Nitrogen isotopic analysis of carbonate-bound organic matter in modern and fossil fish otoliths

The nitrogen isotopic composition (δ15N) of otolith-bound organic matter (OM) is a potential source of information on dietary history of bony fishes. In contrast to the δ15N of white muscle tissue, the most commonly used tissue for ecological studies, the δ15N of otolith-bound OM (δ15Noto) provides a record of whole life history. More importantly, δ15Noto can be measured in contexts where tissue is not available, for example, in otolith archives and sedimentary deposits. The utility and robustness of otolith δ15N analysis was heretofore limited by the low N content of otoliths, which precluded the routine measurement of individual otoliths as well as the thorough cleaning of otolith material prior to analysis. Here, we introduce a new method based on oxidation to nitrate followed by bacterial conversion to N2O. The method requires 200-fold less N compared to traditional combustion approaches, allowing for thorough pre-cleaning and replicated analysis of individual otoliths of nearly any size. Long term precision of δ15Noto is 0.3‰. Using an internal standard of Atlantic cod (Gadus morhua) otoliths, we examine the parameters of the oxidative cleaning step with regard to oxidant (potassium persulfate and sodium hypochlorite), temperature, and time. We also report initial results that verify the usefulness of δ15Noto for ecological studies. For three salmonid species, left and right otoliths from the same fish are indistinguishable. We find that the δ15Noto of pink salmon (Oncorhynchus gorbuscha) is related to the size of the fish for this species. We find that intra-cohort δ15Noto standard deviation for wild pink salmon, farmed brown trout (Salmo trutta), and farmed rainbow trout (Oncorhynchus mykiss) are all 0.4‰ or less, suggesting that δ15Noto will be valuable for population-level studies. Lastly, our protocol yields reproducible data for both δ15Noto and otolith N content in 17th century Atlantic cod otoliths. We find that 17th century cod are approximately 2 ‰ higher than modern cod, arguably consistent with either the larger size of the otoliths (and thus inferred for the fish) or with changes in baseline (primary producer) δ15N in the modern coastal ocean compared to the past. All told, the results of this study bode well for the utility of otolith-bound δ15N for investigating the environment and ecology of modern and past fish.