Forensic techniques: These papers were recommended to us and were links from our module

Stable Isotope Measurement for Wildlife Forensics

Dr W Meier-Augenstein, Queen's University Belfast

Background Stable isotope analysis is based on measuring natural variation in the chemical elements present in biological samples. Many common elements, such as hydrogen, oxygen and carbon occur in different forms, known as isotopes. The presence or relative abundance of different isotopes allows isotope profiles to be generated for individual samples, which can then be compared to each other, or to reference data. For any single element, the proportion of one isotope to another is known as the isotope ratio. Isotope ratios vary in the environment due to a number of physical, geological and biological factors. These factors may correspond to different geographic localities and therefore isotope ratios can be used to infer the geographical origin of a sample. In addition to measuring variation between isotope ratios, it is also possible to measure differences within an isotope. For example, in the units of stable isotope measurement, the minor hydrogen isotope (2H) can assume about 700 different values while the minor carbon isotope (13C) can assume about 110 different values. Analysing organic material such as animal hair and bird feathers for their precise isotopic composition with regard to hydrogen (H), carbon (C), nitrogen (N) oxygen (O) and sulphur (S) will theoretically produce a very specific profile.Isotopes are measured using mass spectrometry techniques, allowing quantitative comparisons to be made between test samples and reference samples. With individual samples, it is possible to differentiate between them on the basis of exclusion, if they do not have matching isotope profiles. If sufficient isotope data is available from multiple samples, it is possible to identify which region or population a sample belongs to, through the use of multivariate statistical analysis.

Stable isotopes as forensic tools:
Stable isotope analysis was first applied as a forensic technique for the analysis of foods, in order to identify illegal labelling and illegal trade. Analytical methods traditionally applied in forensic science laboratories establish a degree of identity between one substance and another by identifying its constituent elements, functional groups, and by elucidating its chemical structure. For example, when comparing two samples of sugar, all of the these measures would correspond and it would be concluded that they are chemically indistinguishable, as they are indeed both sugar. However, although the two samples of sugar are chemically indistinguishable they may not be the same isotopically if they do not share the same origin or are derived from a different source. The two main sources of sugar are sugar cane and sugar beet. With the help of stable isotope fingerprinting it is perfectly straight forward to determine if a sugar sample is either cane sugar or beet sugar. In addition, it is even possible to say where approximately in the world the sugar cane or sugar beet was grown and cultivated. This type of technique has been widely applied in the food industry to examine wines, spirits, high-quality single seed vegetable oils, natural flavourings and honey in order to determine / verify authenticity or to detect fraudulent labelling and misrepresentation.These principles have also been applied over recent years to behavioural and ecological studies examining the feeding and migration patterns of animals and birds and stable isotopes are now being used as a tool in wildlife forensics. For example, isotope analysis of feathers may be used as means of determining the origin and movement of traded birds. The heavy isotope content of water varies widely and systematically across the globe, providing a marker that is incorporated through diet into the bird’s feathers. As a result, these isotopes are potentially ideal tracers of geographic origin. This isotope method has excellent potential where strong variation or difference of isotopes exist between two potential points of geographic origin, e.g. bred and reared in captivity versus illegally trapped in the wild.The use of stable isotopes in wildlife forensics is in its infancy, but the technique offers the potential for producing powerful enforcement data, complimentary to that provided by genetic analysis.

References:
Angerosa, F., Breas, O., Contento, S., Guillou, C., Reniero, F., & Sada, E. 1999, "Application of stable isotope ratio analysis to the characterization of the geographical origin of olive oils", Journal of Agricultural and Food Chemistry, vol. 47, no. 3, pp. 1013-1017.

Angerosa, F., Camera, L., Cumitini, S., Gleixner, G., & Reniero, F. 1997, "Carbon stable isotopes and olive oil adulteration with pomace oil", Journal of Agricultural and Food Chemistry, vol. 45, pp. 3044-3048.

Bearhop, S., Fiedler, W., Furness, R. W., Votier, S. C., Waldron, S., Newton, J., Bowen, G. J., Berthold, P., & Farnsworth, K. 2005, "Assortative mating as a mechanism for rapid evolution of a migratory divide", Science, vol. 310, no. 5747, pp. 502-504.

