Schematic diagram showing the overall experimental design for the study. The upper panel shows the timing and workflow to conduct cardiovascular assessments in zebrafish. The bottom panel shows the physiological and molecular endpoints used to evaluate the potential adverse effect of the tested chemicals.

The morphology of 72 hpf zebrafish larvae after acute exposure in 0 (A), 0.5 (B), and 2.5 ppm (C) of PON (blue arrow show normally developed cardiac chamber while red arrow show abnormal development of cardiac chamber). Zebrafish cardiac physiology parameters (D–G, blue box) and cardiac rhythm parameters ((H–M), red box) after exposure to PON. Data are presented as mean ± standard deviation, and the statistical significance was calculated using ordinary one-way ANOVA with Dunnet multiple comparison test. (** p < 0.01, *** p < 0.001, **** p < 0.0001).

The morphology of 72 hpf zebrafish larvae after acute exposure in 0 (A), 5 (B), and 10 ppm (C) of ASC (blue arrow shows the normally developed cardiac chamber). Zebrafish cardiac physiology parameters ((D–G), blue box) and cardiac rhythm parameters ((H–M), red box) after exposure to ASC. Data are expressed as mean ± standard deviation, and the statistical significance was calculated using ordinary one-way ANOVA with Dunnet multiple comparison test (degree of significance was set to p < 0.05).

The morphology of 72 hpf zebrafish larvae after acute exposure in 0 (A), 0.5 ppm of PON (B), 10 ppm of ASC (C), and a combination of 0.5 and 10 ppm of PON and ASC (D) (blue arrow show the normally developed cardiac chamber). Zebrafish cardiac physiology parameter ((E–H), blue box) and cardiac rhythm parameter ((I–N), red box) after exposure to PON and ASC. Data are expressed as mean ± standard deviation, and the statistical significance was calculated using ordinary one-way ANOVA with Dunnet multiple comparison test (degree of significance was set to p < 0.05).

Representative of zebrafish larvae blood flow oscillation pattern after incubation in 0.05% DMSO (grey), 0.5 (green), 1 (yellow), and 1.5 (red) ppm of PON (dash line show the average velocity) (A). Maximum (B) and average (C) blood flow velocity in the dorsal aorta of zebrafish larvae after exposure to PON. Data are presented as mean ± standard deviation, and the statistical significance was calculated using ordinary one-way ANOVA with Dunnet multiple comparison test. (** p < 0.01, *** p < 0.001, **** p < 0.0001).

Representative zebrafish larvae blood flow oscillation pattern after incubation in 0.05% DMSO (grey), 10 ppm of ASC (yellow), 0.5 ppm of PON (red), and a combination of both compound (orange) (dash line show the average velocity) (A). Maximum (B) and average (C) blood flow velocity in the dorsal aorta of zebrafish larvae after exposure to ASC and PON. Data are presented as mean ± standard deviation, and the statistical significance was calculated using ordinary one-way ANOVA with Dunnet multiple comparison test. (** p < 0.01, *** p < 0.001).

Relative mRNA level of cardiovascular-related genes in zebrafish after exposure to ASC, PON, and combining both compounds. Some molecular makers associated with cardiac development (A–E), vascular development (F–H), and red blood cell development (I–L) were tested. The value was expressed as mean ± standard deviation, and the statistical difference was calculated using ordinary one-way ANOVA with Dunnet multiple comparison test. (**** p < 0.0001).