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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
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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.
