The levels of pharmaceuticals have been continuously increasing in aquatic ecosystems over the past decade due to their extensive use in human and veterinary medicine. Traditional water monitoring methods have been proven insufficient to capture pollution due to pharmaceuticals because of sensitivity limits, their inability to monitor every pollutant, and their failure to provide mechanistic insights into the actions of pollutants. To combat this, new approach methodologies (NAMs) are increasingly introduced as supplementary tools that can provide sensitive metrics for pollution assessment. In addition, NAMs can bridge the gap in ecotoxicological information regarding mixtures of pharmaceuticals, which are more commonly found in aquatic ecosystems versus pharmaceuticals existing alone. In this context, freshwater sentinel species, such as the water flea known as daphnids, were used to assess the acute effects of a typical pharmaceutical mixture on various physiological and molecular responses. In addition to mortality, phenotypic and molecular endpoints such as ingestion rate and enzyme activity were measured to determine the impact of a cocktail of commonly prescribed pharmaceuticals. Specifically, diclofenac, metformin, gabapentin, amoxicillin, trimethoprim, and erythromycin were mixed in equal amounts and tested at 0.1, 1, and 10 mg/L. Results showed differences in mortality, a decrease in feeding, and changes in enzyme activities, thus supporting a distinct pattern in the physiological responses of daphnids

The assessment of pollution is a serious issue and a major consequence of the overgrowing human population and its activities. Focusing on the aquatic ecosystem, traditional approaches of water chemistry mainly provide minimal monitoring with the detection of pollutants, while they fail to produce mechanistic or predictive insight. As such, effect-based methods have gained significant attention for the better mechanistic understanding of aquatic pollution. Among the key species used, daphnids have acquired a central position in aquatic toxicology and ecology. In this study, a novel feeding assay was developed and applied in a battery of exposures to different pollutants. Furthermore, in combination with biochemical markers and sensitive metabolomic analyses, the responses of daphnids following exposures were uncovered in molecular detail. Specific categories of metabolites were identified as significant indicators to predict pollution.

The great increase in nanotechnology in the last 20 years has led to the alarming presence of nanomaterials in the environment as a new category of pollutants. Given the fact that legislation on nanomaterials is not concrete, the monitoring of their toxicity responses remains central. Focusing on tests in aquatic environments, daphnids are commonly employed as a bioindicator species for experiments with nanomaterials. However, until now, there has not been a unified and agreed approach to nanotoxicity testing, while research among different laboratories has been performed with significantly different setups, which may affect the reproducibility of the results. In this study, daphnids were exposed to silver nanoinks and the impact of surface to volume was assessed by comparing shallow vessels such as Petri dishes with deeper exposure vessels. Furthermore, in an attempt to assess whether the tests can be performed in smaller volumes, and thus in miniaturized versions, experiments compared larger and smaller volume setups. Finally, another parameter explored was the crowding of animals in exposure, and therefore their absolute number. Mortality was affected by both surface to volume and miniaturization, and significantly with crowding, supporting the implication of the number of animals in the tests. Further investigation with molecular and phenotypic endpoints confirmed these changes.