*2.3. Zebrafish Exposure*

An overview of the exposure procedures is shown in Figure 1A,B. In the control groups, we added 0.1% DMSO in 5 mL 1 × EM medium. We chose 3 dpf zebrafish embryos and 6 dpf zebrafish larvae for TFZ exposure. Firstly, they were exposed to TFZ ranging from 0 to 150 mg/L for 24, 48 and 72 h. the TFZ exposure solutions were replaced per 24 h. Based on the morphological changes and mortality data, 3 dpf zebrafish embryos were exposed to 0, 1, 2, and 3 mg/L TFZ for 48 h. Likewise, 6 dpf zebrafish larvae were exposed to 0, 0.5, 1, and 1.5 mg/L TFZ for 24 h. These were individually dispensed into the 6-well plates, thirty embryos/larvae per well with 5 mL exposure solution, and biological triplicate was collected for each exposure. In order to retain the suit concentrations and water quality, the TFZ exposure solutions were exchanged daily. The 6-well plates were incubated at 28 ◦C for light/dark cycles of 14/10 h during the exposure period. The survival rates of embryos and larvae were counted in each exposed group every 24 h.

**Figure 1.** Exposure patterns and the survival percentage of zebrafish after triflumizole exposure. (**A**) Three dpf embryos were exposed to 1, 2, and 3 mg/L triflumizole for 48 h, (**B**) Six dpf larvae were exposed to 0.5, 1, and 1.5 mg/L triflumizole for 24 h. (**C**) The percentage of survival of 3 dpf embryos after triflumizole exposure for 48 h. (**D**) The percentage of survival of 6 dpf larvae after triflumizole exposure for 24 h. dpf: d post-fertilization; LC50: lethal concentration 50.

#### *2.4. Fish Intestinal Cell Line Exposure*

The intestinal cell line obtained from a marine fish, silver pomfret (*Pampus argenteus*), was obtained and cultured as our previous method [26]. Cells were plated on 96-well plates, then exposed to 0, 5, 10, 15, 20, 25, 30, 35, and 40 mg/L TFZ in triplicate for 24 h. We used a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) to assess cell viability. Briefly, cells were incubated with CCK-8 solution for 4 h at 28 ◦C. Finally, samples were detected in the absorbance at 450 nm by a microplate reader (BioTek, Winooski, VT, USA). Based on cell viability data, the intestinal cell line was exposed to 0, 5, 10, and 20 mg/L TFZ for 24 h to evaluate its toxicity. Then cells were trypsinized at 37 ◦C for 1 min and collected for the next analysis.
