Fig. 1

Effects of exposure to the NA/EE Mixture upon Survival and Development of Zebrafish Embryos Are Time and Dose Sensitive Zebrafish embryos at 24 hpf were treated with either DMSO, NA 6.25 μg/mL + EE 12.5 ng/mL or NA 12.5 μg/mL + EE 25 ng/mL, incubated until 96 hpf and overall body size, PF, OV and eye size measured. (A) The survival rate decreases as the dose increases. Survival rate starts to decrease at a concentration of NA 6.25 µg/mL + EE 12.5 ng/mL. (B) The rate of gross defects occurrence increases as the dose increases. Embryos start to present defects at a concentration of NA 6.25 µg/mL + EE 12.5 ng/mL (C–G) NA and EE mixture causes reduction of body size, PF, OV and eye size in a dose-dependent manner. Damage caused by the drugs is less severe in embryos treated with lower dose (D–D”) when compared to DMSO controls (C–C” and F). NA: Norethisterone acetate. EE: Ethinyl Estradiol. H: heart; YS: yolk sac; OV: otic vesicle; PF: pectoral fin. Black arrowhead indicates yolk sac and extension oedema. Black arrow indicates otic vesicle is smaller. Black asterisk denotes bent spine. White asterisk denotes reduced (D”) or missing pectoral fin (E”). White arrow indicates pericardial oedema. Vertical black line compares the eye diameter between NA/EE-mixture and DMSO treated embryos in C’, D’ and E’, and indicates diameter of the eye is altered in NA/EE-treated embryos. Relative length: compared to WT. Statistical significance was analysed using Student t-test. Graphs represent mean ± SEM. **p < 0.01, ***p < 0.001 and ****p < 0.0001. Scale bars: 250 µm.