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Appendix B The Forensics Potential of Stable Isotope Analysis Typically, elements in nature behave the same chemically and biologically, independent of their isotopic identity. However, there are differences in a num - ber of physical, chemical, and biological processes that produce small variations in the ratios of minor isotopes of elements to their major components. These differences are small, but with good analytical instrumentation they can be detected. One example is the process of evaporation and condensation. Water that has been evaporated from a large source, such as the ocean, tends to be deficient in the heavier isotopes of hydrogen and oxygen, relative to the original source. In the reverse process of condensation the heavier isotopes will con - dense more readily, yielding rain that is enriched in the heavier isotopes. This continual cycle of evaporation and condensation makes the ratio of 2H/1H and 18O/16O lower for water sources that are farther removed from the oceans (the major ultimate water source). The results are also a function of temperature (summer versus winter precipitation) and altitude. An extensive sampling of river waters in the United States was performed to provide a baseline for further studies (Kendall and Coplen, 2001). River water can come from a number of sources—groundwater as well as surface runoff—but even with this complexity there are some clear geographical trends in the resultant maps of the deviation of the 2H and 18O composition of the water when compared to mean ocean water, expressed as δ2H and δ18O in ‰ (parts per thousand). Negative values mean that the water is depleted in the heavier isotopes relative to ocean water. Since this process affects both the 2H/1H and 18O/16O ratios, a plot of δ2H versus δ18O gives a roughly straight line called the meteoric water line (MWL). Samples that fall below this line are usually from arid regions with low humidi - ties. While different regions have different local linear relationships, the overall trend can be used to infer an average value of δ18O from a given δ2H value. The isotopes of carbon are influenced by the biological processes that are used in the synthesis of the organic compounds by an organism. There are very different ratios of 13C/12C for the C3 and C4 photosynthetic pathways. A C3 plant will typically exhibit a δ13C around −25‰, when referenced against a 183

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184 APPENDIX B standard limestone, while a C4 plant will give δ13C values around −9‰. A δ13C of about −16‰ might represent either a mixture of the two sources or a plant that has grown in water (algae or a hydroponically grown plant). Animals will acquire the δ13C signature of their food sources. In the case of bacteria cultured in the laboratory, the signature of the growth medium will be reflected in that of the spores produced. Tests of this expectation for liquid growth medium bear this out (Kreuzer-Martin and Jarman, 2007). In the case of δ2H and δ18O, cultured microorganisms record the isotopic signature of the water in the culture medium as well the nutrients in the medium. Since the anthrax attacks of 2001 there have been extensive studies testing these relationships using the nonpathogenic Bacillus subtilis. On aver- age about 70 percent of the oxygen atoms in spores produced come from the water, while only about 30 percent of the hydrogen atoms come from the water (Kreuzer-Martin et al., 2003, 2005) Results show a strong positive correlation between the δ2H and δ18O content of the culture water and resultant spores for liquid cultures (Kreuzer-Martin and Jarman, 2007; Kreuzer-Martin et al., 2003). Samples grown on an agar medium are also subjected to isotopic fractionation from evaporation of water from the medium. It was found that the influence of evaporation was small for δ2H, but significant for δ18O (Kreuzer-Martin et al., 2005). Exchange of water vapor with the ambient atmosphere could also be important if the agar was prepared in one location and used in another, or anytime there is a different isotopic signature of the water used and that of the surrounding water vapor, as in the case of studies at Lawrence Livermore National Laboratory, where the source of the tap water is largely the Sierra Mountains and the ambient atmosphere can have a significant marine input (Kreuzer-Martin et al., 2005) The forensic value of isotopic measurements on bacteria cultures depends on the individual circumstances. It has the potential to rule out certain combi - nations of water and growth media as well as to provide a distinguishing marker for discrimination between production batches of spores.