View source for Compound Specific Isotope Analysis (CSIA)


Compound specific isotope analysis (CSIA) usually refers to measurement of the [[wikipedia: Stable isotope ratio | 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 (H<sub>2</sub>, CO<sub>2</sub>, CO, N<sub>2</sub>, O<sub>2</sub>, SO<sub>2</sub>, CH<sub>3</sub>Cl) 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.
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'''Related Article(s)''': 
*[[Molecular Biological Tools - MBTs]]


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


'''Key Resource(s)''': 
*[[Media:Hunkeler-2008-A_Guide.pdf|A Consensus Guide For Assessing Biodegradation and Source Identification Of Organic Contaminants In Groundwater Using Compound Specific Stable Isotope Analysis (CSIA)]]<ref name = "Hunkeler2008">Hunkeler, D., Meckenstock, R. U., Sherwood Lollar, B., Schmidt, T. C. and Wilson, J. T., 2008. A Guide for Assessing Biodegradation and Source Identification of Organic Groundwater Contaminants Using Compound Specific Isotope Analysis (CSIA). U.S. Environmental Protection Agency, Washington, D.C., EPA/600/R-08/148. [[Media:Hunkeler-2008-A_Guide.pdf|Report pdf]]</ref>

==Introduction==
Compound Specific Isotope Analysis (CSIA) refers to measurement of the [[wikipedia: Stable isotope ratio | 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 [[wikipedia: Source rock | source rock]] identification and [[wikipedia: Hydrocarbon exploration | hydrocarbon exploration]], and remains a foundation of the oil and gas industry. In the 1980s, [[wikipedia: John M. Hayes (scientist) | John M. Hayes]] at Indiana University Bloomington and collaborators<ref>Merritt, D.A., Brand, W.A. and Hayes, J.M., 1994. Isotope-ratio-monitoring gas chromatography-mass spectrometry: methods for isotopic calibration. Organic Geochemistry, 21(6-7), 573-583. [https://doi.org/10.1016/0146-6380(94)90003-5 doi: 10.1016/0146-6380(94)90003-5]</ref><ref>Brand, W.A., 1996. High precision isotope ratio monitoring techniques in mass spectrometry. Journal of Mass Spectrometry, 31(3), 225-235. [https://doi.org/10.1002/(sici)1096-9888(199603)31:3<225::aid-jms319>3.0.co;2-l doi: 10.1002/(SICI)1096-9888(199603)31:3<225::AID-JMS319>3.0.CO;2-L]</ref> introduced the era of continuous flow compound specific mass spectrometry by interfacing a gas chromatograph via a sample preparatory oxidation system to a stable [[wikipedia:Isotope-ratio mass spectrometry | 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 stable isotope ratios for compounds of environmental concern at various spatiotemporal scales in [[wikipedia: Environmental chemistry | environmental chemistry]], [[wikipedia: Biogeochemistry | biogeochemistry]], and contaminant [[wikipedia: Hydrogeology | 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 [[wikipedia:Isotope | isotopes]], <sup>12</sup>C and <sup>13</sup>C. Typically occurring under natural conditions in the ratio of 99:1, the small relative differences in the ratio of <sup>13</sup>C/<sup>12</sup>C for a given compound provide a wealth of information relevant to the investigation and remediation of contaminated sites and the environment<ref name = "Hunkeler2008"/>. The measured <sup>13</sup>C/<sup>12</sup>C ratio is normalized with respect to international isotopic standard reference materials and expressed in delta notation (e.g. δ<sup>13</sup>C), in units of permil (parts per thousand or [[wikipedia: Per mille | 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 [https://www.iaea.org/ International Atomic Energy Agency], the [https://www.nist.gov/ National Institute of Standards and Technology] and the [https://www.usgs.gov/ 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<ref name = "Hunkeler2008"/>.

