Title: Introduction Text: When managing contaminated sites, it is important to understand the source of the contamination as well as the fate and transport of the contaminant. Specifically, all potential degradation pathways should be evaluated at the site. If the contaminant is being degraded, understanding the mechanism by which it is being degraded aids in making site management decisions for monitoring natural attenuation and active remedies at the site. The main method by which contaminant degradation has been monitored is by measuring contaminant concentrations over time. However, this has led to some uncertainty as to whether or not contaminants are actually being degraded or simply being diluted by the groundwater or adsorbed by the soil matrix. Compound specific isotope analysis (CSIA) is a novel approach that provides information on whether the contaminant is being degraded and also the reaction mechanism. It works by tracking the change in the ratio of isotopes in the organic contaminant as degradation takes place. This tool presents an overview of the uses and applications of CSIA. It provides information on collecting samples for CSIA and interpreting data and also presents some case studies where CSIA has been used for various applications at contaminated sites.

Title: Theory (1 of 2) Text: Atoms are made up of protons, neutrons and electrons. An atom usually has the same number of protons and electrons, but may vary in the number of neutrons. When atoms of the same element vary in the number of neutrons, they are referred to as isotopes of each other. Some isotopes of an element are radioactive, meaning that the nucleus is unstable and therefore emits alpha, beta, and/or gamma rays until they are stable. Isotopes that are not radioactive are referred to as stable isotopes. Since the mass of an atom is the sum of the protons and neutrons, isotopes have different masses. The difference in mass is what allows the isotopes to be distinguished from each other and be measured using a mass spectrometer (MS).

Title: Theory (2 of 2) Text: For environmental analysis the isotopes measured are of the following elements H, C, N, O Cl, and S. The isotopes of each element can be used to identify specific compounds or processes that occur in nature as seen in the table. In the study of CSIA there are several terms that must be well understood in order to make appropriate use of the data. These include: Isotope Abundance: Usually represented as a percentage, isotope abundance refers to the natural occurrence of an isotope or the likelihood of finding this isotope in nature. Isotope Effect: This is the difference between the chemical and physical properties of isotopes of an element. These differences are caused by the varying number of neutrons in each isotope and therefore difference in mass observed between isotopes. Isotope Ratio: Since within one compound or molecule more than one isotope of an element is present, the ratio of its occurrence can be measured; that ratio is known as the isotope ratio. For example, both the 18O and 16O isotopes of oxygen are present in water molecules. The isotope ratio of oxygen in water is therefore 1:2000 for 18O:16O. Usually, the heavier isotope appears first in the ratio and is described as the ratio of the heavy to light isotope. Isotope Fractionation: The isotope ratio of an element in a compound changes during physical or chemical processes because, during most reactions, the bonds to the lighter isotope are broken more easily. This leads to an enrichment in the heavier isotope within the remaining parent compound. The change in the isotope ratio is known as isotope fractionation and is denoted as alpha (as described in the Data Evaluation page). Isotope Enrichment: The increase in the abundance of an element's isotope in a compound is known as the isotope enrichment. Isotopic Signature: This is the ratio of stable isotopes of a specific element present in a compound.

Title: Applicability (1 of 4) Text: CSIA is typically applied to groundwater samples for the measurement of the isotope signature of different contaminants of concern. Enrichment of the compound's heavier isotopes can be determined for contaminants including: benzene, toluene,, ethylbenzene and xylene (BTEX) polycyclic aromatic hydrocarbons (PAHs) [such as naphthalene, fluoranthene] chlorinated ethenes [tetrachloroethene (PCE), trichloroethene (TCE), cis-1,2 dichloroethene (cis-DCE), trans-DCE, 1,1-DCE, and vinyl chloride (VC)] chlorinated ethanes chlorinated benzenes (chlorobenzene, trichlorobenzene) methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), tertiary-amyl-ether (TAME), and tertiary butanol (TBA)

