Fig. 1. Chemical structures of BPA and its 10 analogues.

Fig. 2. Concentration-response analysis for malformation and mortality. Zebrafish embryos were exposed to BPA and its 10 analogues at a series of concentrations from 8 to 120 hpf. Malformation and mortality were assessed at 120 hpf. The concentration-response curves for malformation (A) and mortality (B) were used to calculate the EC₅₀ and LC₅₀. Values are plotted as the mean ± SEM (N = 60 with 20 embryos in triplicate). (C) Representative malformation phenotypes for exposure at different concentrations. SB, swim bladder; USB, uninflated swim bladder; PE, pericardial edema; YSE, yolk sac edema; BS, bent spine; MT, malformed tail. SC, scoliosis; KY, kyphosis. Scale bar, 100 µm.

Fig. 3. Sublethal concentration exposure causes shoaling behavior deficits. Zebrafish embryos exposed to BPA and its 10 analogues at 0 (0.1 % DMSO), 1 %, 10 %, and 100 % EC₂₅ from 8 hpf to 4.5 dpf were assessed for shoaling at 11 dpf. NND, nearest neighbor distance; IID, inter-individual distance. Values are plotted as the mean ± SEM (N = 20), and the asterisk (*) indicates a significant difference from the vehicle control (0.1 % DMSO) at P < 0.05.

Fig. 4. BPA, BPF, and BPS produce macrocephaly, hyperactivity and ASD-like behavior deficits. Zebrafish embryos exposed to BPA, BPF, and BPS at 0 (0.1 % DMSO), 1 %, 10 %, and 100 % EC₂₅ from 8 hpf to 4.5 dpf. VPA at 5 μM was included as the positive control. (A) Head size t at 5 dpf measured through alcian blue staining with parameters: intraocular distance (ID), lower jaw length (LJL), and ceratohyal cartilage length (CCL), (N = 27–42). (B) Larval locomotion at 5 dpf in a 10 min light period followed by a 10 min dark period (N = 24). (C) Light/dark exploration for larvae at 10 dpf (N = 24). (D) Social contact for larvae at 13 dpf (N = 24). Values are plotted as the mean ± SEM, and the asterisk (*) indicates a significant difference from the vehicle control (0.1 % DMSO) at P < 0.05.

Fig. 5. BPA, BPF, and BPS accelerate neurogenesis. Zebrafish embryos exposed to 0.1 %DMSO (vehicle control), BPA (2 μM), BPF (3 μM), BPS (1 μM), and VPA (5 μM; positive control) from 8 to 108 hpf were assessed for neurogenesis at 4.5 dpf. (A) Staining patterns of mature newborn neurons (HuC/D⁺; green) and mature neurons (NeuN⁺; red) on tissue sections from the anterior telencephalon position. (B) The region of interest in the anterior telencephalon used for fluorescence intensity quantification. (C) The ratio of fluorescence intensity of HuC/D⁺ to NEUN⁺ (N = 6–10). (D) Staining patterns of neural stem cells (GFAP⁺; red) and cell proliferation marker (PCNA⁺; green) on tissue sections from the anterior telencephalon position. (E) The region of interest in the anterior telencephalon used for counting GFAP⁺PCNA⁺ cells. Arrows indicate double positive cells. (F) Quantification of GFAP⁺PCNA⁺ cells (N = 4–10). Values are plotted as the mean ± SEM, and the asterisk (*) indicates a significant difference from the vehicle control (0.1 % DMSO) at P < 0.05. Scale bar, 25 µm.

Fig. 6. BPA, BPF, and BPS upregulate K⁺ ion channel related genes. Zebrafish embryos exposed to 0.1 %DMSO (vehicle control), BPA (2 μM), BPF (3 μM), BPS (1 μM), and VPA (5 μM; positive control) from 8 to 120 hpf were collected at 120 hpf for gene expression analysis. Gene expression fold change (Log₂) for larvae exposed to VPA (A), BPA (B), BPF (C), and BPS (D). Genes related to K⁺ channel: K⁺ channel tetramerization domain containing 4 (kctd4), kctd12, kctd13, K⁺ voltage-gated channel shaker-related subfamily, beta member 1 a(kcnab1a), K⁺ voltage-gated channel, subfamily H (eag-related), member 4a (kcnh4a), K⁺ inwardly-rectifying channel, subfamily J, member 6 (kchj6). Genes related to calcium ion channel: ryanodine receptor 1a (ryr1a), ryr1b, ryr2, ryr3, troponin C type 1a (tnnc-1), and tnnc-2. Sodium ion channel gene: voltage-gated, type IV, beta b (scn4bb). Acetylcholine receptor gene: cholinergic receptor nicotinic alpha 1 subunit (chrna1). Values are plotted as the mean ± SEM (N = 3, pooled sample from 20 larvae per replicate), and the asterisk (*) indicates a significant difference from the vehicle control (0.1 % DMSO) at P < 0.05.