Multilevel assessment of carbamazepine effects: An integrative approach using zebrafish early-life stages.

Carbamazepine (CBZ) is the most commonly used drug in epilepsy treatment, and its metabolites are commonly detected among persistent pharmaceuticals in the aquatic environment. This study aimed to investigate CBZ effects on early-life-stage zebrafish (Danio rerio) (from 2 to 168 hpf) by employing of an integrative approach linking endpoints from molecular to individual level: (i) development; (ii) locomotor activity; (iii) biochemical markers (lactate dehydrogenase, glutathione-S-transferase, acetylcholinesterase and catalase) and (iv) transcriptome analysis using microarray. A 168 h - LC50 of 73.4 mg L-1 and a 72 h - EC50 of 66.8 mg L-1 for hatching were calculated while developmental effects (oedemas and tail deformities) were observed at CBZ concentrations above 37.3 mg L-1. At the biochemical level, AChE activity proved to be the most sensitive parameter, as evidenced by its decrease across all concentrations tested (∼25% maximum reduction, LOEC (lowest observed effect concentration) < 0.6 µg L-1). Locomotor behaviour seemed to be depressed by CBZ although this effect was only evident at the highest concentration tested (50 mg L-1). Molecular analysis revealed a dose-dependent effect of CBZ on gene expression. Although only 25 genes were deregulated in organisms exposed to CBZ when compared to controls, both 0.6 and 2812 µg L-1 treatments impaired gene expression related to development (e.g. crygmxl1, org, klf2a, otos, stx16 and tob2) and the nervous system (e.g. Rtn3, Gdf10, Rtn3), while activated genes were associated with behavioural response (e.g. prlbr and taar). Altogether, our results indicate that environmentally relevant CBZ concentrations might affect biochemical and genetic traits of fish. Thus, the environmental risk of CBZ cannot be neglected, especially in a realistic scenario of constant input of domestic effluents into aquatic systems.

Fluoxetine chronic exposure affects growth, behavior and tissue structure of zebrafish.

Fluoxetine (FLX) is among the top 100 pharmaceutical prescribed annually worldwide and consequently is often detected in wastewater treatment plant effluent and surface waters, in concentrations up to 2.7 and 0.33 µg/L, respectively. Despite the presence of FLX in surface waters, little is known about its chronic effects in fish. Thus, this study aimed at investigating the chronic toxicity of FLX to Danio rerio adults. Rate of weight gain, behavior (feeding and swimming activity) and tissue organization (liver and intestine) were evaluated, after 30 days exposure. A lower rate of weight gain was observed at 100 µg/L FLX. The food intake time decreased, showing a decrease in fish appetite. The preference for the upper aquarium layer was observed at 10 and 100 µg/L of FLX, indicating an inhibition of the stress level (anxiolytic effect). Mild to moderate damage of hepatic tissue and a decrease epithelium height and increase in villus height of intestine were observed in fish exposed to concentrations as low as 0.01 µg/L. Based on obtained results, chronic exposure of fish to FLX could affect swimming and feeding behavior and alter morphological structure of liver and intestine tissues at environmental levels.

Exposure to low concentration of fluoxetine affects development, behaviour and acetylcholinesterase activity of zebrafish embryos.

Fluoxetine (FLX) is a selective serotonin reuptake inhibitor (SSRI) antidepressant widely used in clinics and very often found in environmental samples of urban aquatic ecosystems in concentrations ranging from ng/L to µg/L. Fish populations might be especially susceptible to FLX due to the presence of conserved cellular receptors of serotonin. Neurotoxic effects on fish biota of polluted water bodies may be expected, but there are no sufficient studies in the current literature to elucidate this hypothesis. Batteries of embryo larval assays with zebrafish were performed to evaluate the potential effects of FLX exposure, including environmentally relevant concentrations. Evaluated parameters included survival, development, behaviour and neuronal biochemical markers. Regarding acute toxicity, a 168 h-LC50 value of 1.18 mg/L was obtained. Moreover, hatching delay and loss of equilibrium were observed, but at a concentration level much higher than FLX measured environmental concentrations (>100 µg/L). On the other hand, effects on locomotor and acetylcholinesterase activity (≥0.88 and 6 µg/L, respectively) were found at levels close to the maximum reported FLX concentration in surface waters. Altogether, these results suggest that FLX is neurotoxic to early life stages of zebrafish, in a short period of time causing changes in important ecological attributes which can probably be linked from molecular to population level.