Timeline of the sex developmental period in ZF. Exposure 1 was performed from 0 to 15 dpf during early sexual development while exposure 2 was conducted from 15 to 42 dpf during the sex differentiation period (Orban et al., 2009; Luzio et al., 2015).

Whole-body cortisol levels (ng^⋅g^–1 fish) in adult females and males (6 months of age). (A) Adult ZF exposed to FLX from 0 to 15 dpf (period 1, early sexual development) during early development. (B) Adult ZF exposed to FLX from 15 to 42 dpf (period 2, late sexual development) during the period of sex differentiation in ZF. (C) Stress response (stress-induced cortisol levels – unstressed cortisol levels) in females and males from exposure period 1 and period 2. The two concentrations of FLX used were 0.54 and 54 μg^⋅L^–1 denoted as LOW and HIGH, respectively. The stress response was induced using a standardized net handling stress protocol. The data from the three panels are presented as mean ± SEM and analyzed by two-way ANOVA (and on ranks for data on panel (A) both graphs, and panel (C) period 1), P < 0.05. The letters represent statistical difference when interactions are present. P-values shown above the bars represent significant difference compared with unstressed [for (A,B)] or significant difference between sex [for (C)]. The symbols represent significant difference within the FLX treatments: ^∗P < 0.05 compared with CTR, ^∗∗P < 0.005 compared with CTR and ^#P < 0.05 compared with HIGH. n = 4–8 per group in females and n = 6–10 in males.

Behavioral response following the novel tank diving test in adult ZF (6 months of age). (A) PC1 of females and males exposed to FLX (0.54 and 54 μg^⋅L^–1, LOW and HIGH, respectively) from 0 to 15 dpf (period 1) during early sexual development. (B) PC1 of females and males exposed to FLX (0.54 and 54 μg^⋅L^–1, LOW and HIGH, respectively) from 15 to 42 dpf (period 2) during the period of sex differentiation (late sexual development) in ZF. (C) The behavioral response of females and males exposed to metyrapone (MET; 325 μM) as adults for 1 week. In all panels, PC1 represents the exploratory behavior of the fish, where positive scores are associated with high exploratory activity, and negative scores are linked to reduced behavior. Data were analyzed using two-way ANOVA on ranks [and two-way ANOVA for panel (A)]. Data are presented as box plots showing the median (solid line), the mean (dashed line), the interquartile range (box) and the whiskers embracing data within the 10 and 90th percentiles; all data outside the range of the whiskers are presented as individual data points. n = 12–14 fish per group in panel (A), n = 8–13 per group in panel (B) and n = 15 per group in panel (C). The letters in all panels represent statistical difference when interactions are present.