Exposure to chemical and commercial forms of NSAIDs at environmentally relevant concentrations exert transgenerational metabolic responses in daphnids

Pharmaceuticals, including non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin and ibuprofen, are prevalent pollutants in freshwater ecosystems, raising concerns about their ecological impact. While there is literature about pharmaceuticals in aquatic environments, the effects of both chemical and commercial forms of NSAIDs on aquatic organisms, particularly across generations, remain insufficiently explored. This study aims to bridge this gap by assessing the impact of pure and commercial forms of indomethacin, ibuprofen, and their mixture on Daphnia magna, a key freshwater species. Toxicity curves, enzyme activities, and metabolomics were used as endpoints to assess physiological responses to chronic and transgenerational exposures at an environmentally relevant concentration of 5 ​μg/L. Results showed changes in the activities of β-galactosidase and lipase, while a targeted LC-MS/MS approach revealed distinct metabolic fingerprints as a result of exposure for four generations to the chemical and commercial indomethacin and ibuprofen. Notably, exposure to chemical mixture of the two NSAIDs increased amino acids and biogenic amines, a trend also observed with commercial NSAIDs and their mixture, while the chemical NSAIDs did not have the same impact. This work emphasizes the necessity of ecotoxicological studies with transgenerational exposures as an approach to comprehend the effect of pharmaceutical stressors at low exposure concentrations, using molecular responses in physiology. This approach contributes to the broader mechanistic understanding of the ecological implications of these pharmaceuticals in freshwater ecosystems. • Activities of β-galactosidase and lipase were impacted in the fourth generation. • Daphnids recovered by the fifth generation from commercial but not from the pure chemical forms of NSAIDs. • Commercial NSAIDs significantly altered the metabolic profile of daphnids.