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Rationale The use of sulfur isotopes to study trophic ecology in marine ecosystems has increased in the past decade. Unlike other commonly used isotopes (e.g., carbon), sulfur can better discriminate benthic and pelagic productivity. However, how lipid extraction affects sulfur isotopic values has not been assessed, despite its frequent use to remove lipid effects on δ13C values. Methods We used white muscle and liver samples from two species of sharks and skin samples from two species of pinnipeds (sea lion and fur seal) to assess the effects of lipid extraction on stable isotope values for δ34S, δ13C, and δ15N. Isotopic values were determined using a continuous flow‐isotope ratio mass spectrometer coupled to an elemental analyzer. Results Lipid extraction significantly decreased δ34S values in shark tissues, more so for liver than muscle (−4.6 ± 0.9‰ vs −0.8 ± 0.3‰, average change), with nearly no change in their standard deviations. Lipid extraction did not affect δ34S values from pinniped skin samples (0.2 ± 0.8‰, average change). After lipid extraction, consistent increases in δ13C values (0.2‰–7.3‰) were detected as expected, especially in tissue with high lipid content (C:N >4). After lipid extraction, significant increases in δ15N values (0.5‰–1.4‰) were found in shark muscle and liver tissues. For pinniped skin samples, δ15N values were not significantly lower after lipid extraction (−0.4‰ to –0.1‰). Conclusions Lipid extraction did not have a strong impact on δ34S values of shark muscle and pinniped skin (≤1‰). However, our results suggest it is essential to consider the effects of lipid extraction when interpreting results from δ34S values of shark liver tissue, as they significantly depleted values relative to bulk tissue (~5‰). This may reflect selective removal of sulfolipids and glutathione present in higher concentrations in the liver than in muscle and skin and requires further investigation.

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