Zebrafish Embryo Acute Toxicity Test

The OA potential toxicity (715 µg/mL) was examined in the zebrafish embryo acute toxicity test in accordance with the Organization for Economic Cooperation and Development (OECD) recommendation for the testing of chemicals (Test No. 236 [107 (link),108 ]). Zebrafish embryos were screened for viability, shape, and transparency one hour after fertilization. Only the eggs meeting all criteria, i.e., being viable, completely transparent, and round, were chosen and moved to 12 well plates (Sarstedt, Germany). They were randomly divided into two groups, the control and experimental ones, and kept in each well until the time of experiment elapsed, i.e., up to 96 h. Two batches of embryos (3 wells per batch, n = 5–7 per well) were kept in 3000 µL of the zebrafish medium (control group) or supplemented with 715 µg/mL of OA (experimental group). The mortality rate was scored after 23, 47, 71, and 95 h of incubation. Hatchability was assessed after 71 and 95 h of incubation. The morphological abnormalities were assessed after 95 h of incubation: heartbeat, heart/yolk edema, yolk sac necrosis, hemorrhage [108 ], jaw development, eye size, and posture. Additionally, for assessing muscle function and performance, the touch-evoked response assay was conducted as described in detail previously [109 (link),110 (link)].