Sample acidification significantly alters stable isotope ratios of sulfur in aquatic plants and animals

Sulfur stable isotopes are increasingly being used as tracers of material processing in studies of both modern and historical food webs. Preparation of plant and animal material for isotope analysis routinely includes steps that remove inorganic material not normally assimilated by consumers. Whereas acidification of samples is known to assist with this for some elements (carbon), it can produce unwanted effects for others (nitrogen). Here we tested the effects of acidification on sulfur isotopes by comparing isotope ratios of paired acidified and nonacidified samples of seagrass, epiphytic algae growing on seagrass and animal consumers (3 types of crustaceans: amphipods, copepods and isopods). Acid treatment resulted in significant losses of elemental sulfur from the tissues and changes in sulfur isotope ratios of samples. The artificial depletion of the heavy sulfur isotope decreased sulfur isotope ratios by 2.6‰ on average, and by as much as 7.0‰ in individual samples. Acidification of samples prior to sulfur isotope analysis results in invalid ratios and should not be used.