Assessing pH-dependent toxicity of fluoxetine in embryonic zebrafish using mass spectrometry-based metabolomics.

While it is well known that fluoxetine is more toxic to aquatic organisms at high pH, the metabolic dysregulations related to observed pH-dependent effects are still poorly understood. In the present study, we utilized a gas chromatography mass spectrometry (GC-MS) based metabolomics approach to assess metabolomic profile changes in developing zebrafish embryos following exposure (2 hpf-96 hpf) to different concentrations of fluoxetine at three environmentally relevant pH values (7.0, 8.0, and 9.0). Multivariate data analyses and pathway analyses were used to assess metabolomic profile changes and elicit important biochemical information regarding pH-dependent toxicity of fluoxetine. Overall, the affected biochemical functions related to fluoxetine exposure included amino acid metabolism, energy metabolism, nitrogenous waste excretion and osmolyte functions. While fluoxetine exposure (56 μg/L, 70 μg/L and 500 μg/L) caused no significant changes at pH 7, 500 μg/L and 70 μg/L fluoxetine was differentiated from the controls at pH 8 and pH 9 respectively. Three, eight and seven metabolites were identified as the most adversely affected at pH 7, 8 and 9, respectively. The altered metabolites associated with fluoxetine toxicity at high pH included urea, glycine and d-glucose 6-phosphate. Exposure to 70 μg/L fluoxetine, did not cause significant metabolomic profile changes at pH 7, However, the results indicate that this exposure concentration at pH and 9 can cause significant metabolic dysregulation related to apoptosis and oxidative stress. Increasing aqueous pH progressively enhanced fluoxetine induced toxicity for the 70 μg/L exposure group. The observed impacts included higher energy consumption at pH 7, a breakdown of reserve energy to supplement energy demand at pH 8 and impaired lipid metabolism at pH 9. This study provides important information regarding molecular-level effects related to pH-dependent exposure of fluoxetine in embryonic zebrafish.