Effects of lipid removal and preservatives on carbon and nitrogen stable isotope ratios of squid tissues: Implications for ecological studies

Stable isotope analysis of carbon (C) and nitrogen (N) in animal tissues is an important approach to investigate the tropic status and habitat of marine species. Some biases due to lipid extractions and preservation can hinder the interpretation of results, yet their effects have not been investigated in squid. In this study, we evaluated the effects of lipid extraction and preservatives (dimethylsulfoxide (DMSO), 70% ethanol, and 10% buffered formaldehyde) on the delta C-13, delta N-15 and C:N ratios in squid muscle. Beaks were placed under the same treatments with the exception of DMSO. Muscle and beak samples remained under treatment for 375 days and 416 days, respectively. Our results indicate that lipid extractions increased the mean values of unpreserved samples by 0.8 parts per thousand. for delta C-13 and by 0.68 parts per thousand. for delta N-15. Preservatives also affected the isotopic composition in muscle at different magnitudes. DMSO remarkably reduced and increased the variability for delta C-13 and delta N-15 values among samples, formalin mainly reduced delta C-13 values by 1.5 parts per thousand, whereas ethanol increased both delta C-13 and delta N-15 by <= 0.8 parts per thousand. Lipid extractions eliminated the effect of DMSO and ethanol for delta C-13 and delta N-15, and formalin only for delta N-15. In beak, negligible shifts in delta C-13, delta N-15 and C:N ratios were recorded after preservation in ethanol and formalin. Although lipid extractions can be recommended to reduce the effect of preservation, further research is needed to develop correction models for isotopic shifts associated with both lipid extractions in unpreserved and preserved muscle tissues. Lipid extractions per se could introduce a bias that may have important implications for ecological studies. Published by Elsevier B.V.