Title: Applicability (2 of 4) Text: CSIA of organic contaminants can play a role in the investigation of contaminated sites. The information provided by CSIA analysis includes: 1) Contaminant Degradation: When contaminants degrade, the ratio of stable isotopes changes. For example, during anaerobic microbial degradation, there is enrichment in the heavier 13C isotope in the parent contaminant because the lighter 12C bonds are preferentially broken. The extent of degradation can be determined from the change in the ratio of the stable isotopes. Studies have shown that the magnitude of fractionation tends to decrease as the number of chlorine atoms on the contaminant increases. Understanding the behavior of the contaminant in the soil and groundwater helps to determine if there is a need for active remediation. 2) Mechanism of Degradation: The different mechanisms of contaminant removal include biodegradation (aerobic and anaerobic), abiotic transformation, and/or dissolution. Each mechanism produces a specific isotope ratio that once measured provides sufficient information to conclude the general mechanism of degradation. For example, the d13C isotope enrichment for reductive dechlorination lies in the range 200/00 to 300/00 (units defined in Data Evaluation slide). Microbes prefer the lighter isotope (12C) so enrichment in 13C is seen during biotic degradation. Biotic aerobic dechlorination produces smaller 13C enrichments, usually 30/00 to 50/00. Abiotic transformation of chlorinated ethenes, however, shows a lower enrichment at 20/00 to 40/00, whereas dilution and dissolution produces little to no enrichment, usually around 10/00. Different mechanisms show different isotope fractionation because of changes to different chemical bonds within the contaminants involved. CSIA can therefore be used to identify the degradation pathways. 3) Progress of Monitored Natural Attenuation: CSIA provides a fast and definitive method for monitoring the progress of natural attenuation. CSIA can also replace lengthy microcosm experiments since CSIA analysis of the contaminant can show if the contaminant is being degraded and by what mechanism. Therefore, CSIA has added to the lines of evidence and improved the support for MNA.

Title: Applicability (3 of 4) Text: Additional information provided by CSIA analysis includes: 4) Chemical Fingerprinting: CSIA can be used to determine the origin of a contaminant and therefore the responsible party for a release. The same compound can have different isotope ratios depending on whether the compound occurs naturally or is manmade (anthropogenic). If anthropogenic, the process and raw materials used to manufacture the compound can also determine the isotopic signature of the compound. Therefore, the same compound can have different isotope ratios depending on the manufacturer. Crude oil has different isotope factors depending on depositional environments and source material. Chlorinated ethenes have different isotope signatures depending on manufacturer and also the batch which depends on the material source. 5) Age Dating: CSIA can also tell the age of a contaminant spill. Tritium, 3H, is usually used as the marker to determine the age of the groundwater. This is possible because it is assumed that the H that has been incorporated into groundwater comes from the atmosphere and the H isotope ratio of the atmosphere at specific times is known. Age dating by stable isotope analysis is usually combined with other techniques such as hydraulic conductivity. Due to the reliability of the results of CSIA, it is now accepted as a line of evidence during litigation procedures.

Title: Applicability (4 of 4) Text: CSIA can also be applied to solid samples such as plant matter and soil. Innovative techniques have been created to measure isotope fractionation in solid media including pyrolysis-gas chromatography, combustion, and isotope mass spectrometry. CSIA can be used to detect isotope composition of plants (lignin, pentoses, alkanes), microbial-derived residues in plants (amino acids, phospholipid fatty acids), and soils (black carbon and hydrophobic contaminants like PAHs). CSIA applications for solid media include: 1) quantifying sequestration and turnover of organic compounds, 2) tracing the origin of organic substrates, and 3) studying the evolution of landscape and paleo-climate.

Title: Methods Text: Sample collection and analysis for stable isotopes are very similar to the process followed when analyzing samples for contaminant concentration. It is important to first confirm what specific information is required from CSIA since isotope enrichment data can provide information on mass loss, degradation rates, and degradation mechanisms. The sample collection should have both a temporal and spatial design. Temporal Design. Temporal design is necessary to obtain the required information from the analysis. It is recommended that isotope fractionation is initially taken during a routine sampling event in a few wells for justification of the use of CSIA. This should be followed by more comprehensive analysis every two to three months in more wells. Once the data have been analyzed and the contribution of biodegradation determined, samples can be taken every one to three years to determine the long-term stability and fractionation within the plume. It is very important that the concentration of contaminants are determined before CSIA because this information is necessary to obtain the greatest sensitivity from the instrument by choosing the most ideal method of sample preparation. Spatial Design. Spatially there should be adequate understanding of the location and extent of the source area, direction and velocity of groundwater flow, and the extent of the plume. The samples should be taken in the source zone along the center line of the plume, and throughout the plume area. Vertically one to four levels should be monitored depending on the depth of the contamination.

