Influence of tissue preservation techniques on δ13C and δ15N signatures in selected marine taxa

Stable isotopes, particularly δ13C and δ15N, are widely used in ecological research to infer trophic interactions, energy flow, and resource use. However, preservation methods can introduce biases in isotopic measurements that complicate interpretation. The study quantifies the effects of freezing, oven-drying, repeated freezing and thawing cycles and ethanol preservation on δ13C and δ15N values of six marine taxa spanning Osteichthyes, Elasmobranchia, Cephalopoda, and Crustacea, over a 24-month period. The isotopic changes exhibited variability in accordance with the preservation method, duration, and taxon. Short-term freezing (5 days) and repeated freeze–thaw cycles caused minimal isotopic changes. In contrast, ethanol preservation (12 months) and long-term freezing (24 months) generated consistent and significant changes in both isotopic values across taxa. Δ values were computed relative to pooled control samples to isolate treatment effects from interspecific baseline differences. The application of these methods resulted in an increase in the δ13C and δ15N values by approximately 0.2–1.9‰ and up to 2.3‰, respectively. δ13C responses were more variable across taxa, highlighting challenges in interpreting carbon-based metrics in samples stored for extended periods or preserved in ethanol. Overall, the results show that preservation-induced isotopic shifts are strongly method-dependent and can be large enough to influence ecological interpretation. Careful selection of preservation protocols, and explicit consideration of their potential biases, is therefore essential for generating reliable stable isotope data in marine ecological research.