Fig. 1. Behavioral changes in adult zebrafish waterborne exposed to 0.3 and 3 μg/L glyphosate for 2 weeks. (A) Behavioral parameters assessed in standard 6-min novel tank test (NTT), as well as a cartoon of the experimental tank divided into two equal virtual zones, top and bottom; (B) Behavioral parameters assessed in standard 6-min dark-light test (DLT), as well as a cartoon of the experimental tank divided into two equal zones, white and black; (C) Behavioral parameters of zebrafish shoaling behavior in control and glyphosate-treated zebrafish. Data reported as scatter plot with the median (n = 23–29 for NTT, n = 20–22 for DLT and n = 19–20 for shoaling test), *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Dunnett’s multiple comparison test (Total distance, Distance in top, Time in top, Transitions to top, Erratic bouts, High mobility frequeny, Average interfish distance and Farthest distance) or Kruskal Wallis test with Bonferroni correction (Latency to the top, Erratic duration) Data from 2 to 4 independent experiments.

Fig. 2. Increased dopaminergic activity in the anterior brain region of zebrafish exposed to 0.3 and 3 μg/L glyphosate for 2 weeks. Levels of L-tyrosine, DOPAC, HVA, and the ratios DOPAC/DA and HVA/DA increased significantly in the anterior brain of zebrafish exposed to 3 μg/L glyphosate for 2 weeks. The cartoon on the upper left shows a dorsal view of an adult zebrafish brain, with the three regions collected for the neurotransmitter analysis. Data reported as scatter plot with the median. *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA with Dunnett’s multiple comparison test (NE) or Kruskal Wallis test with Bonferroni correction (L-tyrosine, DA, DOPAC, HVA, 3-MT, DOPAC/ DA, HVA/ DA). Data from 2 independent experiments.

Fig. 3. Effect of two weeks exposure to 0.3 and 3 μg/L glyphosate on the expression of genes associated with the dopaminergic system (th1, th2, comta, comtb, mao, scl6a3, scl18a2 and gfap) in the anterior-middle brain regions of adult zebrafish. Data reported as scatter plot with the median. *P < 0.05, **P < 0.01; one-way ANOVA with Dunnett’s multiple comparison test (th1, th2, comta, mao, scl6a3, gfap) or Kruskal Wallis test with Bonferroni correction (comtb, scl18a2). Data from 2 independent experiments (n = 8).

Fig. 4. Effect of two weeks exposure to 0.3 and 3 μg/L glyphosate on the antioxidant status and lipid peroxidation levels in the brain of adult zebrafish. Whereas catalase and superoxide dismutase (SOD) activities increased, the gamma-glutamate cysteine ligase (γ-GCL) activity was strongly inhibited in the brain of glyphosate-treated fish. Moreover, total glutathione stores decreased and lipid peroxidation levels increased in the treated fish. Data reported as scatter plot with the median. *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA with Dunnett’s multiple comparison test (catalase, γ-GCL, GSx) or Kruskal Wallis test with Bonferroni correction (SOD, LPO). Data from 2 independent experiments (n = 11–13).