Title: Sample Collection (1 of 2) Text: Several measures must be taken to ensure proper collection, storage, and analysis of a sample for isotopes. These processes should be clearly outlined in the Sampling and Analysis Plan (SAP). The procedures for well selection, purging, and sample collection are similar to those used for traditional groundwater sampling as outlined below. 1) Well Selection 2) Well Purging 3) Sample Collection Sample preservation techniques are specific to the type of CSIA analysis and are discussed on the next slide.

Title: Well Selection Text: It is important to select appropriate wells from which to collect samples for isotope analysis. Knowing the location and length of the screen is important to understand the composition of the sample collected. Samples collected from long screens may contain water samples across various layers of an aquifer and therefore have an averaged contaminant concentration and a mixture of isotope signatures. Collecting multilayer samples or sampling water from a narrow screen provides water samples with more uniform characteristics to be sampled.

Title: Well Purging Text: It is important to purge wells before a water sample is taken. This is necessary to ensure that the sampled water is from within the aquifer formation. Wells can be purged by removing three well volumes of water before removing water for taking samples. Another and possibly more accurate method is to use low flow sampling to monitor water quality parameters (pH, temp, conductivity) as the water is being purged until the parameter readings have stabilized. This is an indication that the well has been adequately purged and the water being sampled actually originates in the aquifer.

Title: Sample Collection Text: The groundwater can be collected from the wells using peristaltic pumps or down-hole pumps. Water samples should be collected such that the sample is not exposed to the atmosphere. The samples should then be collected in vials and the vials sealed so that there is no headspace. This preserves the redox state of the groundwater and prevents any dissolution of air into the water sample.

Title: Sample Collection (2 of 2) Text: Additional sample preservation measures are often required for CSIA samples. For example, sample preservation is often done by storing the sample at 4oC. However, studies have shown that bacterial activity may continue at this low temperature and, if biodegradation of the contaminant continues in the sample vial, this changes the isotope fractionation of the contaminant. For this reason, it is recommended that 3 to 5 drops of a 1:1 solution of 36% hydrochloric acid to water is added to the sample to lower the pH to less than 2 and to stop bacterial activity. Sodium hydroxide (at 0.1%) can also be used to raise the pH to 10 to preserve the sample. Samples for isotope analysis can be stored from weeks to months. Samples for BTEX analysis can be stored for 4 weeks, while samples for chlorinated ethene analysis can be stored for 4 months.

Title: GC-IRMS Text: To measure the isotope fractionation of a contaminant in a groundwater sample, a gas chromatograph-isotope ratio mass spectrometer (GC-IRMS) is used. This works under the principle that the sample is first separated into compounds. Compounds are then transformed to a gas. Finally, the isotope ratio of the gaseous compound is measured relative to a standard of known isotope ratio.

Title: Data Evaluation Text: GC-IRMS analysis gives the abundance of the stable isotopes of the element of interest. For instance, the results might state the relative abundance of the stable isotopes of carbon (13C and 12C) or hydrogen (2H and 1H). A ratio of the abundance of the heavy to the light isotope is calculated (13C/12C). To ensure accuracy, the ratios are expressed relative to a standard. For carbon the standard is Vienna Peedee Belemnite (VPDB) and for hydrogen the standard is Vienna Standard Mean Ocean Water (VSMOW). The delta value (d) is obtained, which is the comparison of the isotope ratio for the sample to the isotope ratio for the standard. The delta value is very small and therefore multiplied by 1000 to obtain a value units of 0/00. A sample with a delta value of 00/00 has the same isotope ratio as the standard. d13C of -300/00 means that 13C/12C of sample is 300/00 lower than 13C/12C of the lab standard. The bond attached to the lighter isotope breaks faster during a reaction due to the fact that lighter isotopes bind less strongly than heavier isotopes when a reaction proceeds. This causes a shift in the ratio of the heavier to lighter isotope. This isotope fractionation is defined as alpha. Alpha can then be used to determine the enrichment factor, which is represented by epsilon. The more negative the enrichment factor, the more fractionation that is occurring during the reaction. The enrichment factor can also be determined graphically. The delta value can be plotted against the fraction of contaminant remaining. Plots of degradation of contaminant and formation of product are created. The difference between these lines is the enrichment factor.