==Applications to Environmental Remediation and Restoration – Forensics==
Both naturally sourced contaminants (e.g., petroleum hydrocarbons) and man-made industrial organic contaminants (e.g., [[wikipedia: Organochloride | organochlorides]], [[wikipedia: Chlorofluorocarbon | chlorofluorocarbons (CFCs)]], [[wikipedia: Nitro compound | nitroaromatics]], and gasoline additives such as [[wikipedia: Methyl tert-butyl ether | 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 [[wikipedia: Forensic science | forensic science]] tool<ref>Mancini, S.A., Lacrampe-Couloume, G. and Sherwood Lollar, B., 2008. Source differentiation for benzene and chlorobenzene groundwater contamination: A field application of stable carbon and hydrogen isotope analyses. Environmental Forensics, 9(2-3), 177-186. [http://dx.doi.org/10.1080/15275920802119086 doi: 10.1080/15275920802119086]</ref>. 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 <ref name = "Hunkeler2008"/>. 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 [[wikipedia: Isotopes of hydrogen | hydrogen]], [[wikipedia: Isotopes of nitrogen | nitrogen]] and/or [[wikipedia: Isotopes of chlorine | chlorine]] isotope ratios)<ref name = "Hunkeler2008"/><ref name= "Hunkeler2001">Hunkeler, D., Andersen, N., Aravena, R., Bernasconi, S.M. and Butler, B.J., 2001. Hydrogen and carbon isotope fractionation during aerobic biodegradation of benzene. Environmental Science & Technology, 35(17), 3462-3467. [https://doi.org/10.1021/es0105111 doi: 10.1021/es0105111]</ref><ref>Mancini, S.A., Ulrich, A.C., Lacrampe-Couloume, G., Sleep, B., Edwards, E.A. and Sherwood Lollar, B., 2003. Carbon and hydrogen isotopic fractionation during anaerobic biodegradation of benzene. Applied and Environmental Microbiology, 69(1), 191-198. [https://doi.org/10.1128/aem.69.1.191-198.2003 doi: 10.1128/AEM.69.1.191-198.2003]</ref><ref>Shouakar-Stash, O., Frape, S.K. and Drimmie, R.J., 2003. Stable hydrogen, carbon and chlorine isotope measurements of selected chlorinated organic solvents. Journal of Contaminant Hydrology, 60(3), 211-228. [https://doi.org/10.1016/s0169-7722(02)00085-2 doi: 10.1016/S0169-7722(02)00085-2]</ref><ref name= "Kuder2005">Kuder, T., Wilson, J.T., Kaiser, P., Kolhatkar, R., Philp, P. and Allen, J., 2005. Enrichment of stable carbon and hydrogen isotopes during anaerobic biodegradation of MTBE: microcosm and field evidence. Environmental Science & Technology, 39(1), 213-220. [https://doi.org/10.1021/es040420e doi: 10.1021/es040420e]</ref><ref name= "Zwank2005">Zwank, L., Berg, M., Elsner, M., Schmidt, T.C., Schwarzenbach, R.P. and Haderlein, S.B., 2005. New evaluation scheme for two-dimensional isotope analysis to decipher biodegradation processes: Application to groundwater contamination by MTBE. Environmental Science & Technology, 39(4), 1018-1029. [https://doi.org/10.1021/es049650j doi: 10.1021/es049650j]</ref><ref>Sessions, A.L., 2006. Isotope‐ratio detection for gas chromatography. Journal of Separation Science, 29(12), 1946-1961. [https://doi.org/10.1002/jssc.200600002 doi: 10.1002/jssc.200600002]</ref><ref>Hartenbach, A., Hofstetter, T. B., Berg, M., Bolotin, J., Schwarzenbach, R. P., 2006. Using nitrogen isotope fractionation to assess abiotic reduction of nitroaromatic compounds. Environmental Science & Technology, 40(24), 7710-7719. [http://pubs.acs.org/doi/abs/10.1021/es061074z doi: 10.1021/es061074z]</ref><ref name= "Penning2007">Penning, H. and Elsner, M., 2007. Intramolecular carbon and nitrogen isotope analysis by quantitative dry fragmentation of the phenylurea herbicide isoproturon in a combined injector/capillary reactor prior to GC separation. Analytical Chemistry, 79(21), 8399-8405. [https://doi.org/10.1021/ac071420a doi 10.1021/ac071420a]</ref><ref>Meyer, A.H., Penning, H., Lowag, H. and Elsner, M., 2008. Precise and accurate compound specific carbon and nitrogen isotope analysis of atrazine: critical role of combustion oven conditions. Environmental Science & Technology, 42(21), 7757-7763. [https://doi.org/10.1021/es800534h doi: 10.1021/es800534h]</ref>.

