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Compound specific isotope analysis (CSIA) usually refers to the measurement of the isotope ratios (typically, the stable isotope ratios of carbon, hydrogen, oxygen, nitrogen, sulfur or chlorine) of individual volatile and semi-volatile compounds extracted from complex environmental mixtures. These compounds (or their derivatization products) are generally separable by conventional gas or liquid chromatography and amenable to high-temperature oxidation or pyrolysis to produce simple gases (H₂, CO₂, CO, N₂, O₂, SO₂, CH₃Cl) suitable for analysis by a gas-source isotope-ratio mass spectrometer. The CSIA methodology provides a quantitative means to differentiate reaction pathways for abiotic and biotic degradation, including different pathways of biodegradation, and may serve as a basis for identification of distinct pollutant sources. CSIA can also provide a powerful line of evidence for monitored natural attenuation of sites contaminated with a wide variety of common organic pollutants including petroleum hydrocarbons, fuel oxygenates, BTEX, chlorinated solvents, and nitroaromatics.

Related Article(s):

• Molecular Biological Tools - MBTs

CONTRIBUTOR(S): Dr. Barbara Sherwood Lollar, F.R.S.C.

Key Resource(s):

• A Consensus Guide For Assessing Biodegradation and Source Identification Of Organic Contaminants In Groundwater Using Compound Specific Stable Isotope Analysis (CSIA)
• Stable Isotope Fractionation to Investigate Natural Transformation Mechanisms of Organic Contaminants: Principles, Prospects and Limitations

Introduction

Compound Specific Isotope Analysis (CSIA) refers to the measurement of the isotope ratios (typically carbon, hydrogen, oxygen, nitrogen, sulfur or chlorine) of individual organic compounds extracted from complex environmental mixtures. The approach was developed initially during the post-WWII era for application to source rock identification and hydrocarbon exploration, and remains a foundation of the oil and gas industry. In the 1980s, John M. Hayes at Indiana University Bloomington and collaborators introduced the era of continuous flow compound-specific mass spectrometry by interfacing a gas chromatograph via a sample preparatory oxidation system to a stable isotope ratio mass spectrometry system, which lowered detection limits by up to 5 orders of magnitude and reduced analytical and sample preparation time. This continuous flow technique allowed CSIA to become widely applied by providing the ability, with instrumentation that became commercially available around 1990, to measure and compare stable isotope ratios for compounds of environmental concern at various spatiotemporal scales in environmental chemistry, biogeochemistry, and contaminant hydrogeology.

The CSIA Method

The first isotope ratio to which CSIA was widely applied was that of carbon’s stable isotopes. The element carbon has two stable isotopes, ¹²C and ¹³C. Typically occurring under natural conditions in the ratio of 99:1, the small relative differences in the ratio of ¹³C/¹²C for a given compound provide a wealth of information relevant to the investigation and remediation of contaminated sites and the environment. The measured ¹³C/¹²C ratio is normalized with respect to international isotopic standard reference materials and expressed in delta notation (e.g. δ¹³C), in units of permil (parts per thousand or per mille). Isotopic standard reference materials for stable isotope ratio measurements of carbon and other light elements (H, N, O, S, Cl and others) have been administered since the 1950s through several sources including the International Atomic Energy Agency, the National Institute of Standards and Technology and the U.S. Geological Survey. All stable isotope laboratories worldwide must report their isotopic data normalized to the appropriate standard reference materials to ensure global consistency and inter-comparability of results.

Applications to Environmental Remediation and Restoration – Forensics

Both naturally sourced contaminants (e.g., petroleum hydrocarbons) and man-made industrial organic contaminants (e.g., organochlorides, chlorofluorocarbons (CFCs), nitroaromatics, and gasoline additives such as methyl tert-butyl ether (MTBE)) can have distinct isotopic compositions due to differences in the raw materials and industrial synthesis processes used in their manufacture. This provides the basis for using CSIA as a forensic science tool. Different contamination sources or different spills from the same source may have distinct isotopic compositions that can be used to apportion responsibility in a mixed contaminant plume, such as in a situation where there is off-site migration and impact. In many cases, knowledge of the isotopic composition of the initial spill material is not available, but the forensic utility of the isotope ratios does not depend on that precondition. Distinguishing between potential source areas at a site can be done by comparing isotopic data from different areas of the site in the context of the geologic and hydrogeologic conceptual models to test source zone apportionment . Although there may be significant overlap in isotopic compositions, successful forensic applications have taken advantage of the additional constraints afforded by coupling carbon isotope ratio measurements with other isotopes (usually hydrogen, nitrogen and/or chlorine isotope ratios).

Quantifying and Monitoring Remediation Processes

Abiotic and biotic degradation reactions that transform contaminant compounds in the environment generally affect their isotopic compositions in systematic ways. This overall process is called isotope fractionation and is the key to CSIA providing insight and information on transformation pathways. While both degradative (e.g. chemical or biological transformation of contaminant to degradation products) and non-degradative processes (e.g. phase changes such as volatilization, sorption, diffusion) have the potential to result in carbon isotope fractionation, to date the largest fractionation signals are related to degradative processes in which bonds are broken. This results from the kinetic isotope effect and the fact that bonds involving a heavy stable isotope (e.g. ¹³C-¹²C bond) have a lower zero-point energy and a larger activation energy than bonds containing exclusively light isotopes (e.g. ¹²C-¹²C bond). Effectively this means that the rate of transformation of molecules containing exclusively light isotopes at the reactive site is faster than the rate of transformation of compounds containing a heavy isotope at the reactive site .