Title: Costs Text: CSIA analysis is offered through commercial labs as well as academic labs. Prices vary depending on the laboratory, contaminant being tested, and isotopes being analyzed. The cost of CSIA is usually significantly higher than concentration analysis due to the cost of the equipment used to perform the analysis, the number of standards that must be run, the level of training required by the analytical chemist, and the cost of standards. However, the information obtained from CSIA can result in cost avoidance by eliminating the need for a microcosm study or additional site investigations. When deciding to conduct CSIA analysis talk or coordinate with your Navy Quality Assurance Officer to choose the most appropriate tests and samples for your site.

Title: Advantages & Limitations Text: CSIA has several advantages that make it a valuable tool to aid in understanding contaminated sites. It can be used for source correlation and source discrimination. CSIA is extremely valuable with single compound analysis. For more complex mixtures, CSIA is often used as supporting or confirmatory evidence. For natural attenuation, CSIA can replace time-consuming microcosm experiments. CSIA can provide a conservative estimate on extent of contaminant removal. A 2-D approach provides stronger evidence and also provides information on reaction mechanism. CSIA fulfills the need to discriminate non-degradative effects. CSIA has a few limitations that should be considered when interpreting enrichment data. There is currently only a relatively narrow range of source signatures available. Enrichment may not always be evident until a significant amount of contaminant has been removed. CSIA can only measure natural attenuation of smaller molecules (i.e., organic compounds with less than 10 carbon atoms).

Title: Case Studies Text: CSIA has been studied extensively in order to determine the conditions under which it can be used to understand contaminated sites. CSIA measurements have also been taken from field samples and used for routine analysis. The use of CSIA has increased substantially in recent years and will continue to increase as understanding of the usefulness of the analysis continues to grow. Three case studies have been highlighted in this tool that showcase where CSIA was used to investigate natural attenuation, source differentiation, and enhanced degradation.

Title: Dover AFB - Natural Attenuation Text: The groundwater at Dover Air Force Base (AFB) is contaminated with PCE and TCE that was used for aircraft/vehicle maintenance, painting, stripping and welding. The concentration data revealed that biodegradation of PCE and TCE was occurring since the daughter product plumes were contained within the PCE and TCE plumes. There were also elevated chloride, hydrogen, and methane concentrations and depleted oxygen concentrations within the plume supporting the theory that biological reductive dechlorination was taking place within the plume. The objective of this study was to use CSIA to provide additional evidence for natural reductive dechlorination of the chlorinated ethenes PCE and TCE. The study was conducted in wells in the source area as well as upgradient (but still close to the source area), and downgradient of the source area.

Title: Dover AFB - Natural Attenuation Text: The source area wells had the highest concentrations of PCE and TCE and the lowest concentration of daughter products. These source area wells had the least enriched or most negative d13C isotopic signature compared to upgradient and downgradient wells. The downgradient wells had a higher ratio of daughter products and more enrichment in PCE and TCE 13C compared to the source area wells. This higher enrichment is synonymous with biological degradation of PCE and TCE. The upgradient well enrichments were similar to the source area, indicating that little dechlorination was taking place. This is due to the fact that the upgradient wells were very close to the source area. Upgradient wells further away from the source area had enrichments similar to the downgradient wells.

Title: Dover AFB - Natural Attenuation Text: The 13C enrichments seen in the downgradient and upgradient wells agree with the concentration data and suggest that biological reductive dechlorination is occurring at the Dover AFB by natural attenuation.

Title: Perchlorate - Source Identification Text: Perchlorate has been detected in groundwater in 35 states nationwide. Perchlorate is formed naturally in the atmosphere and deposited. It can also be found in the Chilean desert (the soil of which was imported for use as a fertilizer in the US) and as mineral deposits in Southwest US. Anthropogenic sources include fireworks, road flares, perchloric acid and reagents, chlorate (herbicide), or chlorine. Perchlorate contamination may also have originated at military sites where rocket testing was conducted. However, sites have been found where there is little or no military activity, but there is perchlorate contamination that could have originated from other sources.

Title: Perchlorate - Source Identification Text: CSIA can be used to differentiate perchlorate sources. Perchlorate consists of oxygen and chlorine, both of which have stable isotopes and can be analyzed to determine whether perchlorate contamination was natural or manmade.

