Comparison of PBZ effects on survival rate after exposure of zebrafish embryos beginning at different stages. Survival rates at 5 dpf were calculated for embryos exposed to 0.1% DMSO (control), and 0.34, 3.4, and 17 μM PBZ. Exposures began at 24, 36, 48, 60, 72 or 96 hpf. Data represent the mean (±standard deviation, SD) of three independent experiments; 80 embryos were assessed in each treatment (N = 3. n = 240 embryos). Bars not sharing a common letter are significantly different. Data were compared by ANOVA followed by Fisher’s least significance difference test (p < 0.05).

Pericardial edema is induced by PBZ in zebrafish embryos exposed at early stages. Zebrafish embryos were exposed to a range of PBZ concentrations (0.34, 3.4, and 17 μM) beginning at different stages (24, 36, 48, 60, 72, and 96 hpf). The pericardiac phenotype was observed at 5 dpf, and ratios of affected individuals were calculated. Data represent the mean of three independent experiments; 30 embryos were assessed in each treatment (N = 3. n = 90 embryos).

Pharyngeal arch development is affected according to the initial PBZ exposure stage in zebrafish embryos. Zebrafish embryos were exposed to a range of PBZ concentrations (0.34, 3.4, and 17 μM) beginning at different stages (24, 36, 48, 60, 72, and 96 hpf). The head skeleton of 5 dpf fish was analyzed by Alcian blue staining. Pharyngeal arch phenotypes were categorized as (A) ‘Normal’, ‘Mild’ defects or ‘Severe’ defects. Images were taken from the dorsal view. (B) Frequency of pharyngeal arch phenotypes was compared in zebrafish embryos treated with different concentrations of PBZ at different exposure stages. Data represent the mean of three independent experiments; 30 embryos were assessed in each treatment (N = 3. n = 90 embryos).

Intestine defects are more severe when exposure to PBZ begins at early stages. Zebrafish embryos were exposed to a range of PBZ concentrations (0.34, 3.4, and 17 μM) beginning at different stages (24, 36, 48, 60, 72, and 96 hpf). Embryos were collected at 5 dpf and analyzed by whole-mount in situ hybridization with an ifapb antisense riboprobe; ifapb is specifically expressed in intestine. The intestinal phenotype was categorized as (A) ‘Normal’, ‘Mild’ defects or ‘Severe’ defects. Images were taken from ventral view. (B) Frequency of intestine phenotypes was compared in zebrafish embryos treated with different concentrations of PBZ at different exposure stages. Data represent the mean of three independent experiments; 30 embryos were assessed in each treatment (N = 3. n = 90 embryos).

Liver defects are more severe when exposure to PBZ begins at early stages. Zebrafish embryos were exposed to a range of PBZ concentrations (0.34, 3.4, and 17 μM) beginning at different stages (24, 36, 48, 60, 72, and 96 hpf). Embryos were collected at 5 dpf and analyzed by whole-mount in situ hybridization with an lfpb antisense riboprobe; lfpb is specifically expressed in liver. The liver phenotype was categorized as (A) ‘Normal’, ‘Mild’ or ‘Severe’ defects. Images were taken from ventral view. (B) Frequency of liver phenotypes was compared in zebrafish embryos treated with different concentrations of PBZ at different exposure stages. Data represent the mean of three independent experiments; 30 embryos were assessed in each treatment (N = 3. n = 90 embryos).

Pancreas defects are more severe when exposure to PBZ begins at early stages. Zebrafish embryos were exposed to a range of PBZ concentrations (0.34, 3.4, and 17 μM) beginning at different stages (24, 36, 48, 60, 72, and 96 hpf). Embryos were collected at 5 dpf and analyzed by whole-mount in situ hybridization with an insulin antisense riboprobe; insulin is specifically expressed in pancreas. The intestinal phenotype was categorized as (A) ‘Normal’, ‘Mild’ or ‘Severe’ defects. Images were taken from dorsal view. (B) Frequency of pancreas phenotypes was compared in zebrafish embryos treated with different concentrations of PBZ at different exposure stages. Data represent the mean of three independent experiments; 30 embryos were assessed in each treatment (N = 3. n = 90 embryos).