Compound Specific Isotope Analysis (CSIA)
Compound specific isotope analysis (CSIA) refers to measurement of the isotopic signatures (typically, the stable isotopes of carbon, hydrogen, oxygen, nitrogen or sulfur) of individual compounds from a complex environmental mixture. The approach provides information about source differentiation, a quantitative means to delineate reaction pathways, including biodegradation, and an additional line of evidence for remediation monitoring of sites contaminated with hydrocarbons.
CONTRIBUTOR(S): Dr. Barbara Sherwood Lollar, F.R.S.C.
- A Consensus Guide For Assessing Biodegradation and Source Identification Of Organic Contaminants In Groundwater Using Compound Specific Stable Isotope Analysis (CSIA)
Compound Specific Isotope Analysis (CSIA) refers to measurement of the isotopic signatures (typically, carbon, hydrogen, oxygen, nitrogen or sulfur in an environmental context) of individual compounds from a complex environmental mixture of components. The approach was developed most extensively 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. By lowering detection limits by up to 5 orders of magnitude and reducing analytical and sample preparation time from hours to minutes, continuous flow techniques allowed CSIA to become widely applied. CSIA provides the ability to measure stable isotope ratios for compounds of environmental concern at various spatiotemporal scales in environmental chemistry, biogeochemistry, and contaminant hydrogeology.
Carbon Isotope CSIA
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 (called the stable isotope ratio) 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 standards and expressed in delta notation (e.g. δ13C), in units of permil (parts per thousand or per mille). Isotopic standards have been administered centrally since the 1950s through the International Atomic Energy Agency, and in the U.S. through the National Institute of Standards and Technology. This ensures that all stable isotope laboratories worldwide are cross-calibrated to the same standards ensuring global consistency of results within both public and private laboratories.
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), gasoline additives such as methyl tert-butyl ether (MTBE)) can have different δ13C values due to differences in mechanisms and source materials used in their synthesis. This provides the basis for using CSIA as a forensic science tool. Both different contamination sources and different spills from the same source may potentially have distinct CSIA signatures that can be applied to attribute responsibility in a mixed contaminant plume, such as in a situation where there is off-site migration and impact. In most cases, knowledge of the initial spill material is not possible, but the approach is not dependent on that precondition. Differentiating different potential source areas at a site relative to each other can be achieved by comparing different areas of the site in the context of the geologic and hydrogeologic conceptual models to test source zone apportionment. CSIA does not provide a silver bullet, as there can be significant overlap in carbon isotope signatures, but successful applications have taken advantage of the additional certainty afforded by coupling carbon isotope variations with additional constraints provided by dual isotope (2D) or triple isotope (3D) signatures (usually hydrogen isotope signatures and/or chlorine isotope signatures). Forensic applications of CSIA can include nitrogen isotope signatures (e.g. nitroanilines and other N-containing compounds or other elements relevant to the contaminant of concern.
Quantifying and Monitoring Remediation Processes
Many processes that act on contaminants once they enter the environment can affect the relative abundance of isotopes in the compound of interest and hence the 13C/12C ratio. This effect is called isotope fractionation and is the key to CSIA providing insight and information on those processes. 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 processes in which bonds are broken. This results from the wikipedia: Kinetic isotope effect | kinetic isotope effect]] and the fact that bonds containing a single heavy stable isotope (e.g. 13C) have a lower zero-point energy and corresponding larger activation energy than bonds containing exclusively light isotopes (e.g. 12C). Effectively this means bonds containing a heavy isotope are harder to break, and the rate of transformation then of compounds containing exclusively light isotopes is faster than the rate of transformation of compounds containing a heavy isotope.
CSIA Signals of Transformation and Remediation
The net outcome of fractionation is that a contaminant that has been undergoing degradation can provide a dramatic signal of transformation, as its isotopic signature can have a higher 13C/12C ratio than before transformation. The obvious corollary is that the products of degradation will be preferentially enriched in the light isotopes (lower 13C/12C ratios) than 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, and chlorine as well.
Laboratory experiments have shown that not only does fractionation during transformation provide a strong signal of degradation, but also that signal is highly reproducible. For many organic contaminants of interest, the relationship between the change in carbon isotope signature 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 carbon isotope signatures can be quantitatively related to a specific degree of transformation (e.g. fraction or percentage of remaining contaminant) by the equation:
Equation 1: Rt = R0 f(α-1)
where Rt is the stable isotope ratio (13C/12C) of the compound at time t, R0 is the initial isotope value of the compound and f is the fraction of remaining contaminant expressed as f = 0. The stable isotope fractionation factor is the factor alpha (α) where α = (1000 + δ13Ca)/(1000+ δ13Cb). 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 a compound in a downgradient well for instance.
Equation 1 can be rearranged to produce Equation 2 (see Hunkeler et al. 2008 for details):
where δ13Cgroundwater is the measure of the isotope ratio in the organic contaminant in the sample of groundwater, where δ13Csource is the isotopic ratio in the un-fractionated organic contaminant before biodegradation has occurred, and epsilon (ε), the stable isotope enrichment factor, is defined as ε = (α-1) * 1000.
Implications for Remediation
The quantitative relationships controlling carbon isotope fractionation during transformation means that CSIA not only provides a signal of whether transformation is taking place, but can provide 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 contaminant 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 transport and dispersal processes are largely neutral with respect to carbon isotope signals, a carbon isotope enrichment signal in the contaminants of concern provides a direct line of evidence that transformation is occurring and as outlined above, a second independent quantification of transformation rates that can provide constraints on conventional approaches to derive remediation rates and timelines.
CSIA provides additional value to environmental investigation and remediation – the ability to pinpoint which of a variety of possible degradation mechanisms may be dominating at a contaminated site – because the degree of fractionation is reaction specific. 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 by 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 two 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 transformation of a compound occurs via different pathways and mechanisms, CSIA can contribute to differentiating between the relative contributions of chemical versus biological transformation.
Not all transformation processes result in significant fractionation. Fractionation factors can be small to non-existent simply due to the decreased significance of fractionation related to one carbon in a molecule with many carbon atoms (naphthalene paper), or due to the highly stable nature of, for instance, carbon atoms in an aromatic ring structure. In such cases the development of models to determine intrinsic versus apparent kinetic isotope effects; use of multi-isotope analysis (2D or 3D) or novel techniques related to position specific isotope analysis targeted to specific individual atoms on the compound can be applied. The presence of additional rate-limiting steps in the transformation reaction can suppress the observed fractionation in ways which may complicate the above quantification of transformation governing Equation 1, yet yield other important information for instance about transport effects or the efficiency of the enzymes involved in biodegradation.
Applications of CSIA are dependent on background information about the degree of fractionation associated with a specific chemical reaction or biodegradation pathway. While fractionation can be calculated ab initio (from the beginning), 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.
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