How bone degradation, age, and collagen extraction methods affect stable isotope analysis

Stable isotope analysis from bone collagen and carbonate is a crucial tool in archaeometry for dietary and provenance studies. However, an expedient evaluation and interpretation of results have to rely on the integrity of the isotopic values, reflecting the in vivo signal of the tissue. In this context, postmortem bone breakdown can affect measurement results and add uncertainty. This study investigates the effects of bone breakdown and the use of two different extraction methods on the isotopic composition of carbon, nitrogen, and oxygen from collagen and the structural carbonate. The main goal of this study was to test the efficiency of commonly applied quality markers to identify alterations of the isotopic in vivo signal in the organic and inorganic bone fraction. Three fresh human femur diaphyses from different age groups with a known in vivo signal were degraded experimentally to simulate diagenetic processes. One batch was heated in water to induce hydrolytic breakdown; the other one was inoculated with several aerobic and anaerobic bacterial strains for biodeterioration. Furthermore, 60 long-bone samples from natural burial conditions, representing different age groups and burial times, were analyzed. The collagen extraction method proved to have a relevant effect. For bones already compromised by degradational processes, the common gelatin extraction protocol using pulverized bone and a 1 M HCl demineralization step was shown to be too harsh and damaging to the extract. Additionally, the age of the individual might play a role in the resistance of the tissue against degradational factors. The investigation of the commonly used quality criteria revealed that the C/N molar ratio is well suited to identify diagenetically modified isotopic data from collagen carbon and nitrogen. However, no valid marker could be assessed for the reliable prediction of altered δ18O values from the structural carbonate fraction, but oxygen isotopic values proved to be considerably affected by in vitro degradation.