Effect of EPN in zebrafish embryos at 96 h post-fertilization (hpf). (a), Phenotypes of EPN-treated zebrafish embryos at serial concentrations of 62.5, 125, 250, and 500 μg/L; (b), survival rates (%) of EPN-treated zebrafish embryos at serial concentrations of 62.5, 125, 250, and 500 μg/L; (c), hatching rates (%) of EPN-treated zebrafish embryos at serial concentrations of 62.5, 125, 250, and 500 μg/L; (d), deformity rates (%) in the EPN-treated zebrafish embryos at the different concentrations of 62.5, 125, 250, and 500 μg/L. PE means pericardial edema, and YSE indicates yolk sac edema. BP means blood pooling. The scale bar represents 200 µm. A lowercase letter indicates a significant difference (p < 0.05).

Acridine orange staining on EPN-treated zebrafish embryos to display cell death. Cell deaths (red arrowhead) in zebrafish embryos after the exposure to EPN at the concentration of 500 μg/L were examined at 48, 72, and 96 hpf. These photographs are focused on the forehead’s spots, and cell deaths on the rest of the body are displayed in Fig. S2. The scale bar represents 200 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Effect of EPN observed on the vascular system in transgenic zebrafish Tg(fli1a:EGFP) embryos. Vascular observations are placed in the control group and EPN-treated (500 μg/L) group at 48 hpf. The white arrowhead indicates the intersegmental vessels (ISV). The white double arrow shows the total body length. The red and blue double arrows represent the head’s middle cerebral vessel (MCeV) and the maximum width of the head’s vessels. Formation of the vascular system on 72 and 96 hpf is displayed in Fig. S3. The scale bar represents 200 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Effects on cardiac development of EPN-treated zebrafish embryos using transgenic zebrafish Tg(cmlc2:EGFP) embryos. (a), Heart shape in transgenic zebrafish embryos with or without treatment of EPN (500 μg/L) at 48, 72, and 96 hpf; (b), average heart rate per minute (n = 8) in transgenic zebrafish embryos with or without treatment of EPN (500 μg/L) at 48, 72, 96 hpf. The scale bar represents 200 µm. A different lowercase letter indicates a significant difference (p < 0.05, a > b > c > d).

mRNA expression levels of myl7 and nppa related to heart development in EPN-treated zebrafish embryos. Changes in mRNA expression levels of genes related to the heart of 48 hpf (a), 72 hpf (b), and 96 hpf (c) measured by RT-qPCR. myl7, myosin, light chain 7; nppa, natriuretic peptide A. A different lowercase letter indicates a significant difference (p < 0.05, a > b > c > d).

Blood pooling (red arrow) was observed in the EPN-exposed transgenic zebrafish at 48, 72, and 96 hpf. The control group without exposure to EPN showed normal blood flow circulation, whereas blood flow proceeded irregularly in the EPN-treated groups. The scale bar represents 200 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

EEG recording results for EPN treatment in zebrafish embryos. (a) EEG recording method. EPN was treated at 6 hpf in zebrafish larvae (n = 7 or 8) until 96 hpf. PTZ was treated to zebrafish larvae for 15 min before 96 hpf; (b) Number of events; (c) Cumulative duration of events; (d) Mean duration of events. Results were expressed as mean ± SEM. Significant differences in the comparison to control expressed by the symbols *,**,***, and **** for expressing p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Significant differences in the comparison to PTZ-treated group (positive control) expressed by the symbols #,##,###, and #### for expressing p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively.

Acknowledgments
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