Effective crop protection is essential to safeguard food production and quality, but crop resistance to plant pathogens is heavily dependent on the use of fungicides in addition to varietal resistance in the face of diverse and evolving pathogens [1]. However, repeated use of fungicides with the same mode of action can lead to the evolution of resistance and loss of effective fungicides [1,2]. Also, new diseases, market needs, toxicity for other organisms, and risk to humans drive companies to invest heavily in fungicide innovation [2]. In the past decade, the novel succinate dehydrogenase inhibitor (SDHI) fungicides have become the fastest-growing class of fungicides due to the novel mechanism of action [[3], [4], [5], [6]]. SDHI fungicides inhibit fungal respiration by explicitly blocking the ubiquinone binding sites in mitochondrial complex II, thus which has excellent efficacy against various plant diseases [7].

Furthermore, approximately 30% of SDHI fungicides contain at least one chiral center, including penthiopyrad, pydiflumetofen and penflufen, etc. [[8], [9], [10]]. Generally, most chiral fungicides exhibit enantioselective biological activity, ecotoxicological, and environmental behavior in chiral environments, such as metconazole [11], epoxiconazole [12], and penconazole [13]. However, most commercially available chiral SDHI fungicides were produced in racemates (i.e., 1:1 ratio of enantiomers), and their ecological risk knowledge was provided by the research on the racemate. Therefore, the enantioselective effects should be considered when conducting a risk assessment of SDHI chiral fungicides.

Penthiopyrad, as one of the chiral SDHI fungicides, has been registered as the racemic form in thirty-five countries worldwide to control and prevent verticillium wilt on sugar beet, peanut leaf spot and strawberry gray mold, etc [7,[14], [15], [16]]. A dietary risk assessment study for penthiopyrad indicated that penthiopyrad residues were below the recommended MRL values (2000 μg/kg) for tomato and cucumber, and the half-life of penthiopyrad in cucumber and tomato samples was less than eight days [17]. However, penthiopyrad exhibits longer half-life (from 38.9 to 97.6 days) in soil [18]. Thus, inappropriate use may induce soil to carry more penthiopyrad residues and transport them to an aquatic system through various channels (e.g., runoff, spray drift, etc.). Previous research indicated penthiopyrad has low acute toxicity to mammals and poultry but high acute toxicity for freshwater fish, as the LC50-96 h (lethal concentration 50%; LC50) for Common Carp (Cyprinus carpio) was 572 μg/L and rainbow trout (Oncorhynchus mykiss) was 386 μg/L [4]. The 96 h-LC50 values of penthiopyrad for zebrafish embryos were 2.77 mg/L and 2.38 mg/L for larvae [4]. For adult zebrafish, the 96 h-LC50 value was 2.841 mg/L [9]. In addition, penthiopyrad also exhibits a longer hydrolysis half-life (range from 46.8 to 67.3 days) in aquatic environments [19]. This could have a deep and lasting negative impact on aquatic organisms and could be a potential threat to human health through the food chain. On the other hand, many SDHI fungicides has proven negatively impact to aquatic organisms by affecting energy metabolism and lipid metabolism, such as thifluzamide [20], flutolanil [21,22], boscalid [23], and fluxapyroxad [24], etc. Importantly, energy metabolism and lipid metabolism play an irreplaceable role on the early stage of aquatic organisms, ensuring normal physiological functions. Thus, assessing the toxic effects of penthiopyrad on the early life stages of aquatic organisms is essential for their growth and development. Zebrafish embryos enable an analysis of multiple toxicological endpoints from the determination of acute and developmental toxicity to complex genetic and physiological analyses, thus, which is increasingly being used as a model for biomedical research and ecotoxicology [25]. A number of evidences also suggest that fungicides (fluxapyroxad [26], mefentrifluconazole [27] and oxathiapiprolin [28], etc.) induce deeper effects on early life stages of zebrafish.

Moreover, previous research indicated that S-(+)-penthiopyrad was preferentially degraded in the soil and aquatic environment, and S-(+)- penthiopyrad demonstrates higher toxicity to HepG2 cells [18,29,30]. Our previous study indicated that S-(+)-penthiopyrad was preferentially bioaccumulation in adult zebrafish and significantly affected mitochondrial function and induced oxidative stress and apoptosis in adult zebrafish [9]. Thus, we hypothesize that Rac-/R-(+)-/S-(+)-penthiopyrad have enantioselective toxic to early life stage of zebrafish and mitochondrial function barrier could be the potential toxic mechanism. Thus, the embryonic stage of zebrafish was selected to investigate the toxicological endpoints (including autonomous movement, heart rate, malformation and death) of penthiopyrad racemate and enantiomers; as well as the expression of genes related to respiratory chain complex II, heart development and lipid metabolism in zebrafish early life stages by molecular biology means; and deeply explored its enantioselective toxic effects. This study contributed to improve the comprehensive risk assessment and enantiomeric research system of penthiopyrad to early life stage of zebrafish.