Effects of sample treatment on the analysis of stable isotopes of carbon and nitrogen in zooplankton, micronekton and a filter-feeding shark
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Stable isotope analysis is often used to investigate the trophic ecology of marine systems. However, a lack of standardization of the treatment of samples prior to analysis, hampers comparisons of results within and among studies. This study examined the effects of lipid extraction (LE), acidification for the removal of inorganic carbonate (RIC) and rinsing with deionized water (DIW) on δ13C and δ15N values and C:N ratios in sub-dermal tissue of whale sharks (Rhincodon typus), zooplankton (>200–1000 μm) and a wide range of micronekton (>1000 μm) taxa collected in 2013 and 2014 at Ningaloo Reef (Western Australia). For whale shark tissue, lipid extraction (LE and LE + DIW) increased values of δ13C, whereas LE, LE + DIW and DIW treatments increased values of δ5N and C:N ratios. These results confirm the need to remove lipids and 15N-depleted nitrogenous waste from elasmobranch tissues. The LE + DIW treatment was the most efficient at achieving this goal. For zooplankton and micronekton, LE and RIC treatments had consistent effects on δ13C values, however, effects on δ15N values were more unpredictable. Therefore, zooplankton and micronekton samples should be split into two portions, one subjected to LE or LE + RIC treatments to standardize δ13C values, and a second untreated portion used for analysis of δ15N values. For these taxa, the RIC + DIW treatment resulted in the greatest change in δ15N values, which may confound results. Mathematical normalization models used to predict outcomes of treatments on values of δ13C and δ15N were not found to be suitable for all the taxa in this study.
This work was funded by the Save Our Seas Foundation, Winifred Violet Scott Charitable Trust, Holsworth Wildlife Research Endowment Grants and the Australian Institute of Marine Science. We also thank captain Terry Maxwell and his crew and all the people who contributed to fieldwork, with special mention to Kim Brooks, Michelle Thums, Dani Rob and the WA Department of Parks and Wildlife. We thank Peter Mansour and Diana Davies at CSIRO, James Horne at the Central Science Laboratory, University of Tasmania and Andrea Walters and Jorge Mardones at the Institute for Marine and Antarctic Science for their help during analysis. The manuscript was improved by the assistance and helpful comments from the editor Chris Harrod and two anonymous journal reviewers.
Compliance with Ethical Standards
Conflict of interest
All authors declare they have no conflict of interest.
All applicable national and institutional guidelines for the care and use of animals were followed. All work was conducted after ethical approval by the University of Tasmania Animal Ethics Committee (A13102) and under permits and exemptions from the Department of Park and Wildlife (SF009814, SF009227) and the Western Australian Department of Fisheries (2255, 2307).
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