CSIA Signals of Transformation and Remediation

The net outcome of isotope fractionation is that a contaminant that has been undergoing degradation can provide a significant signal of transformation, as its isotopic composition generally (but not always) increases to heavier values as a function of reaction progress. The obvious corollary is that the products of degradation will generally be preferentially enriched in the lighter isotopes relative to the instantaneous isotopic composition of the parent compound from which they are derived. This principle holds for both chemical transformation and biologically mediated transformation reactions, and the principles described above apply to other elements such as hydrogen, nitrogen, oxygen, sulfur, and chlorine as well. The largest isotope effects have always been found at the reactive sites where bonds are broken or formed.

Laboratory experiments have shown that not only does fractionation during transformation provide a strong signal of degradation, but also that the signal is highly reproducible. For many organic contaminants of interest, the relationship between the change in isotopic composition and the degree of degradation is governed by a quantitative relationship – the Rayleigh equation. Specifically, for a given compound and degradation pathway or mechanism, the measured difference in isotopic composition can be quantitatively related to the extent of transformation (e.g. fraction or percentage of contaminant remaining) by the equation:

Equation 1:      R_t = R₀ f ^{(α -1)}

where R_t is the stable isotope ratio (¹³C/¹²C) of the compound at time t, R₀ is the initial isotope ratio of the compound and f is the fraction of contaminant remaining where f = 1 at t = 0 and decreases to f = 0 when the reactant compound is fully transformed to products. The stable isotope fractionation factor (α) is defined as:

Equation 2:      α = (1000 + δ¹³C_a)/(1000 + δ¹³C_b)

where subscripts a and b may represent a compound at time zero (t₀) and at a later point (t) in a reaction; or a compound in a source zone, versus the compound in a downgradient well for instance.

Equation 1 can be rearranged to produce Equation 3:

Equation 3:      f = e^{(δ13C_groundwater - δ13C_source) / ε}

where δ¹³C_groundwater is the measure of the isotope ratio in the organic contaminant in the sample of groundwater, δ¹³C_source is the isotopic ratio in the un-fractionated organic contaminant before biodegradation has occurred, and epsilon (ε) is the stable isotope enrichment factor, defined as:

Equation 4:      ε = (α -1) * 1000

Implications for Remediation

The quantitative relationships controlling isotope fractionation during transformation enable CSIA to provide a diagnostic signal that transformation is taking place, as well as a quantitative measure of the extent of transformation independent of conventional metrics based on changes in concentration. In some cases, due to signal sensitivity, changes in stable carbon isotope fractionation can be identified in advance of definitive reduction in contaminant concentrations, or before appearance of daughter products, providing an “early warning system” for confirmation of remediation. This is particularly advantageous for field studies since changes in contaminant concentration result not only from transformation processes, but from physical transport and dispersal. For this reason, decreasing concentrations of contaminants alone are insufficient evidence that a site is undergoing transformation towards clean-up goals. In contrast, as physical transport and dispersal processes without concomitant degradation produce negligible changes in isotopic composition, any significant isotope effects measured in the contaminants of concern provide a direct line of evidence that transformation is occurring, and also yields independent quantification of the rate of the transformation.

CSIA provides additional value to environmental investigation and remediation in that the degree of fractionation is reaction specific, giving researchers the ability to pinpoint which of a variety of possible degradation mechanisms may be dominating at a contaminated site. A specific example of this is 1,2-dichloroethane, an industrial chemical used in PVC production, production of furniture, upholstery and automobile parts and a common environmental contaminant of concern. Microbial biodegradation of this compound in the environment is common, but different organisms degrade the compound via different pathways (e.g. involving a C-Cl bond cleavage, or a C-H bond cleavage). As a result, CSIA can be used to positively identify which of the biodegradation pathways is operative at a site – information that can be critical to optimizing a remediation strategy. In other examples, CSIA has been a critical tool in deciphering the biodegradation potential and remediation mechanisms for benzene, methyl tert-butyl ether (MTBE) and other priority pollutants. In related applications, where abiotic and biotic transformations of a compound occur via different pathways and mechanisms, CSIA can differentiate between the relative contributions of chemical versus biological transformations.

Not all transformation processes necessarily result in measurable isotopic fractionation. Fractionation factors can be small simply due to isotope dilution, e.g., where a C isotope effect occurs at a single reactive site in a molecule that has many carbon atoms. In such cases there is need for the development of models to disambiguate intrinsic versus apparent kinetic isotope effects , for use of multi-isotope analysis, and for the use of novel techniques such as ¹³C NMR that can allow position-specific isotope analysis. The presence of additional rate-limiting steps in the transformation reaction can complicate the measurement of isotope fractionation in ways that may obscure the extent of transformation, yet may also yield other important information about transport effects or the efficiency of the enzymes involved in biodegradation.

Summary

Applications of CSIA are dependent on background information about the isotope fractionation factors associated with specific chemical reactions or biodegradation pathways. While fractionation can be calculated ab initio (from the beginning) by using molecular modeling methods, fractionation factors are typically empirically derived from laboratory experiments and other approaches. Recent guidance documents and review papers provide an essential resource with database compilations of this knowledge to date.

See Also

• Validation of Chlorine and Oxygen Isotope Ratio Analysis to Differentiate Perchlorate Sources and to Document Perchlorate Biodegradation
• Integrated Stable Isotope-Reactive Transport Model Approach for Assessment of Chlorinated Solvent Degradation
• Online Lecture Course - Stable Isotopes