INVESTIGATION OF HEAT-TREATED BONES AND TEETH AND AFFECT ON STABLE ISOTOPE RATIOS MARR, POIYUN, HANNAH KOON UNIVERSITY OF BRADFORD Introduction The purpose of this project was to determine which method can extract the most optimal yield of intact collagen and to examine how the carbon and nitrogen isotope ratios change due to the cremation process (Ambrose 1990, DeNiro and Weiner 1988, Meier-Augenstein 2010, Schurr et al 2008). This project is relevant to forensic investigators identifying human remains due to fire-related incidents and for archaeologists studying cultural inferences involving intentional heating rituals, such as cremations or cannibalistic studies (Harbeck et al. 2011, McKinley 1994, Oestigaard 2000, Schultz et al. 2008). Aims and Objectives The aims and objectives of this project were to 1. Conduct a literature review of current bone taphonomic research on heated mammalian or human bone with emphasis on the methods of analysis and previously documented alteration to the bone composition 2. Heat defleshed pig bones at different temperatures 3. Extract intact collagen molecules from the unheated and heated samples 4. Examine the carbon and nitrogen stable isotopes ratios from the heated and compare against the unheated bone This poster highlights the preliminary results of the heating experiments and some early results from the stable isotope analysis of the heated bone Samples and Methods Three corresponding mandible with teeth and femoras of fresh Sus scrofa (pig) from West Yorkshire, UK. The defleshed samples was heated for 1 hour in a muffle furnace at: • Unheated (control) • 250°C • 350°C The heated cortical and trabecular bones was fragmented into 1.00-2.00mm. The unheated cortical and trabecular bones was fragmented into 1.00-5.00mm. One complete or mostly complete tooth was extracted from the mandible for each demineralisation test. The teeth, cortical, and trabecular bone fragments was subdivided and demineralised with: • 0.1 M EDTA, NaOH or deionised H 2 O • 0.5 M HCl acid, NaOH or deionised H 2 O The cortical section from one set of samples was additionally treated with 2:1 chloroform to methanol solution (Logan and Lutcavage 2008). The samples were gelatanised at: • 70°C at neutral pH (pH = 5.8) • 70-75°C with a combination of neutral pH and 3.0 pH level • 75°C at 3.0 pH Preliminary Results References Ambrose SH (1990) Preparation and characterization of bone and tooth collagen for isotopic analysis. Journal of Archaeological Science 17(4): 431-451.DeNiro MJ and Weiner S (1988) Chemical, enzymatic and spectroscopic characterization of “collagen” and other organic fractions from prehistoric bones. Geochimica et Cosmochimica Acta 52(9): 2197-2206. DeNiro MJ, Schoeninger MJ and Hastorf CA (1985) Effect of heating on the stable carbon and nitrogen isotope ratios of bone collagen. Journal of Archaeological Science 12(1): 1-7. DeNiro MJ and Weiner S (1988) Chemical, enzymatic and spectroscopic characterization of “collagen” and other organic fractions from prehistoric bones. Geochimica et Cosmochimica Acta 52(9): 2197-2206. Grupe G and Hummel S (1991) Trace element studies on experimentally cremated bone. I. Alteration of the chemical composition at high temperatures. Journal of Archaeological Science 18(2): 177-186. Harbeck M, Schleuder R, Schneider J, Wiechmann I, Schmahl WW and Grupe G (2011) Research potential and limitations of trace analyses of cremated remains. Forensic Science International 204(1–3): 191-200. Logan JM and Lutcavage ME (2008) A comparison of carbon and nitrogen stable isotope ratios of fish tissues following lipid extractions with non- polar and traditional chloroform/methanol solvent systems. Rapid Communications in Mass Spectrometry 22(7): 1081-1086. McKinley JI (1994) Bone fragment size in British cremation burials and its implications for pyre technology and ritual. Journal of Archaeological Science 21(3): 339-342. Meier-Augenstein W (2010) Stable Isotope Forensics : An Introduction to the Forensic Application of Stable Isotope Analysis. Chichester: Wiley. Munro LE, Longstaffe FJ and White CD (2007) Burning and boiling of modern deer bone: Effects on crystallinity and oxygen isotope composition of bioapatite phosphate. Palaeogeography, Palaeoclimatology, Palaeoecology 249(1–2): 90-102. Oestigaard T (2000) Sacrifices of raw, cooked and burnt humans. Norwegian Archaeological Review 33(1): 41-58. Shipman P, Foster G and Schoeninger M (1984) Burnt bones and teeth: an experimental study of color, morphology, crystal structure and shrinkage. Journal of Archaeological Science 11(4): 307-325. Schurr MR, Hayes RG and Cook DC (2008) Thermally induced changes in the stable carbon and nitrogen isotope ratios of charred bones. In Schmidt C and Symes SA (eds) The Analysis of Burned Human Remains. London: Academic, 95-108. Schultz JJ, Warren MW and Krigbaum JS (2008) Analysis of human cremains: gross and chemical methods. In Schmidt C and Symes SA (eds) The Analysis of Burned Human Remains. London: Academic, 75-94. van Klinken GJ (1999) Bone collagen quality indicators for palaeodietary and radiocarbon measurements. Journal of Archaeological Science 26(6): 687-695. Acknowledgements The first author wish to thank: • J. Hollund and Sons for procuring and supplying the pig samples used in this project • Andy Glenhill, Stuart Fox, and Belinda Hill for technical and laboratory support • Julia Beaumont and Becca Nicholls editing and assistance with analysing the data • Hannah Koon for supervising and assisting with the Master dissertation This project was partly funded by the Andy Jagger Fund (University of Bradford) Discussion and Conclusion Figure 5: No significant difference in the amount of insoluble collagen obtained from the four different demineralisation treatments from the unheated and 250°C samples; p ≤ 0.05 Figure 6: Carbon and nitrogen stable isotope ratios from the unheated and 250°C pig samples • Physical gross bone changes (i.e. colour) matches the expected changes due to heating as observed in other studies (Grupe and Hummel 1991, Munro et al 2007, Shipman et al 1984) • There was no significant difference between the treatment used and the amount of collagen produced in general • There is a negative shift in the carbon isotope values; corroborates with the results of other modern experimental studies (DeNiro et al. 1985) and the archaeological charred remains (Schurr et al. 2008) • The data from Schurr et al. and DeNiro et al. also report a shift in nitrogen • The nitrogen stable isotope values were largely unaffected by heat treatment • Nitrogen values can still be used for investigating diet Figure 1: A set of pig samples heated at 250°C for 1 hour Figure 2: Cortical bone fragments heated at 250°C (left) and 350°C (right) fragmented into 1.00 – 2.00 mm Figure 3: Samples of demineralised organic residue to be placed on a heating block Figure 4: Collagen of the unheated and 250°C samples from a variety of different demineralisation treatments Figure 7: The relationship between the C/N ratios to δ 15 N. The normal values are highlighted in blue (van Klinken 1999). Figure 8: The relationship between the C/N ratios to δ 13 C. The normal values are highlighted in blue (van Klinken 1999)