentration profiles of dissolved TCE and its degradation products cis-DCE, VC, and ETH, and the investigators could not convincingly confirm biodegradation of the chlorinated ethenes. Isotopic analysis of cis-DCE and VC offered the investigators assurance that biodegradation was occurring in the pilot treatment area, as the isotope values of cis-DCE and VC became significantly more enriched in C-13 over time. Quantification of the extent of biodegradation in the pilot treatment area using the Rayleigh model indicated that the decrease in cis-DCE and VC concentrations could be attributed to the effects of biodegradation during this time period, while within each well, the isotope profile of TCE remained relatively constant from continuous input of undegraded TCE due to DNAPL dissolution.

Stable Isotope Fractionation Analysis as a Tool to Monitor Biodegradation in Contaminated Aquifers
Meckenstock, Rainer U. (Inst. of Groundwater Ecology, GSF- National Research Center for Environment and Health, Neuherberg, Germany); Barbara Morasch; Christian Griebler; Hans H. Richnow. Journal of Contaminant Hydrology, Vol 75 Nos 3-4, p 215-255, Dec 2004

In a review of recent laboratory and field studies in which applied stable isotope analysis (SIFA) was applied to assess biodegradation of contaminants, stable isotope enrichment factors were noted that varied from no fractionation to moderate fractionation to larger fractionations. The different isotope enrichment factors can be related to the respective biochemical reactions. The authors discuss under what circumstances SIFA can be used for a qualitative or even a quantitative assessment of biodegradation in the environment, with a focus on the aerobic and anaerobic degradation of aromatic hydrocarbons and chlorinated solvents as the major contaminants of groundwater.

13C/12C Isotope Fractionation of Aromatic Hydrocarbons to Characterise Microbial In Situ Degradation
Richnow, H.H.; E. Annweiler; W. Michaelis; R.U. Meckenstock, Inst. fur Biogeochemie und Meereschemie Univ. Hamburg. European Geophysical Society (EGS) XXV General Assembly, 25-29 April 2000, Nice, France. Geophysical Research Abstracts, CD-ROM, Volume 2, 2000

The concentration and the isotopic composition of contaminants in a polluted aquifer was used to characterize in situ biodegradation by calculating the contribution of microbial degradation to the total contaminant removal using laboratory-derived compound specific C-13/ C-12 isotope fractionation factors. The researchers evaluated a contaminant plume 800 meters long and estimated that more than 98% of toluene and o-xylene were removed by in situ biodegradation. Isotope fractionation of contaminants can be used to evaluate in situ biodegradation in the context of intrinsic bioremediation and natural attenuation studies.

Use of Stable Isotope Analysis to Assess Biodegradation of Volatile Organic Compounds in the Unsaturated Subsurface
Bouchard, Daniel, Ph.D. dissertation, University of Neuchatel, Switzerland. 141 pp, 2007

The general aim of the project is to evaluate the feasibility of using compound-specific isotope analysis to assess biodegradation of petroleum hydrocarbons in the unsaturated zone. The first objective was to quantify the isotope enrichment factors during biodegradation of several volatile organic compounds (VOCs) commonly found on petroleum hydrocarbon- contaminated sites. These microcosm experiments were carried out with unsaturated soil sediments under aerobic conditions. The results confirmed the possibility to monitor an isotope enrichment in the remaining VOCs in the air phase. The magnitude of isotope enrichment factors was small for carbon and large for hydrogen. A column experiment was carried out to investigate the possible role played by diffusion in the isotope fractionation of migrating VOC vapors. During an initial period after source emplacement, the heavy isotope 13C became increasingly depleted with distance from the source. Later, the isotope profile leveled out, and for some compounds, an enrichment of 13C with distance was observed, indicating biodegradation. Some compounds, however, were also affected by an enrichment of 13C at the source as the compounds were depleting from the source. The source isotope evolution of these compounds followed a Rayleigh trend with the ratio of diffusion coefficients for molecules with a different isotopic composition as isotope fractionation factor. A controlled field experiment provided a unique opportunity to evaluate if similar isotope trends also occur at the field scale. The experiment consisted of burying an artificial fuel source in the unsaturated zone of a sandy aquifer. Isotope evolution similar to the lab study was observed, with an initial depletion of 13C with distance followed by a leveling out of the isotope profile and enrichment of 13C. To evaluate the relative contribution of diffusion and biodegradation on isotope shifts in more detail, the concentration and isotope ratio evolution was simulated using a modified numerical code (MOFAT). The study demonstrates that assessment of biodegradation in the unsaturated zone is more complex than in the saturated zone because the diffusion process influences isotope ratios as well. Under steady-state conditions and as long as the compound does not deplete significantly, isotope data can provide qualitative and possible quantitative information on the degree of biodegradation. While the substantial shift of isotope ratios during source depletion complicates the identification of biodegradation trends, it may help to identify the final stage of the evolution of a VOC source.