Bearhop, S., Furness, R. W., Hilton, G. M., Votier, S. C., & Waldron, S. 2003, "A forensic approach to understanding diet and habitat use from stable isotope analysis of (avian) claw material", Functional Ecology, vol. 17, no. 2, pp. 270-275.

Bowen, G. J., Wassenaar, L. I., & Hobson, K. A. 2005, "Global application of stable hydrogen and oxygen isotopes to wildlife forensics", Oecologia, vol. 143, no. 3, pp. 337-348.

Brooks, J. R., Buchmann, N., Phillips, S., Ehleringer, B., Evans, R. D., Lott, M., Martinelli, L. A., Pockman, W. T., Sandquist, D., Sparks, J. P., Sperry, L., Williams, D., & Ehleringer, J. R. 2002, "Heavy and light beer: A carbon isotope approach to detect C-4 carbon in beers of different origins, styles, and prices", J.Agric.Food Chem., vol. 50, no. 22, pp. 6413-6418.

Calderone, G., Guillou, C., & Naulet, N. 2003, "Official methods based on stable isotope techniques for analysis of food. Ten years' of European experience", Actualite Chimique no. 8-9, pp. 22-24.

Calderone, G., Naulet, N., Guillou, C., Reniero, F., & Cortes, A. I. B. 2005, "Analysis of the C-13 natural abundance of CO2 gas from sparkling drinks by gas chromatography/combustion/isotope ratio mass spectrometry", Rapid Communications in Mass Spectrometry, vol. 19, no. 5, pp. 701-705.

Farmer, N., Meier-Augenstein, W., & Kalin, R. M. 2005, "Stable Isotope Analysis of Safety Matches using IRMS - A Forensic Case Study", Rapid Communications in Mass Spectrometry, vol. 19, pp. 3182-3186.

Fraser, I., Meier-Augenstein, W., & Kalin, R. M. 2006, "The role of stable isotopes in human identification: a longitudinal study into the variability of isotopic signals in human hair and nails", Rapid Communications in Mass Spectrometry, vol. 20, no. 7, pp. 1109-1116.

Hobson, K. A. 2005, "Using stable isotopes to trace long-distance dispersal in birds and other taxa", Diversity and Distributions, vol. 11, no. 2, pp. 157-164.

Hobson, K. A., Bowen, G. J., Wassenaar, L. I., Ferrand, Y., & Lormee, H. 2004, "Using stable hydrogen and oxygen isotope measurements of feathers to infer geographical origins of migrating European birds", Oecologia, vol. 141, no. 3, pp. 477-488.

Hor, K., Ruff, C., Weckerle, B., Konig, T., & Schreier, P. 2001, "Flavor authenticity studies by H-2/H-1 ratio determination using on-line gas chromatography pyrolysis isotope ratio mass spectrometry", Journal of Agricultural and Food Chemistry, vol. 49, no. 1, pp. 21-25.

Jung, J. C., Sewenig, S., Hener, U., & Mosandl, A. 2005, "Comprehensive authenticity assessment of lavender oils using multielement/multicomponent isotope ratio mass spectrometry analysis and enantioselective multidimensional gas chromatography-mass spectrometry", European Food Research and Technology, vol. 220, no. 2, pp. 232-237.

Kelly, S., Parker, I., Sharman, M., Dennis, J., & Goodall, I. 1997, "Assessing the authenticity of single seed vegetable oils using fatty acid stable carbon isotope ratios (C-13/C-12)", Food Chemistry, vol. 59, pp. 181-186.

Kelly, S. D. & Rhodes, C. 2002, "Emerging techniques in vegetable oil analysis using stable isotope ratio mass spectrometry", Grasas y Aceites, vol. 53, no. 1, pp. 34-44.

Meier-Augenstein, W. & Liu, R. H. 2004, "Forensic Applications of Isotope Ratio Mass Spectrometry," in Advances in Forensic Applications of Mass Spectrometry, J. Yinon, ed., CRC Press, Boca Raton, Florida 33431, pp. 149-180.

Meier-Augenstein, W. 2006, "Stable Isotope Fingerprinting - Chemical Element 'DNA'?," in Forensic Human Identification, T. J. T. Thomson & S. M. Black, eds., CRC Press, Boca Raton, FL, USA, pp. 29-53.

