Norfluoxetine and venlafaxine in zebrafish larvae: Single and combined toxicity of two pharmaceutical products relevant for risk assessment

Antidepressant metabolites are found in natural and waste waters. However, investigation of their toxic effects on aquatic animals, single or in mixture with other occurring psychoactive drugs, has been neglected. Here, effects of 80hpf exposure to norfluoxetine (0.64-400 ng/L), venlafaxine (16-10000 ng/L) or their combination (3.2 ng/L +2000 ng/L, respectively) were investigated in embryos and zebrafish larvae. Mortality, embryonic malformations, sensorymotor reflexes and the expression of 34 genes involved in the toxicants mode-of-action (MoA) and metabolism were evaluated (i.e. monoamine receptors and transporters, nuclear receptors, and detoxification transporters and enzymes). Compared to controls, norfluoxetine treatments only caused de pigmentation of embryos and larvae. Venlafaxine-exposed larvae exhibited depigmentation and spinal deformities, impaired sensorymotor reflexes, alterations in the expression of genes belonging to the serotonergic, noradrenergic and dopaminergic pathways, as well as nuclear receptors related to lipid and drug metabolism. The mixture elicited distinct interaction effects, depending on the level of biological organisation analysed and the neurotransmitter pathways affected; synergism (lethality), no interaction (sensorymotor reflexes), antagonism and inverse agonism (gene expression). The results call for investigation of the toxicity of pharmaceutical metabolites single and in mixture, as well as their risk assessment in approaches accounting for possible interactions with other endocrine-disrupting compounds.