*2.5. Morphological Assay*

After TFZ exposure, phenotypic changes were evaluated in 3 dpf zebrafish embryos and 6 dpf zebrafish larvae. After anesthetizing with 0.16% tricaine (Sangon Biotech, Shanghai, China), they were fixed on the culture dish in a lateral view using 3% methylcellulose. A lateral view of the entire zebrafish larvae was observed and photographed with a stereomicroscope (Olympus, Tokyo, Japan).

#### *2.6. Enzyme Activities Assay*

Thirty embryos/larvae (each replicate) were homogenized with 300 μL saline solution, followed by centrifuging at 2500 rpm for 15 min at 4 ◦C to collect the supernatant for use in subsequent experiments. Three replicates were performed. The activities of SOD, CAT, alanine aminotransferase (ALT), and the content of MDA were assayed by commercially available biochemical assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) in accordance with the manufacturer's instructions. The SOD activity was tested by the xanthine oxidase method. In brief, superoxide anions can oxidize hydroxylamine to nitrite and then turn amaranth purple in the presence of a chromogenic agent. Data were recorded by reading the absorbance at 550 nm and computing the activity of the SOD. One unit of SOD activity (U) was defined as the amount of enzyme required to inhibit the oxidation reaction by 50% and was expressed as U/g protein. The CAT activity was tested by the ammonium molybdate method. The decomposition of hydrogen peroxide is quickly terminated by adding ammonium molybdate. The rest of the hydrogen peroxide reacts with ammonium molybdate to form a pale-yellow complex compound, which was detected at 405 nm and used to compute the CAT activity. One unit of CAT activity was defined as the amount of enzyme required to consume 1 μmol H2O2 in 1 min at 25 ◦C and was expressed as μmol/min/g protein. The products of lipid peroxidation (measured MDA content) were tested by the thiobarbituric acid (TBA) method, and the amount of TBA substance that occurred through lipid peroxidation was detected after incubation at 95 ◦C with TBA. The pink color generated in these reactions was detected by spectrophotometry at 532 nm, and MDA content was expressed as nmol/g protein. The pyruvate reacts with 2-4-dinitrophenylhydrazine to generate the hydrazone, which acquires maximum staining by the addition of NaOH to measure ALT activity [27]. It was examined at 505 nm and was expressed as μmol/min/g protein. Protein concentration was tested using the bicinchoninic acid (BCA) protein assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) for Cu2+ following protein-mediated reduction of Cu2+ by an alkaline environment. All measurements were performed on a microplate reader, using the A590 microwell plate protocol.
