Triadimefon in Aquatic Environments: Occurrence, Fate, Toxicity and Risk


 Background：As a triazole fungicide, triadimefon is widely used around the world. The ubiquitous occurrence of triadimefon in aquatic environments and potential adverse effects on aquatic organisms have resulted in global concerns. In this review, the current state of knowledge on occurrence, environmental behavior, and toxic effects are presented and used to conduct an assessment of risks posed by current concentrations of triadimefon in aquatic environments. Results: The key findings from this review are that: (1) triadimefon occurred widely in surface waters, with high rates of detection; (2) abiotic degradation of triadimefon was affected by many factors. Stereo-selectivity was found during biotic degradation and metabolism of triadimefon. Different enantiomers can cause various adverse effects, which complicates the assessment and requires enantiomers-specific considerations; (3) triadimefon exposure can affect organisms by causing multiple toxic effects on the thyroid, reproduction system, liver, nervous system as well as carcinogenicity and teratogenicity, and it can also act synergistically with other pesticides. Long-term, low-dose effects were considered to be the main characteristics of toxic effects of triadimefon; (4) results of the risk assessment based on probabilistic relationships represented by Joint Probability Curves (JPCs) indicated that risk of triadimefon was classified as low risk.Conclusion: The concentration data of triadimefon in surface water is relatively less, more work needed to do to detect it. Reproductive toxicity was observed which indicated that triadimefon might result in adverse effects on the population level or even on the ecosystem level. Risk assessments for pesticides that cause long-term and low-dose effects on aquatic organisms such as triadimefon need to consider higher ecology level risk.

Jiulongjiang River, Tai Lake, Baiyang Lake, and other places in China, with the greatest detectable concentration of 12 µg/L [5,6,7,8]. At the same time, on account of strong stability, good mobility and adsorption, and long-lasting accumulation in aquatic environments, triadimefon can accumulate in bodies of aquatic organisms where it can undergo biotransformation. Exposures of various types and life stages of aquatic organisms to various concentrations of triadimefon can result in a wide range of toxic effects, that poses threats to ecosystems, eventually affecting ecosystem services that can in turn affect health and economic wellbeing of humans.
Triadimefon has attracted extensive international attention. In 1999, the United States "Safe Water Reference and Toxicity Enforcement Act (Proposition 65)" added triadimefon to the list of chemicals, concentrations of which were controlled in drinking water. Triadimefon was thought to induce developmental toxicity, androgen-like and estrogen-like effects [9]. In 2006, the European Union (EU) included triadimefon and transformation products on the list of pesticides with endocrine-reproductive interference toxicity [10]. It is controversial whether triadimefon is an endocrine disruptor or not, but most studies showed that triadimefon caused endocrine disrupting effects and could affect the balance of hormones in bodies of animals [11][12][13]. According to the World Wide Fund for Nature and World Wildlife Fund (WWF), triadimefon was an estrogen receptor agonist, which had certain effects on estrogen in vitro. Regardless, its potential toxic potencies cannot be ignored. Results of several studies have demonstrated that triadimefon affected development [12,14,15], reproduction [16,17] of organisms and caused neurotoxicity [18], hepatotoxicity [19], cardiovascular toxicity [20] and oxidative stress [11,13]. In addition, triadimefon resulted in malformations of branchial arches in developing amphibians [21] and zebra sh (Danio rerio) or adversely affected development of embryos of mammals [22]. Several previous studies have demonstrated that triadimefon altered expressions of genes related to the hypothalamicpituitary-thyroid axis (HPT) and disrupted thyroid endocrine function by delaying thyroid hormonedependent metamorphic development [12]. Those results con rmed that the use of pesticides was one of the major factors in declines of populations of some amphibians [12]. In recent years, studies demonstrating effects of triadimefon on endocrine function and oxidative stress in vertebrates have been increasing. The results have shown that triadimefon can cause oxidative stress and interfere with endocrine, eventually in uencing reproduction [11,23,15]. Therefore, the long-term effect of triadimefon on aquatic ecosystems is worthy of attention. As for assessment of risks studies, Liu et al. (2018) [24] estimated the PNEC based on reproductive tness of triadimefon and calculated hazard quotients (HQs), which con rmed risk of triadimefon on reproductive function. Based on the results summarized above, it is believed that more surface water concentration and toxicity data are needed for more advanced risk assessment.
Over the past few years, several studies have summarized detailed information on concentrations and toxic effects of triadimefon [20,24]. However, there has been no systematic review of the current state of knowledge regarding the presence, environmental behavior, toxic effects and potencies or a critical assessment of risks posed by triadimefon in aquatic environments. This review summarized results of research that have been conducted on triadimefon. In the present synthetic review, environmental behavior, toxic effects and ecological risk assessment of various concentrations of triadimefon in surface waters from 2000 to 2020 were reviewed. The main objectives of this review were to: 1) obtain a clear understanding of the occurrence and environmental behavior of triadimefon in aquatic environments; 2) identify triadimefon exhibiting toxic effects and potencies and then to combine exposure and responses to assess current risks in aquatic environments; 3) identify knowledge gaps and propose future research needs, to provide a reference for management of triadimefon in aquatic environments, particularly in China.

