2.3. Acute Toxicity Experiment of Zebrafish Embryos

The water used in embryo cultivation and embryo exposure experiment was oxygenated saturated standard embryo culture medium (E3 solution: 5 mmol/L NaCl, 0.17 mmol/L KCl, 0.33 mmol/L CaCl₂ and 0.33 mmol/L MgSO₄, containing no methylene blue, and the pH value adjusted to about 7.2 with NaHCO₃ solution.). Normally-developed fertilized embryos were selected and cultivated in a 24-hole plate, with each hole infilled with 3ml blank control E3 solution, regular TiO₂ contrast solution (100 mg/L), TiO₂ NPs exposure solution of different concentrations (10, 50 and 100 mg/L) and 4 fertilized embryos, 10 replicates for each concentration. Group experiment was completed 2 h after the passing out of fertilized eggs. The culture plate was sealed with a lid, and placed in an incubator with a temperature of 28 ± 0.5 °C, and under a light/dark period of 14/10 h. During the exposure process, the growth of the fertilized eggs was monitored every 12 h, keeping records of the death and incubation of the eggs within 0–96 hpf (hour postfertilization, hpf) and getting rid of the whitening and condensation eggs to prevent contamination. The test solutions were changed every 24 h, and mixed by ultrasonic for 30 min before use. The embryo acute toxicity experiment was replicated quintically, with each concentration containing 200 embryos. The embryo hatching rate = (hatched embryos/the number of zebrafish embryos in total) × 100%.