*3.2. Zebrafish Larvae Morphology after TFZ Exposure*

To investigate the developmental toxicity of TFZ, morphological changes were scored. After TFZ exposure for 48 h in 3 dpf zebrafish embryos, it was discovered that TFZ induced pericardium edema in the 3 mg/L group, and induced yolk sac retention and liver degeneration in the 2 and 3 mg/L groups (Figure 2A). After TFZ exposure for 24 h in 6 dpf zebrafish larvae, liver degeneration was also noted in 1 and 1.5 mg/L groups, pericardial edema was not observed in 0.5, 1, and 1.5 mg/L groups (Figure 2B).

**Figure 2.** Effects of development after triflumizole exposure in zebrafish. (**A**) 3 dpf zebrafish embryos with morphological changes after triflumizole exposure for 48 h. (**B**) 6 dpf zebrafish larvae with liver degeneration after triflumizole exposure for 24 h. Blue arrows and red arrows indicate pericardial edema and liver degeneration, respectively. H: heart, the green dotted line; L: liver, the red dotted line; I: intestine, the blue dotted line; Y: yolk sac, the yellow dotted line. Scale bars = 500 μm; Scale bars = 100 μm.

#### *3.3. Enzyme Activities after TFZ Exposure*

The SOD activity in 3 dpf zebrafish embryos was markedly decreased in the 3 mg/L TFZ group relative to the control group (*p* < 0.01, Figure 3A). Compared to the control group, the CAT activity in 3 dpf zebrafish embryos was of no significance in 3 mg/L TFZ group (*p* > 0.05, Figure 3B). Meanwhile, the MDA content in 3 dpf zebrafish embryos was significantly higher in 3 mg/L TFZ group than the control group (*p* < 0.01, Figure 3C). The ALT activity in 3 dpf zebrafish embryos was significantly decreased in 3 mg/L TFZ group compared to the control group (*p* < 0.01, Figure 3D).

**Figure 3.** Effects of triflumizole on enzyme activities in zebrafish. For 3 dpf zebrafish embryos, SOD activity (**A**), CAT activity (**B**), MDA content (**C**), and ALT activity (**D**) after triflumizole exposure for 48 h. For 6 dpf zebrafish larvae, SOD activity (**E**), CAT activity (**F**), MDA content (**G**), and ALT activity (**H**) after triflumizole exposure for 24 h. Data are expressed as the mean of three replicates ± standard error (SEM). Asterisks denote significant differences between the control group and TFZ groups (determined by Dunnett post hoc comparison, \* *p* < 0.05, \*\* *p* < 0.01). SOD: superoxide dismutase; CAT: catalase; MDA: malondialdehyde; ALT: alanine transaminase.

The SOD activity in 6 dpf zebrafish larvae was dramatically increased in 0.5 mg/L TFZ group relative to the control group (*p* < 0.01, Figure 3E). However, compared to the control group, the SOD activity in 6 dpf zebrafish larvae was of no significance after 1 mg/L TFZ exposure (*p* > 0.05, Figure 3E). The CAT activity in 6 dpf zebrafish larvae was significantly increased in 0.5 and 1 mg/L TFZ groups compared with the control group (*p* < 0.01, Figure 3F). Meanwhile, the MDA content in 6 dpf larvae was significantly higher in 0.5 (*p* < 0.01) and 1 mg/L (*p* < 0.05) TFZ groups than the control group (Figure 3G). The ALT activity in 6 dpf zebrafish larvae was significantly increased in 0.5 and 1 mg/L TFZ groups compared to the control group (*p* < 0.01, Figure 3H).

#### *3.4. Heat Shock Response in Zebrafish Larvae after TFZ Exposure*

The expression of glucose-regulated protein 94 (*grp94*) in 3 dpf zebrafish embryos was markedly increased in 1 mg/L TFZ group relative to the control group (*p* < 0.05, Figure 4A). Furthermore, compared to the control group, the expression of heat shock protein 70 (*hsp70*), *grp78*, *hsp90*, and *grp94* in 3 dpf zebrafish embryos was significantly increased in 2 and 3 mg/L TFZ groups (*p* < 0.01, Figure 4A).

**Figure 4.** The expression of heat shock response genes (including *hsp70*, *grp78*, *hsp90*, and *grp94*) in zebrafish. (**A**) For 3 dpf zebrafish embryos, the expression of *hsp70*, *grp78*, *hsp90*, and *grp94* after triflumizole exposure for 48 h. (**B**) For 6 dpf zebrafish larvae, the expression of *hsp70*, *grp78*, *hsp90*, and *grp94* after triflumizole exposure for 48 h. Data are expressed as the mean of three replicates ± standard error (SEM). Asterisks denote significant differences between the control group and TFZ groups (determined by Dunnett post hoc comparison, \* *p* < 0.05, \*\* *p* < 0.01). *hsp70*: heat shock protein 70; *grp78*: glucose-regulated protein 78; *hsp90*: heat shock protein 90; *grp94*: glucose-regulated protein 94.