==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  [[wikipedia: Isotope fractionation | isotope fractionation]] and is the key to CSIA providing insight and information on transformation pathways<ref name= "Faure2004">Faure, G. and Mensing, T.M., 2005. Isotopes: principles and applications. John Wiley & Sons Inc.</ref>. 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<ref name = "Hunkeler2008"/>. This results from the [[wikipedia: Kinetic isotope effect | kinetic isotope effect]] and the fact that bonds involving a heavy stable isotope (e.g. <sup>13</sup>C-<sup>12</sup>C bond) have a lower [[wikipedia: Zero-point energy | zero-point energy]] and a larger [[wikipedia: Activation energy | activation energy]] than bonds containing exclusively light isotopes (e.g. <sup>12</sup>C-<sup>12</sup>C 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 <ref name= "Faure2004"/>.

==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<ref>Meckenstock, R.U., Morasch, B., Warthmann, R., Schink, B., Annweiler, E., Michaelis, W. and Richnow, H.H., 1999. 13C/12C isotope fractionation of aromatic hydrocarbons during microbial degradation. Environmental Microbiology, 1(5), 409-414. [https://doi.org/10.1046/j.1462-2920.1999.00050.x doi: 10.1046/j.1462-2920.1999.00050.x]</ref><ref>Hunkeler, D., Aravena, R. and Butler, B.J., 1999. Monitoring microbial dechlorination of tetrachloroethene (PCE) in groundwater using compound-specific stable carbon isotope ratios: microcosm and field studies. Environmental Science & Technology, 33(16), 2733-2738. [https://doi.org/10.1021/es981282u  doi: 10.1021/es981282u]</ref><ref>Sherwood Lollar, B., Slater, G.F., Ahad, J., Sleep, B., Spivack, J., Brennan, M. and MacKenzie, P., 1999. Contrasting carbon isotope fractionation during biodegradation of trichloroethylene and toluene: Implications for intrinsic bioremediation. Organic Geochemistry, 30(8), 813-820. [https://doi.org/10.1016/s0146-6380(99)00064-9 doi: 10.1016/S0146-6380(99)00064-9]</ref>. 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<ref name = "Hunkeler2008"/>. 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<ref>Mariotti, A., Germon, J.C., Hubert, P., Kaiser, P., Letolle, R., Tardieux, A. and Tardieux, P., 1981. Experimental determination of nitrogen kinetic isotope fractionation: some principles; illustration for the denitrification and nitrification processes. Plant and Soil, 62(3), 413-430. [https://doi.org/10.1007/bf02374138 doi: 10.1007/BF02374138]</ref>. 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:'''&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<big>''R<sub>t</sub> = R<sub>0</sub> f<sup> (α -1)</sup>''</big>   

where ''R<sub>t</sub>'' is the stable isotope ratio (<sup>13</sup>C/<sup>12</sup>C) of the compound at time ''t'', ''R<sub>0</sub>'' 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:'''&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<big>''α'' = (1000 + ''δ''<sup>13</sup>C<sub>''a''</sub>)/(1000 + ''δ''<sup>13</sup>C<sub>''b''</sub>)</big> 

Subscripts ''a'' and ''b'' may represent a compound at time zero (''t''<sub>0</sub>) 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 3<ref name = "Hunkeler2008"/>:

