**1. Introduction**

The widespread use of pesticides may endanger aquatic species by contaminating water through surface runoff or leachingOerke [1–3]. Triflumizole (TFZ, C15H15ClF3N3O), a triazole fungicide (Nippon Soda Co., Ltd., Tokyo, Japan), has not been approved by the European Commission. It is mostly used to prevent powdery mildew and rust in cereals, vegetables, fruit trees, and other crops [4–7]. It has the potential to hinder the biological synthesis of ergosterol by limiting C14-demethylation in sterol [8]. As recently as 2009, 56,231 pounds of TFZ were spilled in California, although the actual level of human exposure is unknown [9]. Further, TFZ has been found in pears, apples, and cucumbers [10]. Environmental risk assessments of TFZ to non-target species must be carried out immediately [11].

Several studies have demonstrated the toxicity of TFZ to aquatic species, such as freshwater algae [12] and fish [13]. Xi et al. (2019) have indicated that TFZ is potentially toxic to freshwater algae in hydrophytic ecosystems [12]. Ecological studies have suggested that TFZ be classified as showing medium toxicity to fish compared to other triazoles [5]. It has also been found that the 72-h lethal concentration 50 (LC50) of TFZ for rare minnow (*Gobiocypris rarus*) embryos was 7.11 mg/L, and that it induced abnormal development, extensively modified enzyme activities, and genes expression [10]. Therefore, further toxicological investigation of TFZ for aquatic organisms is required.

Many aquatic species can survive in polluted environments because of defense mechanisms such as antioxidant system and stress response [14]. In fish, toxicant bioaccu-

**Citation:** Bai, L.; Shi, P.; Jia, K.; Yin, H.; Xu, J.; Yan, X.; Liao, K. Triflumizole Induces Developmental Toxicity, Liver Damage, Oxidative Stress, Heat Shock Response, Inflammation, and Lipid Synthesis in Zebrafish. *Toxics* **2022**, *10*, 698. https://doi.org/10.3390/ toxics10110698

Academic Editors: François Gagné, Stefano Magni and Valerio Matozzo

Received: 18 October 2022 Accepted: 15 November 2022 Published: 17 November 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

<sup>1</sup> School of Marine Sciences, Ningbo University, Ningbo 315211, China

<sup>2</sup> Ningbo No. 2 Hospital, Ningbo 315010, China

mulation initiates redox reactions that generate reactive oxygen species (ROS) changing metabolism [15]. Organisms scavenge it using antioxidants including superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA), which are used to assess environmental risk [16]. Furthermore, ROS production is deeply involved in inflammatory reactions [17]. ROS might be accumulated or released due to the increase in oxygen uptake caused by the aggregation of immune cells in damaged tissues. Furthermore, it has been documented that oxidative stress leads to cellular proteins being damaged so that they must subsequently be refolded [18]. Heat shock proteins (HSPs) are the most effectively protective mechanism, and their syntheses increase remarkably with stress. HSPs enable cells to accommodate various xenobiotic factors and naturally derived cytotoxic factors, and previous research has demonstrated the crucial role of HSPs in oxidative stress [19,20].

Some studies have reported that triazoles may affect lipid metabolism, harm embryonic development, and alter the expression of lipid synthetic genes [21–23]. In addition to these target mechanisms, TFZ could also be recognized as an obesogen in mice by acting on the peroxisome proliferator-activated receptor-gamma (PPAR-γ) pathway to increase weight [9], this suggests that TFZ could participate in the lipid synthesis of organisms.

Zebrafish have been commonly used to assess the toxicity of pesticides in the environment [24,25]. Our research aim to study the toxicity and lipid synthesis effects of TFZ by exposing 3 d post-fertilization (dpf) zebrafish embryos and 6 dpf zebrafish larvae. Our results will be conducive to an original understanding of the harmful impacts of TFZ on fish and its mechanism.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

Triflumizole (Aladdin, China, CAS: 99387-89-0, 99% purity) was dissolved in dimethyl sulfoxide (DMSO) to 40 mg/mL as a stock solution, then diluted in 1 × EM medium (20 × EM medium: NaCl, 17.5 g; KCl, 0.75 g; CaCl2 anhydrous, 2.18 g; KH2PO4, 0.41 g; Na2HPO4 anhydrous, 0.142 g; MgSO4-7H2O, 4.9 g) to the exposure concentrations ensuring that the final DMSO volume was less than 0.1%.