Rossmann, A., Lullmann, C., & Schmidt, H. L. 1992, "Mass-spectrometric determination of carbon and hydrogen isotope ratios for honey authenticity control", Zeitschrift fur Lebensmittel-untersuchung und -forschung, vol. 195, pp. 307-311.

Rossmann, A., Schmidt, H. L., Reniero, F., Versini, G., Moussa, I., & Merle, M. H. 1996, "Stable carbon-isotope content in ethanol of ec data-bank wines from italy, france and germany", Zeitschrift fur Lebensmittel-untersuchung und -forschung, vol. 203, pp. 293-301.

Spangenberg, C. E., Macko, S. A., & Hunziker, J. 1998, "Characterization of olive oil by carbon isotope analysis of individual fatty acids: Implications for authentication", Journal of Agricultural and Food Chemistry, vol. 46, no. 10, pp. 4179-4184.

Spangenberg, J. E. & Ogrinc, N. 2001, "Authentication of vegetable oils by bulk and molecular carbon isotope analyses with emphasis on olive oil and pumpkin seed oil", Journal of Agricultural and Food Chemistry, vol. 49, no. 3, pp. 1534-1540.

Weber, D., Robmann, A., Schwarz, S., & Schmidt, H. L. 1997, "Correlations of carbon isotope ratios of wine ingredients for the improved detection of adulterations .1. Organic acids and ethanol", Zeitschrift Fur Lebensmittel-Untersuchung Und-Forschung A-Food Research And Technology, vol. 205, pp. 158-164.

Woodbury, S. E., Evershed, R. P., & Rossell, J. B. 1998, "Purity assessments of major vegetable oils based on delta C-13 values of individual fatty acids", Journal of the American Oil Chemists' Society, vol. 75, pp. 371-379.

Woodbury, S. E., Evershed, R. P., Rossell, J. B., Griffith, R. E., & Farnell, P. 1995, "Detection of vegetable oil adulteration using gas-chromatography combustion isotope ratio mass-spectrometry", Analytical Chemistry, vol. 67, pp. 2685-2690.



Identification of geographic origin using DNA

Rob Ogden Wildlife DNA Services, UK.

Introduction:

Knowing the geographic origin of a sample is often important in wildlife crime investigations, particularly where legislation varies between regions. The collection and trade of an animal or plant may be permitted in certain areas, but prohibited in others. In order to enforce this type of legislation using DNA evidence, it is necessary to demonstrate that a DNA sample is consistent with the genetic variation found in a particular geographic region and is extremely unlikely to have come from anywhere else. From a biological perspective, geographic origin identification is essentially a question of identifying the population that an individual belongs to. Population identification is difficult compared to individual identification or species identification because the unit of measurement, the population, is not precisely defined. A population may consist of a group of inter-related families or an entire sub-species distributed across a whole continent (Figure 1). Therefore at one extreme, population identification can be achieved using DNA sequencing methods, similar to species identification, while at the other extreme, it requires a large number of variable genetic markers such as microsatellites (STRs) that are used in individual DNA profiling. For further information on these techniques, please refer to the DNA identification section.

Population identification via DNA sequencing:

