Effects of pre-treatment, historical age, and sample characteristics on the stable isotope analyses of killer whale (Orcinus orca) bone.

Rationale: Stable isotope analysis of bone provides insight into animal foraging and allows for ecological reconstructions over time, however pre-treatment is required to isolate collagen. Pre-treatments typically consist of demineralization to remove inorganic components and/or lipid extraction to remove fats, but these protocols can differentially affect stable carbon (δC) and nitrogen (δN) isotope values depending on the chemicals, tissues, and/or species involved. Species-specific methodologies create a standard for comparability across studies and enhance understanding of collagen isolation from modern cetacean bone.

Methods: Elemental analyzers coupled to isotope ratio mass spectrometers were used to measure the δC and δN values of powdered killer whale (Orcinus orca) bone that was intact (control) or subjected to one of three experimental conditions: demineralized, lipid-extracted, and both demineralized and lipid-extracted. Additionally, C:N ratios were evaluated as a proxy for collagen purity. Lastly, correlations were examined between control C:N ratios vs. historical age and control C:N ratios vs. sample characteristics.

Results: No significant differences in the δN values were observed for any of the experimental protocols. However, the δC values were significantly increased by all three experimental protocols: demineralization, lipid extraction, and both treatments combined. The most influential protocol was both demineralization and lipid extraction. Measures of the C:N ratios were also significantly lowered by demineralization and both treatments combined, indicating the material was closer to pure collagen after the treatments. Collagen purity as indicated via C:N ratio was not correlated with historical age nor sample characteristics.

Conclusions: If only the δN values from killer whale bone are of interest for analysis, no pre-treatment seems necessary. If the δC values are of interest, samples should be both demineralized and lipid-extracted. As historical age and specimen characteristics are not correlated with sample contamination, all samples can be treated equally.