Testing Assumptions for Stable Isotope Analysis of Marine Mammal Dentin Growth Layer Groups

Stable isotope analysis of growth layer groups (GLGs) in mammal dentin is an increasingly prevalent and noninvasive approach to study animal foraging ecology. However, empirical evidence to support assumed proper methodologies for sampling GLGs is lacking. Here, we examine the effects of intratooth and intertooth variations with respect to targeted GLGs, as well as the effects of common pretreatments (e.g., formic acid and graphite) to enhance GLG visibility, on stable isotope values (δ¹³C and δ¹⁵N) from dentin.

We measured the δ¹³C and δ¹⁵N values of killer whale (Orcinus orca) dentin. We used dentin from 37 teeth to compare stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope values from multiple locations within a GLG (intratooth variation), from corresponding GLGs among teeth of an individual (intertooth variation), and from treated and untreated teeth.

We observed no significant differences in the δ¹³C or δ¹⁵N values when sampling a single GLG from multiple locations (intratooth variation) or when comparing the same GLG across duplicate teeth of individuals (intertooth variation). One tooth in a triplicate set showed a significantly different but likely biologically inconsequential δ¹³C value. Lastly, formic acid and graphite highlighting to accentuate GLGs did not significantly influence measured stable isotope values.

We validate several previous assumptions in this field of study. First, dentin samples for stable isotope analysis can be sampled from different locations across a GLG. Second, researchers can compare stable isotope values from the same GLGs of different teeth collected from the same individual in most cases, as the δ¹³C and δ¹⁵N values did not vary with the sampled tooth. Third, a common protocol of formic acid and graphite treatment to enhance GLG visibility does not bias the δ¹³C and δ¹⁵N values from dentin. We also describe factors to consider and cautions associated with these conclusions.