Where populations have been isolated from one another over evolutionary time, differences between mitochondrial DNA sequences are likely to have arisen. These population differences may correspond to specific geographic regions, such as islands, seas, or land masses divided by mountain ranges. While the different populations have not formed separate species, there is often enough genetic sequence variation to identify a population, and therefore a geographic region of origin. For example, mitochondrial DNA sequences have been used to identify Asian elephants from the island of Borneo (1) due to a characteristic sequence present in the population on Borneo, but not elsewhere. Such research provides tools for conservation of endangered populations and also for wildlife forensic applications, providing evidence on the origin of illegally traded ivory. Where categorical sequence differences occur between populations they provide very powerful markers for geographic origin identification, however for many species, such markers have not been identified. The distribution of individuals within most species is continuous, enabling the flow of genetic material between geographic regions. Separate populations may exist, but the transfer of DNA through individual migration or dispersal prevents the build up of unique genetic markers in populations. From a forensic perspective, this prevents geographic origin from being identified with absolute certainty, as the DNA type observed in a population may also theoretically be found somewhere else. In such instances, population identification relies on measuring the relative frequencies of a genetic marker in a population and then calculating the probability that an individual with that marker originates from that region. Frequency-based approaches to population identification can also use mitochondrial DNA sequence data to measure genetic variation. Characteristic sequences, referred to as haplotypes, can be identified within a species. The frequency of these haplotypes will often vary among populations, allowing the origin of individuals to be inferred. For example, a species may consist of two populations (1 & 2) each displaying three mitochondrial haplotypes (A, B, C) at the following frequencies: Pop 1 (A=49%, B=50%, C=1%); Pop 2 (A=1%, B=50%, C=49%). If an individual of unknown origin carries haplotype A, then we can state that it is 49 times more likely to have come from Pop 1, than Pop 2. However haplotype B individuals are equally likely to have come from Pop 1 or Pop 2. Mitochondrial haplotype frequency differences can be informative indicators of geographic origin, but are not very useful for forensic applications as the resulting probabilities are often not powerful enough to provide convincing evidence in court. Frequency approaches can provide stronger evidence when based on multiple genetic markers, such as microsatellites (STR), or single nucleotide polymorphisms (SNPs), used to create DNA profiles.

Population identification via DNA profiling:

The use of individual DNA profiles to match two samples is an established technique, transferred directly from human forensic methods (see Individual identification using DNA). In contrast, applying DNA profiles to geographic origin identification is less common and has no parallel with human DNA analysis. Instead, the use DNA profiling in forensic genetic population identification is based on population assignment methods developed for evolutionary and conservation research. DNA profiles consist of data from multiple genetic markers. Each marker typically has a number of different states, or alleles. The specific alleles found at each marker in an individual constitute its DNA profile. Different alleles vary in frequency, so that some are very rare, others very common. Allele frequency differences can be distributed geographically, in the same way that sequence haplotype frequencies can vary. By comparing the alleles in an individual profile with the frequency of those alleles in different populations, it is possible to identify which population a sample is most likely to have originated from. There are two principal genetic markers used for constructing DNA profiles. Microsatellites (or STRs) are the best known type of marker that form the basis of human profiling systems and have been developed for a large number of animal and plant species. Microsatellite markers tend to have multiple alleles (ca. 3-10) and an individual profile would normally consist of alleles from 10-15 independent markers. Single nucleotide polymorphisms (SNPs) are a newer type of marker, less well developed for most wildlife species. They have fewer alleles (2-4) and therefore more markers (>50) are required to identify samples with equal confidence to microsatellite profiles, however SNPs do have several technical advantages. Both types of marker are now used for geographic origin identification in wildlife crime investigation. A good example of geographic origin identification using microsatellite DNA profiles is work carried out on the identification of elephant ivory (2,3). Understanding the origins of ivory may help direct anti-poaching efforts and uncover trade routes. Microsatellite analysis has recently been used to track ivory seized in Singapore back to Zambia, by comparing the DNA profiles of the elephant ivory with those of elephants throughout southern Africa. The alleles observed in the ivory allowed it to be matched to a specific population with a high degree of confidence. SNP profiling has also recently been demonstrated for use as an enforcement tool for population identification (4). Assignment of SNP profiles to winter or summer spawning populations in fresh water fish has successfully shown that these markers can be used to identify individuals to their original population. In all applications of DNA profiling to geographic origin identification, it is necessary to employ statistical analysis to calculate the probability of the sample belonging to a certain population. There are several statistical approaches available for this (see 5 for a review) and care must be taken in both the selection and application of the analytical technique.

References:

Fernando P et al. (2003) DNA analysis indicates that Asian elephants are native to Borneo and are therefore a high priority for conservation. Public Library of Science, 1(1): 1102.

Wasser SK et al. (2004) Assigning African elephant DNA to geographic region of origin: Applications to the ivory trade. Proc. Natl. Acad. Sci. USA, 101 (41): 14847-148523.

Wasser SK et al (2007) Using DNA to track the origin of the largest ivory seizure since the 1989 trade ban. Proc. Natl. Acad. Sci. USA4.

Schwenke et al. (2006) Forensic identification of a chinook salmon using a multilocus SNP assay. Conservation Genetics 7:9835.