Compared to the control group, the expression of *hsp70*, *grp78*, *hsp90*, and *grp94* in 6 dpf zebrafish larvae was of no significance in 0.5, 1, and 1.5 mg/L TFZ groups (*p* > 0.05, Figure 4B).

## *3.5. Inflammatory Genes Expression in Zebrafish Larvae after TFZ Exposure*

The expression of tumor necrosis factor α (*tnfα*) in 3 dpf zebrafish embryos was significantly decreased in 2 mg/L TFZ group compared to the control group (*p* < 0.01, Figure 5A). Compared to the control group, the expression of p65-nuclear transcription factor κB (*p65-nfκb*), interleukin 1, beta (*il-1β*), and cyclooxygenase type 2 a (*cox2a*) in 3 dpf zebrafish embryos was significantly increased in 2 mg/L TFZ group (*p* < 0.01, Figure 5A). The expression of *p65-nfκb* and *il-1β* in 3 dpf zebrafish embryos was obviously increased in 3 mg/L TFZ group compared to the control group (*p* < 0.01, Figure 5A).

**Figure 5.** The expression of inflammatory genes (including *tnfα*, *il-1β*, *p65-nfκb*, *cox2a*, and *cox2b*) in zebrafish. (**A**) For 3 dpf zebrafish embryos, the expression of *tnfα*, *il-1β*, *p65-nfκb*, *cox2a*, and *cox2b* after triflumizole exposure for 48 h. (**B**) For 6 dpf zebrafish larvae, the expression of *tnfα*, *il-1β*, *p65-nfκb*, *cox2a*, and *cox2b* after triflumizole exposure for 24 h. Data are expressed as the mean of three replicates ± standard error (SEM). Asterisks denote significant differences between the control group and TFZ groups (determined by Dunnett post hoc comparison, \*\* *p* < 0.01). *tnfα*: tumor necrosis factor α; *il-1β*: interleukin 1, beta; *p65-nfκb*: p65-nuclear transcription factor κB; *cox2a*: cyclooxygenase type 2 a; *cox2b*: cyclooxygenase type 2 b.

Compared to the control group, the expression of *tnfα*, *p65-nfκb*, *il-1β*, *cox2a*, and *cox2b* in 6 dpf zebrafish larvae was of no significance in the 0.5, 1, and 1.5 mg/L TFZ groups (*p* > 0.05, Figure 5B).

#### *3.6. Lipid Synthesis Gene Expression in Zebrafish Larvae after TFZ Exposure*

The expression of sterol regulatory element binding transcription protein 1 (*srebp1*) and *ppar-γ* in 3 dpf zebrafish embryos was markedly increased in 1 mg/L TFZ group relative to the control group (*p* < 0.05, Figure 6A). Compared to the control group, the expression of *srebp1*, fas cell surface death receptor (*fas*), acetyl-coa carboxylase (*acc*), and *ppar-γ* in 3 dpf zebrafish embryos was significantly increased in 2 mg/L TFZ group (*p* < 0.01, Figure 6A). The expression of *srebp1* and *fas* in 3 dpf zebrafish embryos was dramatically increased in 3 mg/L TFZ group compared to the control group (*p* < 0.01, Figure 6A).

**Figure 6.** The expression of lipid synthesis genes (including *srebp1*, *fas*, *acc*, and *ppar-γ*) in zebrafish. (**A**) For 3 dpf zebrafish embryos, the expression of *srebp1*, *fas*, *acc*, and *ppar-γ* after triflumizole exposure for 48 h. (**B**) For 6 dpf zebrafish larvae, the expression of *srebp1*, *fas*, *acc*, and *ppar-γ* after triflumizole exposure for 48 h. Data are expressed as the mean of three replicates ± standard error (SEM). Asterisks denote significant differences between the control group and TFZ groups (determined by Dunnett post hoc comparison, \* *p* < 0.05, \*\* *p* < 0.01). *srebp1*: sterol regulatory element binding transcription protein 1; *fas*: fas cell surface death receptor; *acc*: acetyl-coa carboxylase; *ppar-γ*: peroxisome proliferator-activated receptor gamma.

Compared to the control group, the expression of *srebp1*, *fas*, *acc*, and *ppar-γ* in 6 dpf zebrafish larvae was of no significance in the 0.5, 1, and 1.5 mg/L TFZ groups (*p* > 0.05, Figure 6B).
