Fertilized, undamaged eggs with no malformation, 1 h post fertilization (hpf) and within 3 hpf, were randomly selected from different mates (n = 5) and were distributed in 24-well plates. Embryos (20 for each experimental condition) were exposed to 500 μL of embryo solution (control group) or GNP suspensions in a thermostatic chamber at 26 ± 0.5 °C, under static conditions. Control and treated embryos were observed at 24, 48, 72, and 96 hpf. Lethal endpoints (coagulation, lack of somite formation, lack of detachment of the tail from the yolk sac, lack of heartbeat) indicating acute toxicity were monitored every 24 h. Moreover, sublethal endpoints (such as edemas, and tail and eye malformations) and the hatching rate were analyzed. Embryos were observed, and phenotype modifications were acquired through a M205FA stereomicroscope (Leica Microsystems Srl, Buccinasco, Italy) equipped with the LAS-X Expert software.
For the experiments on dechorionated embryos, the chorion was mechanically removed at 24 hpf, and the developing animals were exposed to control solution or GNP suspensions. FET phenotypic observations were performed at 48, 72, and 96 hpf, as reported above.
Median lethal concentration (LC50) and hatching time (HT50) were calculated using IBM-SPSS Probit Analysis.
For the experiments on dechorionated embryos, the chorion was mechanically removed at 24 hpf, and the developing animals were exposed to control solution or GNP suspensions. FET phenotypic observations were performed at 48, 72, and 96 hpf, as reported above.
Median lethal concentration (LC50) and hatching time (HT50) were calculated using IBM-SPSS Probit Analysis.