Diclofenac affects early embryo development in the marine bivalve Mytilus galloprovincialis

Diclofenac-DCF, one of the most widely prescribed non-steroidal anti-inflammatory drug, is globally detected in environmental compartments. Due to its occurrence in freshwater and potential impact on aquatic organisms, it has been added to the watch list of chemicals in the EU Water Directive; consequently, research on the impact of DCF in model aquatic organisms has great regulatory implications towards ecosystem health. DCF is also detected in coastal waters at concentrations from ng/L to 1 μg/L, as well as in marine organisms, such as the mussel Mytilus. Increasing evidence indicates that environmental concentrations of DCF have multiple impacts in adult mussels. Moreover, in M. galloprovincialis, DCF has been shown to affect early embryo development. The developmental effects of DCF in mussels were further investigated. DFC (1 and 10 μg/L) was added at different times post-fertilization (30 min and 24 hpf) and the effects were compared in the 48 hpf embryotoxicity assay. Shell mineralization and morphology were investigated by polarized light microscopy, X-Ray Spectrometry-XRD and Scanning Electron Microscopy-SEM. Transcriptional profiles of 12 selected genes physiologically regulated across early embryo development were assessed at 24 and 48 hpf. DCF induced shell malformations, irrespectively of concentration and time of exposure. DCF phenotypes were characterized by convex hinges, undulated edges, fractured shells. However, no changes in biomineralization were observed. DCF affected gene transcription at both times pf, in particular at 1 μg/L. The most affected genes were those involved in early shell formation (CS, CA, EP) and biotransformation (ABCB, GST). The results confirm that Mytilus early development represents a significant target for environmental concentrations of DCF. These data underline how the standard embryotoxicity assay, in combination with a structural and transcriptomic approach, represents a powerful tool for evaluating the early impact of pharmaceuticals on mussel embryos, and identification of the possible underlying mechanisms of action.