Experimental design timeline for different life stages. (A) Zebrafish embryos up to 4 h post-fertilization (hpf) were exposed to water (control groups) or to different concentrations of the two herbicides (atrazine or diuron) for 96 h, after which they were transferred to an environment free of herbicides in which they remained until 7 days post-fertilization (dpf). During 7 dpf, morphological parameters, survival, and hatching rates were daily monitored. At 7 dpf, behavioral analyzes were performed and included the exploration of a new environment, aversive behavior, and optomotor response and individuals were photographed. (B) Adult animals aged 12–18 months were exposed to water (control groups) or to different concentrations of atrazine or diuron. Animals were exposed for 96 h, after which they were transferred to an environment free of herbicides where they remained for more than 3 days. On the third day, their performance on a new environment and the social interaction were evaluated. In the following day, an analysis of aggressive behavior was performed.

Kaplan–Meier survival and hatching rates for zebrafish embryos and larvae exposed to the herbicides atrazine and diuron during their first 96 hpf and monitored for 7 days (168 hpf). (A) Atrazine effects on survival rates. Atrazine significantly reduced survival (p < 0.0001) at 5 mg/L when compared to controls; (B) Diuron effects on survival rates. Diuron significantly reduced survival (p < 0.0001) at 2.5 and 5 mg/L when compared to controls; (C) atrazine effects on the hatching rate. Atrazine did not impact hatching on surviving animals (p = 0.1231); (D) diuron effects on the hatching rate. Diuron significantly delayed hatching (p = 0.0359). Rates are expressed as percentage of the total number of animals from each group. Experiments were performed in triplicate, n = 60.

Morphological effects of exposure to the herbicides atrazine and diuron during their first 96 hpf in zebrafish larvae at 7 dpf. (A) Atrazine exposure effects on the body length (µm); (B) atrazine exposure effects on the head length (µm); (C) atrazine exposure effects on the mesencephalon width (µm); (D) atrazine exposure effects on the forebrain width (µm); (E) atrazine exposure effects on the ocular distance (µm); (F) representative images of the most prominent morphology or teratogenic effects observed in individuals exposed to atrazine during the 7 dpf; (G) diuron exposure effects on the body length (µm); (H) diuron exposure effects on the head length (µm); (I) diuron exposure effects on the mesencephalon width (µm); (J) diuron exposure effects on the forebrain width (µm); (K) diuron exposure effects on the ocular distance (µm); (L) representative images of the most prominent morphology or teratogenic effects observed in individuals exposed to diuron during 7 dpf. Data were analyzed by one-way ANOVA followed by a post-hoc Tukey’s test. ** represents significant differences at p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001 in relation to control.

Exploratory behavior of zebrafish larvae at 7 dpf after atrazine or diuron exposure during the initial 96 hpf. Columns depict means ± S.E.M. Sample sizes are n = 27 for each group. (A) Atrazine exposure effects on total distance traveled (m); (B) atrazine exposure effects on mean speed (m/s); (C) atrazine exposure effects on time in movement (s); (D) atrazine exposure effects on time in the outer area (s) of the well; (E) diuron exposure effects on total distance traveled (m); (F) diuron exposure effects on mean speed (m/s); (G) diuron exposure effects on time in movement (s); (H) diuron exposure effects on time in the outside area (s). Data were analyzed by one-way ANOVA followed by a post-hoc Tukey’s test. Asterisks represent significant differences at *p ≤ 0.01 and ***p ≤ 0.001 in relation to controls.

Cognitive effects of atrazine or diuron exposure during the initial 96 hpf at 7 dpf larvae. Columns depict means ± S.E.M. Sample sizes are n = 45 for each group. (A) Atrazine exposure effects on the avoidance from an aversive visual stimulus; (B) Diuron exposure effects on the avoidance from an aversive visual stimulus; (C) Atrazine exposure effects on the optomotor response to moving stripes; (D) Diuron exposure effects on effects on the optomotor response to moving stripes. Data were analyzed by one-way ANOVA followed by a post-hoc Tukey’s test.

Effects of acute exposure to atrazine or diuron on locomotion and exploratory behavior in adult zebrafish. Columns depict means ± S.E.M. Sample sizes are n = 18 for each group (A) Atrazine exposure effects on total distance traveled (m); (B) atrazine exposure effects on mean speed (m/s); (C) atrazine exposure effects on time mobile (s); (D) atrazine exposure effects on time spent in the upper tank zone (s); (E) diuron exposure effects on total distance traveled (m); (F) diuron exposure effects mean speed (m/s); (G) diuron exposure effects time mobile (s); (H) diuron exposure effects time spent in the upper zone (s). Data were analyzed by one-way ANOVA followed by a post-hoc Tukey’s test.

FIGURE 7. Effects of acute exposure to atrazine or diuron on aggression and social interaction in adult zebrafish. Columns depict means ± S.E.M. Sample sizes are n = 18 for each group. (A) Atrazine exposure effects on aggressive behavior estimated by the time spent at the stimulus zone closer to the mirror (s); (B) atrazine exposure effects on the number of bites; (C) diuron exposure effects on aggressive behavior estimated by the time spent at the stimulus zone closer to the mirror (s); (D) diuron exposure effects on the number of bites; atrazine exposure effects on time mobile (s); (E) atrazine exposure effects on social behavior estimated by the time spent at the stimulus zone closer to the mirror (s); (F) atrazine exposure effects on social behavior estimated by the time spent at the stimulus zone closer to the mirror (s). Data were analyzed by one-way ANOVA followed by a post-hoc Tukey’s test.