Fig. 1. Spontaneous movements recorded hourly from 23 to 28 hpf in zebrafish embryos/larvae following exposure to DBDPE from 2 hpf until 120 hpf. Data are expressed as mean ± SEM (60 individuals per group). * p < 0.05, * * p < 0.01 and * ** p < 0.001 indicate significant difference detected between exposure groups and control group.

Fig. 2. Touch response and locomotor behavioral changes in zebrafish embryos/larvae following exposure to DBDPE from 2 hpf until 120 hpf. (A) Touch response measured at 36, 48 and 72 hpf (48 individuals per group). (B) Free-swimming activity detected under 10 min continuous visible light at 120 hpf (24 individuals per group). (C) Locomotor traces during the dark-light-dark photoperiod stimulation test performed at 120 hpf. (D) Average swimming speeds at 120 hpf in response to dark-to-light photoperiod stimulation (24 individuals per group). Data are expressed as mean ± SEM. * p < 0.05 and * * p < 0.01 indicate significant difference detected between exposure groups and control group.

Fig. 3. Changes in AChE and ChAT activity (A) and transcriptions of nAChRs (B) in larval zebrafish at 120 hpf following exposure to DBDPE from 2 hpf until 120 hpf. Data are expressed as mean ± SEM (n = 4). * p < 0.05, ** p < 0.01 and *** p < 0.001 indicate significant difference detected between exposure groups and control group.

Fig. 4. Altered expressions of proteins associated with CNS development in larval zebrafish at 120 hpf following exposure to DBDPE from 2 hpf until 120 hpf. (A) Western blot analyses carried out with antibodies against α1-TUBULIN, MBP, GAP43, SYN2α and GAPDH in larval zebrafish exposed to DBDPE. (B) Quantification of abundances of the four proteins relative to the control. Data are represented as the mean ± SEM value from four replicates. * p < 0.05 indicates significant difference relative to the control.

Fig. 5. Molecular docking of DBDPE with neurodevelopment associated proteins α1-TUBULIN (A, B), Synapsin II (SYN2a) (C, D) and ChAT (E, F). The dashed line represents the interactions between the ligand (DBDPE) and the target proteins.