A Novel Method for the Assessment of Feeding Rate as a Phenotypic Endpoint for the Impact of Pollutants in Daphnids

Traditional approaches for monitoring aquatic pollution primarily rely on chemical analysis and the detection of pollutants in the aqueous environments. However, these methods lack realism and mechanistic insight and, thus, are increasingly supported by effect-based methods, which offer sensitive endpoints. In this context, daphnids, a freshwater species used extensively in molecular ecotoxicology, offer fast and noninvasive approaches to assess the impact of pollutants. Among the phenotypic endpoints used, feeding rate is a highly sensitive approach because it provides evidence of physiological alterations even in sublethal concentrations. However, there has been no standardized method for measuring feeding rate in daphnids, and several approaches follow different protocols. There is a diversity among tests employing large volumes, extensive incubation times, and high animal densities, which in turn utilize measurements of algae via fluorescence, radiolabeling, or counting ingested cells. These tests are challenging and laborious and sometimes require cumbersome instrumentation. In the present study, we optimized the conditions of a miniaturized fast, sensitive, and high-throughput assay to assess the feeding rate based on the ingestion of fluorescent microparticles. The protocol was optimized in neonates in relation to the concentration of microplastic and the number of animals to increase reproducibility. Daphnids, following exposures to nonlethal concentrations, were incubated with microplastics; and, as filter feeders, they ingest microparticles. The new approach revealed differences in the physiology of daphnids in concentrations below the toxicity limits for a range of pollutants of different modes of action, thus proving feeding to be a more sensitive and noninvasive endpoint in pollution assessment. Environ Toxicol Chem 2024;43:2211-2221. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.