A case against acidifying freshwater macrophytes prior to C and N stable isotope analysis

Acidifying organic samples prior to stable isotope analysis is a common practice to eliminate inorganic carbonates; however, it is still unclear what impact this has on δ 13C and δ 15N values. Here we present the first extensive study to test the effects of acidification on freshwater macrophyte carbon and nitrogen isotopic ratios. Focusing on the more productive and ecologically relevant seasons, we collected eleven common macrophyte species (both submerged and emergent), from four lakes in south-central Ontario, Canada. These lakes vary considerably in chemistry, particularly in the inorganic carbon and calcium concentrations. All individual plant samples were equally separated, prepared into acidified and un-acidified treatments, and analyzed for their carbon and nitrogen stable isotope ratios. No significant differences in macrophyte δ 13C or δ 15N were detected between the treatments. Additionally, we found that within-species δ 13C variability was greater in acidified samples. Finally, in assessing the isotopic values of different macrophytes across a chemical gradient at different times throughout the season, it became evident that natural variability exists in both the δ 13C and δ 15N of un-acidified samples within and between species. Based on these results, we assert that pre-analysis acidification of freshwater macrophytes from temperate lakes is unnecessary and not recommended. Additionally, we implore ecologists to acknowledge the macrophyte δ 13C and δ 15N variability in future food web studies.