The effect of preservation on biomass and length estimates and its variation within and between two mayfly species

Biomass estimation offers insight into aquatic ecosystem processes and represents a powerful tool for measuring biomass in communities. However, the accuracy of this estimate depends considerably on the sample preservation method and biological traits of species. We quantified the effect of commonly used preservatives (70% ethanol, 4% formaldehyde, freezing), preservation time and initial body length on the estimation bias of body length, wet mass and dry mass in two mayfly species with dorsoventrally flattened (Rhithrogena carpatoalpina) and cylindrically elongated (Habroleptoides confusa) bodies. Freezing caused the largest relative bias in the length estimates (up to 18%). Both ethanol and freezing led to significant underestimation of wet mass of preserved animals (up to 26%). The most severe biases were observed in dry weights, where all preservation methods reduced body mass by up to 89%. Importantly, initial body mass influenced the bias caused by preservation, and the larger animals were less affected by mass losses. In contrast, larger individuals were subject to greater length reduction. Additionally, the length-mass relationship for R. carpatoalpina, based on fresh mass of living animals, was significantly different from relationships based on preserved animals. Overall, formaldehyde caused the least biased estimates of length and biomass in both examined species. This case study indicates that direct measurements of unpreserved individuals or length-mass equations based on fresh material are likely the best ways to avoid severe underestimation of biomass in aquatic insects. However, more studies covering a broader range of taxa are needed to reach general conclusions.