Environmental Behavior Of Triadimefon In Aquatic Environments
Environmental behavior of pesticides including migration and transformation in various environmental media determines the ultimate exposure to aquatic organisms. Environmental behavior of pesticides included process of chemical behavior (release of residues, degradation, and biotransformation), physical behaviors (leaching and runoff, soil adsorption, volatile, and diffusion migration), and biological effects (bioaccumulation and adverse effects on organisms). Occurrences and sources in surface water, biota sorption, abiotic transformation and degradation, bioaccumulation, metabolism, elimination, and potential for biomagni cation of triadimefon were summarized.
Occurrence and source of triadimefon in surface water Due to the wide-spread use and rates of application and mobility, triadimefon is ubiquitous in the environment, and relatively great concentrations of triadimefon have been detected in soil and food [25]. However, information on concentrations of triadimefon in surface water is relatively less. One day after treatment, residues of triadimefon and its metabolites from a turfgrass facility ranged from 46 to 922 µg/L [26]. A study of pesticide monitoring in Amvrakia Lake (Western Greece) found that after changing use type of agricultural land, the maximum concentration of triadimefon was 0.0371 µg/L [27]. A threeyear monitoring survey (March 2005-February 2008) in Acheloos River (Western Greece) found that the maximum concentration of triadimefon was 0.1923 µg/L [28]. Some studies also involved measuring triadimefon in surface water of China (Table S1). The pollution status of 9 triazole pesticides were investigated at 16 points along the Jiulongjiang River. Results indicated that during the wet season, concentrations of triadimefon ranged from 0.1733 to 0.047 µg/L, with a rate of detection of 43.8%, but during the dry season, triadimefon was never detected at any location [7]. Concentrations of triadimefon in ditches around rice elds ranged from 0.4 to 12 µg/L, and the detection rate was 33.3-100% [8], which indicated that application of triadimefon resulted in relatively great concentrations of triadimefon in surrounding aquatic environments. When samplings of drinking water sources were conducted at Red Maple Lake, Baihua Lake and the Aha Reservoir, in Guiyang province during the dry season from 2014 to 2015, concentrations of triadimefon were 1.1-4.1 µg/L, and the detection rate was 33.3% [5]. During the same period, 11 sampling sites were established by adding one site in the Songbaishan Reservoir. The rate of detection of triadimefon was 33.6%, and concentrations ranged from 0.185 to 5.220 µg/L [3]. The maximum concentration of triadimefon in the Tai Lake Basin was 0.00727 µg/L, but the detection rate was 100%, while the maximum detected concentration in Baiyangdian Lake was 0.13 µg/L [29,30]. Besides, triadimefon was also detected in three rivers, Bai, Chao and Chaobai, and a reservoir, Miyun, in Beijing, and some other waters including Poyang Lake, Ganjiang River and Xiushui River of Jiangxi Province. In those surveys the maximum concentration was 0.054 µg/L (Table S2). Concentrations of triadimefon in rivers of Beijing, Shanghai, and Jiangsu provinces were relatively small, but greater in surrounding watersheds of agricultural land, to which triadimefon was applied. Concentrations of triadimefon in the source of drinking water of Guiyang reached several micrograms per liter [5].
Concentrations in water in locations outside of China tended to be less because the use of triadimefon in those areas is relatively less, but triadimefon has been detected in most surface waters, although at lesser concentrations. Mean rates of removal of triadimefon by municipal sewage treatment plants, after secondary and tertiary treatments were 65% and 93%, respectively [31]. Therefore, wastewater treatment plant discharges might not be primary sources of triadimefon to aquatic environments.

Degradation and metabolism
Transformation and degradation of triadimefon in the environment and the metabolic process in organisms are determinants of whether triadimefon will persist in the environment and whether it poses a threat to aquatic environments and organisms. Triadimefon is composed of a chiral center and two enantiomers R-enantiomer (R-triadimefon, R-TDF) and S-enantiomer (S-triadimefon, TDF), respectively [32]. Triadimenol, which is a more potent fungicide than triadimefon, is the main product of degradation or metabolism of triadimefon. In the transformation process, the carbonyl group linked to the chiral center is converted to a hydroxyl group, giving rise to a new chiral center. Thus, two chiral centers and four stereo-isomers can result [33,34]