Title: Perchlorate - Source Identification Text: Groundwater samples were taken from six wells at a rocket testing facility. Two wells were immediately downgradient of the rocket testing facility, while the other four wells (Wells #3, #4, #5 and #6) were on the outskirts of the facility, but within a perchlorate plume.

Title: Perchlorate - Source Identification Text: Both the 18O and 37Cl isotope signatures of the wells downgradient of the rocket testing facility were similar, but significantly different from those on the outskirts. This suggests that the perchlorate contamination immediately downgradient of the testing facility was from the same source. It was also found that the perchlorate in wells outside the facility were from a single source that was distinct in its isotopic signature from the perchlorate contamination from the rocket testing facility.

Title: NWS Charleston - Enhanced Degradation Text: The Naval Weapons Station (NWS) in Charleston, SC is the site of Solid Waste Management Unit (SWMU) 12, which is contaminated with chlorinated VOCs. A permeable reactive barrier and phytoremediation were the chosen remediation strategies. The objective of the study was to use CSIA to aid in understanding the degradation pathways occurring within the plume.

Title: NWS Charleston - Enhanced Degradation Text: Concentrations of cis-1,2-DCE, VC, 1,1-DCA, 1,1,1-TCA continue to decrease in the source area Well 12MW-10S and also in Well 12MW-03S. While PCE concentrations increased in well 12MW-05S, there was a reduction in TCE and cis-DCE. Substantial decreases in VOC concentrations are seen in downgradient well 12MW-05S. 13C values of PCE, TCE and cis-DCE were obtained from 12MW-10S, 12MW-03S and 12MW-05S. The decrease in concentrations was significant in several wells downgradient of the plume, but the isotope enrichment was smaller than would be expected from degradation. This suggests that the predominant attenuation within the plume was not by reductive dechlorination, but by phytoremediation and sorption.

Title: Conclusion Text: CSIA can be conducted on dissolved organic contaminants including chlorinated solvents, fuel oxygenates, and aromatic petroleum hydrocarbons. CSIA can provide both qualitative and quantitative evidence for biotic and abiotic degradation. Although a powerful analytical tool, it should be kept in mind that CSIA is a new approach to monitoring contaminant degradation and it should be used in conjunction with more established methods. CSIA is also a useful tool for source characterization and differentiation.

Title: References Text: Aravena, Ramon and Hunkeler, D. 2007. Application of Environmental Isotopes in Contaminant Hydrogeology. Short Course presented at The Ninth International In Situ and On-site Bioremediation Symposium, Baltimore, Maryland, May 7-10. Glaser, Bruno. 2005. Compound-specific Stable-isotope (d13C) Analysis in Soil Science. J. Plant Nutr. Soil Sci. 168, 633-648. Hunkeler, Daniel, Rainer U. Meckenstock, Barbara Sherwood Lollar, Torsten C. Schmidt, and John T. Wilson. 2008. EPA 600-R-08-148. December. Lollar, B. Sherwood, G.F. Slater, B. Sleep, M. Witt, G.M. Klecka, M. Harkness and J. Spivack. 2001. "Stable Carbon Isotope Evidence for Intrinsic Bioremediation of Tetrachloroethene and Trichloroethene at Area 6, Dover Air Force Base,"Environ. Sci. Technol. 35(2), pp. 261-269. (Graphics reprinted with permission from Copyright (2009) American Chemical Society. http://pubs.acs.org/journal/esthag) Philp, Paul and Tomasz, Kuder. 2008. Compound-Specific Isotope Analysis Today, Advances in Isotopic Characterization of Contaminants. School of Geology and Geophysics, Oklahoma University, presented at RITS Fall 2008. Plumb, Russell H., Jr. 2004. Fingerprint Analysis of Contaminant Data: A Forensic Tool for Evaluating Environmental Contamination. EPA/600/5-04/054 May. Vlassopoulos, Dimitri. 2008. Application of Stable Isotopes to Site Characterization CVOC Sources, Commingled Plumes, and Groundwater-Surface Water Interactions. Presented at U.S. EPA Ground Water Forum Portland, Oregon July 10.

Title: Contact Text: For more information about this tool, please contact: NAVFAC ESC T2 Point of Contact PRTH_NFESCT2@navy.mil