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Figure 3. Boxplot of δ C and δ N values estimated in core (C), opaque (O) and non-opaque (H) bands in
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spine of T unnus thynnus sections.

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Figure 4. Exponential model f tted to δ N values recorded in spines of T unnus thynnus in relation to FL.
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According to the following equation, δ N = a ∗ 1 − e ( −FL b/ ) ) , the estimated parameters were: a = 7.566‰
(
and b = 26.75 cm.

Estimate Std. Error t value Pr(>|t|) p
a 7.5633 0.2277 33.222 <2e-16 0.001
b 26.7463 3.1649 8.451 3.94E-11 0.001
Table 2. Exponential f tting estimates. Residual standard error: 1.136 on 49 degrees of freedom. Number of
iterations to convergence: 5. Achieved convergence tolerance: 7.88e-06.

δ N values to produce a time series of stable isotope patterns from individual animals. Indeed, both acidif cation
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and lipids removal should af ect only bioapatite carbon and not collagen-bound nitrogen. Furthermore, the lipid
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molecules do not contain nitrogen .
However, supplementary researches are needed to establish if the lipids ef ects are linked only to the inner
medullary cavity or also to the dif erent growth layers, especially for the case in which the C:N is dif cult to deter-
mine due to samples material derived from drilled growth layers. Unfortunately, with our current knowledge, we
are not able to evaluate this aspect. We postpone to future studies the possibility to f nd a method (or an equation)
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that will allow the correction of the lipids ef ects of δ C values from untreated and not delipidated samples (whole
spine and/or powder extracted from growth layers) for this species. However, the obtained results evidenced that
the δ N analysis can be used to produce isotopic prof les useful to detect trophic and foraging habitat changes
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along the individual lifetime, particularly when the amount of sample material is not enough to perform the col-
lagen extraction, such as in small f sh, archeological remains (mainly f sh remains) and/or spines of f sh stored in
the museum (extinct species or in need of conservation).

Scientific RepoRtS | (2020) 10:9899 | https://doi.org/10.1038/s41598-020-66566-w 6