Development and application of a sensitive feeding assay for daphnids based on the ingestion of fluorescent microparticles

The toxicological hazard and safety assessment of chemical substances relies on the outcome of animal testing. A combination of mortality, phenotypic and molecular endpoints are employed to assess this. However, animal welfare considerations, societal concerns, and regulatory action have initiated the need to use new approaches and methodologies in risk assessment. In this context, non-invasive tests and model species not categorized as "animals" can be used to reduce the use of higher animals according to the 3Rs principle. Additionally, such tests could provide comparative conclusions with faster and more economical approaches. Focusing on the freshwater ecosystem, daphnids have been extensively used for toxicological studies, and their feeding rate following exposure to pollutants is a common phenotypic endpoint in ecotoxicology assessment. Feeding impairment indicates early alterations in animal physiology, thus providing insight for further investigation. The feeding rate is usually assessed with extended incubation periods and large volumes of media, resulting in increased waste generation and use of animals, which highlights the need for improved methods. In this study, we developed a robust and sensitive approach based on tracking the ingestion of fluorescent microparticles that requires a low number of animals and incubation times. Parameters such as the total volume, the concentration of microparticles, and the number of daphnids were optimized to study the impact of a selection of pollutants. As indicated by the results, the animal number used per replicate had a significant impact (an increase in the animal number increased the ingestion) on the feeding rate rather than the assay volume and the concentration of microparticles. To assess the effect of exposure to chemicals on the feeding rate of daphnids, a range of metals (lithium chloride, zinc sulfate heptahydrate, zirconium chloride, aluminium sulfate hexadecahydrate, cobalt nitrate hexahydrate), pharmaceuticals (diltiazem hydrochloride, propranolol hydrochloride, diclofenac sodium, metformin), and one stimulant (nicotine) were selected. There was a concentration-dependent decrease in feeding rates for most of the pollutants used, particularly after exposure to metals, indicating their strong effect on the physiology of the animal. This method demonstrates an efficient means of assessing toxicology to guide future studies in working concentrations of chemicals and the assessment of water quality. The toxicological hazard and safety assessment of chemical substances relies on the outcome of animal testing involving phenotypic tests such as feeding.