## 4 Discussion  
The present study evaluated different behavior endpoints associated with anxiety/stress response (locomotion, thigotaxis, and exploratory behavior), sociability (social test), and biochemical changes associated with neurotransmission, biotransformation, and oxidative stress, after chronic exposure of juvenile zebrafish to the SSRI antidepressant fluoxetine. In general, the data showed that, in the low \(\mu\)g/L range, fluoxetine can cause mild biological alterations in the tested endpoints affecting fish locomotor activity which, in the long term, may compromise population fitness. The concentrations tested in this study are considered environmentally relevant, and fluoxetine levels similar to those tested in the present study, and even lower, have already been detected in the environment. For example, in Baiyangdian Lake, Pearl River, Songhua River, Yellow River, Huai River, Hai River, and Liao River, fluoxetine levels up to 0.101 \(\mu\)g/L were detected [79, 80]. Alternating light and dark changes are often used to assess the stress response in zebrafish larvae, where, larvae respond to the shift from light to dark with a burst of activity [81, 82]. However, this assessment is scarcely used in juvenile fish. In the present study, after 12 days of exposure to fluoxetine, zebrafish juveniles altered swimming time during the dark period at the highest concentrations. Curiously, the total distance traveled during this period was unaffected by fluoxetine, which indicates a decrease in swimming velocity and, therefore, a lower effect on the stress reaction to this condition. After 21 days of exposure, the change in locomotor activity during the light period became more evident. In a study carried out by [PERSON] et al. (2020) [60], fluoxetine reduced the swimming distance of embryos in the dark and changed the maximum speed during light periods, after 96 h  
Figure 6: Effects of fluoxetine concentrations on zebrafish enzymatic activities: (**A**)—Glutathione S-transferase (GST) activity; (**B**)—Glutathione peroxidase (GPx) activity; (**C**)—Cholinesterase (ChE) activity; (**D**)—Lactate Dehydrogenase (LDH) activity. Results are expressed as mean values \(\pm\) standard error (Supplementary Material, Table S6). Asterisks (*) indicate differences towards the control (One Way ANOVA, Dunnett’s Method, \(p<0.05\)). exposure. Thigmotaxis is seen as the tendency of an animal placed in a new environment, to remain close to the walls and avoid the center of the aquarium. In fish, the persistence of this activity in the outer zone can be considered a measure of anxiety [69, 83, 84]. Fluoxetine did not affect this behavior following 21 days of exposure. Regarding the measurement of angles during the fish's path, the high amplitude angles (class 1) showed no effects of fluoxetine exposure, after 12 and 21 days of exposure. High amplitude angles are indicators of erratic swimming behavior (stress behavior) and the decrease in its frequency manifests the anxiolytic effect of fluoxetine on the stress behavior of exposed juveniles. The novel tank test aims to assess the exploratory behavior in zebrafish, focusing on measures of \"vertical\" behavior patterns. This test explores the tendency of a fish to dive and stay at the bottom of a new environment (geotaxis), before exploring the upper areas. This test is used extensively to assess the effects of a wide variety of compounds, including pharmacological chemicals [69, 70]. Drugs with antidepressant and anxiolytic properties have the ability to modify this behavior, consequently leading the fish to explore the new space sooner and spend less time in the bottom [71, 85, 86]. In the present study, there was no evidence of effects over fish geotaxis behavior. Similar to this study, [PERSON] et al. (2016) [87] also failed to observe any changes in fish behavior to a novel environment following 7 days of exposure to 10 \(\mu\)g/L of fluoxetine.