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Background  Micromilled fish otoliths ( ear bones ) have been widely used for stable oxygen (δ 18 O) and carbon (δ 13 C) isotope analysis. The first dorsal fin spine is routinely used for ageing in the eastern Atlantic bluefin tuna (ABFT) population; however, stable isotope analysis remains unapplied in this hard structure. The objective of the present protocol is to achieve a sequential sampling of growth layers in the ABFT fin spine at high spatial resolution and along growth trajectory for stable isotope analysis.    Methods  We used a micromilling and micro-powder collecting technique for sequentially sampling annual growth layers of the fin spine bone collected from freshly caught ABFT. We assessed the carbonate content and the optimal drilling amount to ensure enough powder quantity was recovered from each annual growth band to accommodate accurate measurement of the δ 18 O and δ 13 C values.    Results  The optimal drilling path included 20 drilling lines in 49 μm, with 400 μm depth and 900 μm-line width, which represents a time resolution of about 2.5 months. The minimum powder quantity required from each annual growth layer for δ 18 O and δ 13 C isotope analysis was approximately 180 μg because the carbonate contents in the fin spine powder was 5–6%. The δ 18 O profile assayed in the last growth intervals coincide with the surface δ 18 O seawater  for the southern west waters off the Norwegian coast, where the tuna was caught. The fluctuating pattern in the second growth interval may reflect certain fidelity to the Norwegian SE and Swedish SW coast and/or otherwise to the US west coast. The δ 13 C spine  displayed a larger temporal variability along the growth transect that may be explained by several factors including differences in habitat use, behavior, and even trophic level.    Conclusion  Based on the result obtained, we present an optimal standard protocol for the sampling of sequential, annually formed growth layers in the fin spine bone for stable isotope analysis using the micro-milling as a high precision technique. This protocol is particularly useful in endangered and/or protected species for which fin spines represent a non-lethal alternative to otoliths opening new research avenues to improve their management and conservation.

Protocol for Sampling Sequential Fin Spine Growth Intervals for Isotope Analysis in the Atlantic Bluefin Tuna