## 3 Results  
### Locomotor Activity and Thigmotactic Behavior  
After 12 days of exposure to fluoxetine, no differences across treatments were observed in terms of total distance moved during light and dark periods (Figure 1A). On the other hand, differences in the time spent swimming was observed between light and dark conditions for control, 0.1 and 1 ug/L of fluoxetine, with fish swimming for longer periods during the light period (Figure 1B). Furthermore, fluoxetine significantly increased fish swimming time during the dark period at concentrations 1 and 10 ug/L (Two-Way ANOVA, 1 ug/L: \(p\) = 0.002; 10 ug/L: \(p\) = 0.003) (Figure 1B). In terms of thigmotactic behavior, no significant differences were observed between dark and light conditions in the total distance traveled in the outer zone (Figure 1C). When considering the time spent in the outer zone, the control group presented significant differences in response to dark and light conditions (Two-Way ANOVA, Control: \(p\) = 0.003), spending more time in the outer zone during the light period. However, this trend was not observed in fish exposed to fluoxetine, where no differences were observed during dark and light periods (Figure 1D). After 21 days exposure, fish exposed to all fluoxetine concentrations showed significant decreases in the swimming activity during the light period, when compared to control (Two-Way ANOVA, 0.1 ug/L: \(p\) = 0.022; 1 ug/L: \(p\) = 0.010; 10 ug/L: \(p\) = 0.041) (Figure 2A). In terms of total time spent swimming, fish spent more time swimming during the light period (as observed at 12 days exposure), a pattern that was not altered by fluoxetine exposure (Figure 2B). Both parameters analyzed for fish thigmotactic behavior (total distance and time spent in the outer zone) remained similar across dark and light conditions as well as across fluoxetine treatments (Figure 2C,D). The measurement of the angles during the fish path, after 12 days exposure, showed that the frequency of class 1 angles (high amplitude angles) was much higher during the light, where all treatments, including the control, showed \(>\) 2-fold more frequency than that during the dark period (Figure 3A). Contrary to class 1 angles, class 4 angles (low amplitude angles) were significantly more frequent during the dark period (Figure 3B). However, no effect was observed for class 1 and class 4 angle frequency. After 21 days of exposure, profiles of turn angle frequencies, across dark and light periods were similar to those observed at day 12, and not affected by fluoxetine exposure (Figure 3C,D). In the remaining classes of angles, no effects of fluoxetine exposure were observed after 12 and 21 days of exposure (data not shown). Figure 1: Effects of fluoxetine concentrations on locomotor activity and thigmotactic behavior of juvenile zebrafish, in response to dark and light cycles, after 12 days exposure. (**A**)—Total swimming distance traveled by the juvenile zebrafish; (**B**)—Total time spent swimming; (**C**)—Total distance traveled in the outer zone; (**D**)—Total time spent swimming in the outer zone. Results are expressed as mean values \(\pm\) standard error (Supplementary Material, Table S1). Yellow bars represent the light period and grey bars represent the dark period. Asterisks (*) indicate differences towards the control and the symbol “#” indicates differences between dark and light periods in their respective concentrations (Two-way ANOVA, Holm-Sidak method, \(p<0.05\)). Figure 2: Effects of fluoxetine concentrations on locomotor activity and thigmotactic behavior of zebrafish, in response to dark and light cycles, after 21 days exposure. (**A**)—Total swimming distance traveled by the juvenile zebrafish; (**B**)—Total time spent swimming; (**C**)—Total swimming distance traveled in the outer zone; (**D**)—Total time spent swimming in the outer zone. Results are expressed as mean values \(\pm\) standard error (Supplementary Material, Table S2). Yellow bars represent the light period and grey bars represent the dark period.