::'''Equation 3:'''&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<big>''f'' = ''e''<sup>(''δ''<sup>13</sup>C<sub>groundwater</sub> - ''δ''<sup>13</sup>C<sub>source</sub>) '''/''' ''ε''</big> 

where ''δ''<sup>13</sup>C<sub>groundwater</sub> is the measure of the isotope ratio in the organic contaminant in the sample of groundwater, ''δ''<sup>13</sup>C<sub>source</sub> 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:'''&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<big>''ε'' = (''α'' -1) * 1000</big>

==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<ref name= "Morrill2005">Morrill, P.L., Lacrampe-Couloume, G., Slater, G.F., Sleep, B.E., Edwards, E.A., McMaster, M.L., Major, D.W. and Lollar, B.S., 2005. Quantifying chlorinated ethene degradation during reductive dechlorination at Kelly AFB using stable carbon isotopes. Journal of Contaminant Hydrology, 76(3), pp.279-293. [https://doi.org/10.1016/j.jconhyd.2004.11.002 doi: 10.1016/j.jconhyd.2004.11.002]</ref>. 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<ref>Wiedemeier, T.H., Wilson, J.T., Kampbell, D.H., Miller, R.N. and Hansen, J.E., 1995. Technical Protocol for Implementing Intrinsic Remediation with Long-Term Monitoring for Natural Attenuation of Fuel Contamination Dissolved in Groundwater. U.S. Air Force Center for Environmental Excellence, Technology Transfer Division, Brooks Air Force Base, San Antonio, Texas.</ref><ref>Wiedemeier, T.H.,  Swanson, M.A., Moutoux, D.E., Gordon, E.K., Wilson, J.T., Wilson, B.H., Kampbell, D.H., Haas, P.E., Hansen, J.E., and Chapelle, F.H. 1998. Technical protocol for evaluating natural attenuation of chlorinated solvents in ground water.  EPA-600-R-98-128. [[Media:Wiedemeier-1998-Technical_Protocol_for_Evaluating_Natuaral_Attenuation.pdf|Report pdf]]</ref>. 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<ref>Sherwood Lollar, B., Slater, G.F., Sleep, B., Witt, M., Klecka, G.M., Harkness, M. and Spivack, J., 2001. Stable carbon isotope evidence for intrinsic bioremediation of tetrachloroethene and trichloroethene at area 6, Dover Air Force Base. Environmental Science & Technology, 35(2), 261-269. [https://doi.org/10.1021/es001227x doi: 10.1021/es001227x]</ref><ref name= "Morrill2005"/><ref>McKelvie, J.R., Mackay, D.M., de Sieyes, N.R., Lacrampe-Couloume, G. and Lollar, B.S., 2007. Quantifying MTBE biodegradation in the Vandenberg Air Force Base ethanol release study using stable carbon isotopes. Journal of Contaminant Hydrology, 94(3), 157-165. [https://doi.org/10.1016/j.jconhyd.2007.05.008 doi: 10.1016/j.jconhyd.2007.05.008]</ref>.

