Stable Isotope Analysis (δ 13 C, δ 15 N and δ 18 O) of Killer Whale Dentine: Evaluating Treatment Methods and Establishing Best Practices

ABSTRACT Rationale Stable isotope measurements in odontocete teeth can be used to reconstruct lifetime ecology, but some pre‐treatments may introduce bias. Isolating dentine collagen with HCl decalcification, removing the organic matrix or drying dentine prior to the analysis of structural carbonates can introduce unintended variability, limiting comparability among studies and populations. This study tested pre‐treatment methods to quantify their effects. Methods Killer whale teeth were sectioned, annual growth layers microdrilled, and powder allocated to treatments. For collagen δ 13 C and δ 15 N, 75 samples were analysed via EA‐IRMS as untreated bulk dentine and after 0.25 N HCl decalcification. For structural carbonate δ 13 C and δ 18 O, treatment with 30% H 2 O 2 was compared with plasma‐ashing and with no treatment ( n = 16); and we examined the removal of residual water by comparing samples oven‐dried at 80°C or 100°C with untreated samples ( n = 10). All samples for carbonate analysis were injected with 104% orthophosphoric acid to generate CO 2 , which was analysed using continuous‐flow IRMS. Results HCl decalcification caused mass loss (96.6% ± 2.0%) but yielded collagen of an acceptable quality (C: N = 3.3 ± 0.2). Compared with untreated dentine, collagen δ 13 C decreased (Δmedian = −0.34‰) while δ 15 N did not differ significantly. Organic‐matrix removal treatments altered both isotopes: plasma‐ashed samples showed large negative shifts compared with untreated samples in δ 13 C (Δmedian = −9.04‰) and δ 18 O (Δmean = −13.48‰) and reduced measurable carbon. H 2 O 2 treatments lowered δ 13 C relative to untreated samples (Δmean = −0.93‰) but showed no consistent δ 18 O effect. Drying at 80°C minimised δ 13 C and δ 18 O variability. Conclusions HCl decalcification is recommended before δ 13 C and δ 15 N collagen analysis of killer whale dentine, or a δ 13 C correction factor (here −0.34‰) should be applied. Treatments were unsuitable when undertaking structural carbonate analyses of hydroxyapatite: we suggest that untreated powder oven‐dried overnight at 80°C should be used, and we emphasise the importance of pre‐treatment tests prior to any new study.