When zebrafish are relocated to a new environment, they seek protection by diving and staying at the safety home, until they feel safe enough to explore the unfamiliar environment (26). The swimming trajectories showed that zebrafish in the control group can swiftly explore and adapt to the novel environment, but chronic exposure to acrylamide reduces the ability to adapt to the unfamiliar environment (Fig. 3 A). Visualized heatmaps showed significant changes in swimming trajectories of zebrafish in the acrylamide exposure groups compared with those in the control group (Fig. 3 B). Furthermore, we found the swimming time and distance ratios in Zone 1 exhibited a dose-dependent decreasing trend in acrylamide exposure groups. Chronic exposure to acrylamide (0.5 mM) significantly decreased both swimming time and distance in Zone 1 and increased those in Zone 2 (Fig. 3 C and D). We recorded the novel object exploration test to visualize the behavioral alteration between the control and each acrylamide-treated group (Movie S3). The movie displays that zebrafish in the control group could swiftly explore and adapt to the novel environment, but chronic exposure to acrylamide reduced the ability to adapt to the unfamiliar environment, which indicated that acrylamide induces anxiety-and depressive-like behaviors by reducing exploration ability of zebrafish.

Moreover, the social preference test was used to assess sociality of zebrafish. Since the zebrafish are a group preference species, they frequently swim by forming a school and swim closely to one another (27). In the current social preference test, representative radar maps and visualized heatmaps exhibited significant changes of preference in swimming trajectories of zebrafish in acrylamide exposure groups compared to those in the control group, indicating that chronic exposure to acrylamide remarkably impairs the sociality of zebrafish (Fig. 3 E–G). For detailed parameters of behavioral trajectories, chronic exposure to acrylamide (0.5 mM) significantly increased both swimming time and distance ratios in the left zone and decreased those in the right zone (Fig. 3 H and I). Notably, chronic exposure to acrylamide (0.5 mM) significantly elevated traversing times and number of crossing the middle line (Fig. 3 J and K).