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<ref>Hunkeler, D. and Aravena, R., 2000. Evidence of Substantial Carbon Isotope Fractionation among Substrate, Inorganic Carbon, and Biomass during Aerobic Mineralization of 1, 2-Dichloroethane by Xanthobacter autotrophicus. Applied and Environmental Microbiology, 66(11), 4870-4876. [https://doi.org/10.1128/aem.66.11.4870-4876.2000 doi: 10.1128/AEM.66.11.4870-4876.2000]</ref><ref>Hirschorn, S.K., Grostern, A., Lacrampe-Couloume, G., Edwards, E.A., MacKinnon, L., Repta, C., Major, D.W. and Lollar, B.S., 2007. Quantification of biotransformation of chlorinated hydrocarbons in a biostimulation study: Added value via stable carbon isotope analysis. Journal of Contaminant Hydrology, 94(3), 249-260. [https://doi.org/10.1016/j.jconhyd.2007.07.001 doi: 10.1016/j.jconhyd.2007.07.001]</ref>. In other examples, CSIA has been a critical tool in deciphering the biodegradation potential and remediation mechanisms for benzene<ref name= "Hunkeler2001"/><ref>Mancini, S.A., Lacrampe-Couloume, G., Jonker, H., van Breukelen, B.M., Groen, J., Volkering, F., Sherwood Lollar, B., 2002. Hydrogen isotope enrichment: An indicator of biodegradation at a petroleum hydrocarbon contaminated field site. Environmental Science & Technology, 36(11), 2464-2470. [http://pubs.acs.org/doi/abs/10.1021/es011253a doi: 10.1021/es011253a]</ref><ref>Mancini, S.A., Devine, C.E., Elsner, M., Nandi, M.E., Ulrich, A.C., Edwards, E.A. and Sherwood Lollar, B., 2008. Isotopic evidence suggests different initial reaction mechanisms for anaerobic benzene biodegradation. Environmental Science & Technology, 42(22), 8290-8296. [https://doi.org/10.1021/es801107g doi: 10.1021/es801107g]</ref><ref>Fischer, A., Gehre, M., Breitfeld, J., Richnow, H.-H., 2009. Carbon and hydrogen isotope fractionation of benzene during biodegradation under sulphate-reducing conditions: A laboratory to field site approach. Rapid Communications in Mass Spectrometry, 236, 2439-2447. [https://doi.org/10.1002/rcm.4049 doi:10.1002/rcm.4049]</ref>, methyl tert-butyl ether (MTBE)<ref name= "Zwank2005"/><ref>McKelvie, J.R., Hyman, M.R., Elsner, M., Smith, C., Aslett, D.M., Lacrampe-Couloume, G. and Sherwood Lollar, B., 2009. Isotopic fractionation of methyl tert-butyl ether suggests different initial reaction mechanisms during aerobic biodegradation. Environmental Science & Technology, 43(8), 2793-2799. [https://doi.org/10.1021/es803307y doi: 10.1021/es803307y]</ref><ref>Elsner, M., McKelvie, J., Lacrampe Couloume, G. and Sherwood Lollar, B., 2007. Insight into methyl tert-butyl ether (MTBE) stable isotope fractionation from abiotic reference experiments. Environmental Science & Technology, 41(16), 5693-5700. [https://doi.org/10.1021/es070531o doi: 10.1021/es070531o]</ref><ref name= "Kuder2005"/> 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<ref>Elsner, M., Chartrand, M., VanStone, N., Lacrampe Couloume, G. and Sherwood Lollar, B., 2008. Identifying abiotic chlorinated ethene degradation: characteristic isotope patterns in reaction products with nanoscale zero-valent iron. Environmental Science & Technology, 42(16), 5963-5970. [https://doi.org/10.1021/es8001986  doi: 10.1021/es8001986]</ref><ref>Elsner, M., Couloume, G.L., Mancini, S., Burns, L. and Lollar, B.S., 2010. Carbon isotope analysis to evaluate nanoscale Fe (O) treatment at a chlorohydrocarbon contaminated site. Groundwater Monitoring & Remediation, 30(3), 79-95. [https://doi.org/10.1111/j.1745-6592.2010.01294.x doi: 10.1111/j.1745-6592.2010.01294.x]</ref>. 

