Effects of sample treatment on the analysis of stable isotopes of carbon and nitrogen in zooplankton, micronekton and a filter-feeding shark

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.