Hauser L. et al. (2006) An empirical verification of population assignment methods by marking and parentage data: hatchery and wild steelhead (Oncorhynchus mykiss) in Forks Creek, Washington, USA, Mol. Ecol. 15 (11): 3157-3173




Radiocarbon Dating for Forensic Science

Tom Higham Oxford Radiocarbon Accelerator Unit.

Radiocarbon dating is something of a blunt instrument in the modern period (between c. 1650-1950 AD), but is much more useful for the post-1950 period. From the mid-1950s and over the course of a decade, the atmospheric radiocarbon concentration increased dramatically because radiocarbon was produced artifically through thermo-nuclear bomb testing. Atmospheric nuclear testing resulted in a doubling of radiocarbon concentration over natural proportions by 1963 in the northern hemisphere. Since then, with occasional perturbations, the levels of 'bomb carbon' have declined as this artificial radiocarbon enters the biosphere. In 2007 we are now almost back to the levels of radiocarbon in the early 1950s. One advantage of this nuclear bomb signal is that it is possible to derive radiocarbon ages for material that was once alive in this very recent period. Radiocarbon can therefore be useful in providing ages for forensic specimens.

Advantages of Accelerator Mass Spectrometry:
Accelerator measurement differs from the conventional method in that it enables samples a thousand times smaller to be dated. The ability to obtain a radiocarbon date using only a small quantity of material has made possible the direct dating of forensic samples with the minimum of damage. Consequently, the last ten years have seen a great deal of interest in radiocarbon dating this kind of material. The precision of the measurements is similar to conventional radiocarbon measurements and for the post-1950 era can provide results to within two years at best, if the material dated is not subject to the vagaries of bone collagen turnover effects or other sample related problems. Sample Material and Type A variety of organic materials is suitable for dating in human forensic cases including:* Human bone (although much depends on the age of the human, since bone turnover rates slow with age, influencing the accuracy).* Human hair * Human skin* Clothing of an organic nature, such as cotton.
In addition, radiocarbon may be used in examining issues related to wildlife crime and the trafficking of illegal animals and birds. Suitable sample types include;
Animal or bird bone
Antler, tusk and horn
Hair
Feathers
Egg carbonates

Oxford Radiocarbon Accelerator Unit (ORAU)
There are several accelerator facilities worldwide performing radiocarbon analyses, with about six in Europe. The Oxford Laboratory is unique in putting the majority of our effort into the dating of archaeological samples. We have an international reputation for this and have specialised both in developing chemical treatment processes, and in providing archaeological advice, scrutiny and interpretation.Much of the expertise in this area is directly applicable to forensic science questions. We measure about 1000 dates per year in house with a further 1000 AMS measurements on samples prepared elsewhere. About 25-40 of these are related to forensic cases, mostly for local UK police forces investigating human remains found by the general public to determine whether they are of forensic interest or not. ORAU has considerable expertise in these areas and encourages potential submitters to contact us. In addition to our dating programme, we undertake research into methods that improve the accuracy and usefulness of radiocarbon dating. We have relevant interest and expertise in these areas amongst others:
Chemical pre-treatment of bone
Removal of preservatives and contaminants from objects
Stable isotope analysis and interpretation - including the identification of contaminants
Dating of extremely small samples
Radiocarbon calibration
General interpretation of radiocarbon dating evidence

We perform regular quality control tests both through the main international laboratory inter-comparisons (TIRI, FIRI etc), informal laboratory inter-comparisons and with regular in-house measurements on known-age material. We are also ISO-9001 accredited.


Other wildlife crime topics covered in my degree:

Species at Risk: Bats

The Illegal Wildlife Trade

Bees

Biodiversity Action Plans (BAPS)

The Wildlife and Countryside Act 1981 (WCA81)

Twelve Fish Protected Under WCA81

Breeding and/or Catching Animals for fur: Debate

Traditional Alternative Medicines (TAMs)

Seals

Environmental Ethics and Ecopsychology

The Climate Change Conference 2009

Biological Diversity

Conservation Strategies

Conventions, Legislation and Contributing Bodies

Example Papers of Forensic Analysis used in Wildlife Crimes

Researching Wildlife Crime - My Research Idea

Climate Change Affecting Wildlife

Coral Reefs