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<ref>Morasch, B., Richnow, H.H., Schink, B. and Meckenstock, R.U., 2001. Stable hydrogen and carbon isotope fractionation during microbial toluene degradation: mechanistic and environmental aspects. Applied and Environmental Microbiology, 67(10), 4842-4849. [https://doi.org/10.1128/aem.67.10.4842-4849.2001 doi: 10.1128/AEM.67.10.4842-4849.2001]</ref>. In such cases the development of models to determine intrinsic versus apparent kinetic isotope effects<ref>Elsner, M., Zwank, L., Hunkeler, D. and Schwarzenbach, R.P., 2005. A new concept linking observable stable isotope fractionation to transformation pathways of organic pollutants. Environmental Science & Technology, 39(18), 6896-6916. [https://doi.org/10.1021/es0504587 doi: 10.1021/es0504587]</ref>; use of multi-isotope analysis (2D or 3D)<ref name= "Penning2007"/> or novel techniques related to position specific isotope analysis targeted to specific individual atoms on the compound<ref>McKelvie, J.R., Elsner, M., Simpson, A.J., Sherwood Lollar, B., Simpson, M.J., 2010. Quantitative site-specific 2H NMR investigation of MTBE: Potential for investigating contaminant sources and fate. Environmental Science & Technology, 44(3), 1062-1068. [http://pubs.acs.org/doi/abs/10.1021/es9030276 doi: 10.1021/es9030276]</ref><ref>Julien, M., Parinet, J., Nun, P., Bayle, K., Höhener, P., Robins, R.J. and Remaud, G.S., 2015. Fractionation in position-specific isotope composition during vaporization of environmental pollutants measured with isotope ratio monitoring by <sup>13</sup>C nuclear magnetic resonance spectrometry. Environmental Pollution, 205, 299-306. [https://doi.org/10.1016/j.envpol.2015.05.047 doi: 10.1016/j.envpol.2015.05.047]</ref><ref>Gilbert, A., Yamada, K., Suda, K., Ueno, Y. and Yoshida, N., 2016. Measurement of position-specific <sup>13</sup>C isotopic composition of propane at the nanomole level. Geochimica et Cosmochimica Acta, 177, 205-216. [http://dx.doi.org/10.1016/j.gca.2016.01.017 doi: 10.1016/j.gca.2016.01.017]</ref> 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<ref>Nijenhuis, I., Andert, J., Beck, K., Kastner, M., Diekert, G., Richnow, H-H., 2005. Stable isotope fractionation of tetrachloroethene during reductive dechlorination by sulfurospirillum multivorans and desulfitobacterium sp. Strain PCE-S and abiotic reactions with cyanocobalamin. Applied and Environmental Microbiology, 71(7), 3413-3419. [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1169044/ doi: 10.1128/AEM.71.7.3413-3419.2005]</ref> or the efficiency of the enzymes involved in biodegradation<ref>Mancini, S.A., Hirschorn, S.K., Elsner, M., Lacrampe-Couloume, G., Sleep, B.E., Edwards, E.A. and Sherwood Lollar, B., 2006. Effects of trace element concentration on enzyme controlled stable isotope fractionation during aerobic biodegradation of toluene. Environmental Science & Technology, 40(24), 7675-7681. [https://doi.org/10.1021/es061363n doi: 10.1021/es061363n]</ref><ref>Lollar, B.S., Hirschorn, S., Mundle, S.O., Grostern, A., Edwards, E.A. and Lacrampe-Couloume, G., 2010. Insights into enzyme kinetics of chloroethane biodegradation using compound specific stable isotopes. Environmental Science & Technology, 44(19), 7498-7503. [http://dx.doi.org/10.1021/es101330r doi: 10.1021/es101330r]</ref>.

==Summary==
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<ref>Meckenstock, R.U., Morasch, B., Griebler, C. and Richnow, H.H., 2004. Stable isotope fractionation analysis as a tool to monitor biodegradation in contaminated acquifers. Journal of Contaminant Hydrology, 75(3), 215-255. [https://doi.org/10.1016/j.jconhyd.2004.06.003 doi: 10.1016/j.jconhyd.2004.06.003]</ref><ref name = "Hunkeler2008"/>.

==References==

<references/>

==See Also==
*[https://www.serdp-estcp.org/Program-Areas/Environmental-Restoration/Contaminated-Groundwater/Emerging-Issues/ER-200509  Validation of Chlorine and Oxygen Isotope Ratio Analysis to Differentiate Perchlorate Sources and to Document Perchlorate Biodegradation]
*[https://www.serdp-estcp.org/Program-Areas/Environmental-Restoration/Contaminated-Groundwater/ER-201029 Integrated Stable Isotope-Reactive Transport Model Approach for Assessment of Chlorinated Solvent Degradation]
*[https://www.coursera.org/learn/natural-attenuation-of-groundwater-contaminants/lecture/C0EN7/types-of-isotope-analysis-and-sampling-techniques-lab-analysis   Online Lecture Course - Stable Isotopes]