Abiotic and Biotic transformation and degradation
There are several pathways and mechanisms of transformation and degradation of triadimefon and it has different durations of persistence in various environmental matrices (Table S3). Hydrolysis of triadimefon is the primary abiotic mechanism of transformation [37][38][39]. Triadimefon was stable under acidic conditions and not easy to hydrolyze. However, at higher pH or higher temperatures, hydrolysis was accelerated [39]. In simulated aquatic environments, the half-life of triadimefon was 5.3 days and 1.15 days at pH 7.18 and pH 9.25, respectively. Those results were similar to results in a buffer solution [37]. In an alkaline aqueous environment, hydrolysis might be one the primary mechanism by which triadimefon disappears, but in an acidic aqueous environment, it is di cult to remove triadimefon by hydrolysis.
There is slightly more information about photolysis of triadimefon than there is on hydrolysis. Under natural sunlight conditions, the half-life of triadimefon in ultrapure water was 11.7 days. Half-lives of sterilized river water and seawater were 16.5 days and 22.3 days, respectively [7]. It was signi cantly delayed in river water, compared to ultrapure water. Solvent and irradiation wavelength were the major factors affecting photo-degradation of triadimefon [7]. Triadimenol, the main metabolite of triadimefon, was more stable to photolysis than triadimefon [40]. With electron-acceptor sensitizers and longer durations of irradiation signi cant degradation of triadimenol can occur [41]. Homolytic cleavage of the C-N and C-O were major mechanism to photolytic attack of triadimefon during photo-degradation in methanol and cyclohexane [42,43]. 4-Chlorophenol and 1,2,4-triazole were two major products of photodegradation of triadimefon or triadimenol [42]. However, studies of photolysis of triadimefon have focused primarily on mechanisms and factors in uencing degradation. Information on photolysis in natural waters which might attenuate light or contain photosensitizing compounds such as humic acid and fulvic acids are lacking. Quantum yields of photo-degradation of triadimefon indicated that rates of photo-chemical reactions observed in the presence of organic solvents could not be applied to natural aquatic systems [40]. Therefore, more studies based on natural aquatic systems are needed to explore the photo-chemical behavior and persistence of triadimefon under more realistic natural conditions. In dry soils, the primary degradation product of triadimefon is triadimenol, due to reduction of the carbonyl group to an alcohol, but in ooded soils, triadimefon is degraded to the diol derivative [1-(1H-1,2,4-triazol-1-yl)-3,3-dimethylbutan-2-one-1,4-diol] [34,44]. Thus, the rates of transformation and products formed are in uenced by soil moisture content. In addition, degradation of triadimefon is also affected by organic carbon content and temperature [44]. The biotransformation rate of triadimefon in the soil can be affected by pH, such as the rate of biotransformation in soil with a pH of 6.3 was approximately twice that of soil with a pH of 4.8 [34]. By contrasting the result of sterilized soil and non-sterilized soil, the degradation of triadimefon in soil was mainly biologically-mediated. The microorganisms Arthrobacter and Halomonas played important roles in converting R-TDF to S-TDF and S-TDF to R-TDF in neutral and alkaline soil, respectively [45]. Thus, triadimefon would not persist very long in soil, with observed halflives of 8 days in sandy-loam and 13 days in clay soils [46], 5.2 days in facility soil [47] and nally 39.4 days in sterilized soil [45]. But, triadimenol, the degradation product of triadimefon, is much more persistent, with a half-life exceeding 240 days [33,44,48]. Concentrations of triadimenol in surface water can be several micrograms per liter. Therefore, the persistence of triadimefon and its degradation production triadimenol in the environment cannot be ignored. Whether triadimefon can be transport longrange to polar regions has not been determined.
Degradation of triadimefon in soil exhibits signi cant stereo-selectivity. Biotic transformations of triadimefon to triadimenol in soil, resulted in different relative abundances of the four stereo-isomers compared with commercially synthesized triadimenol [32,47]. Degradation of triadimefon into triadimenol in a model soil showed stereo-selectivity, and the content of R-triadimenol was 4 times greater than that of S-triadimenol. In addition, under different soil or physical conditions, the stereo-isomer components of triadimenol formed by conversion of triadimefon can result in different ratios, and abiotic racemization contributed to different chiral pro les [33]. Triadimefon is a chiral pesticide, and during its degradation process, abiotic racemization would occur [33,43], which would affect the isomer composition and proportions of products. Different stereo-isomers might have different biological properties. For example, acute toxic potency of triadimenol diastereomer A to rats (oral LD 50 ) is 10 times greater than that of diastereomer B [49]. Therefore, it is essential to take into account stereo-selectivity and con gurational stability when assessing risks of triadimefon and triadimenol to aquatic organisms.

Metabolism and Elimination
Triadimefon has one chiral center and two enantiomers, R-TDF and S-TDF. The metabolite of triadimefon in organisms, which is triadimenol, was found in several studies [36,50,51,52]). During metabolic transformation, the carbonyl group attached to the chiral center is reduced to a hydroxyl group, which results in a new chiral center. As a result, triadimenol possesses four enantiomers, including SS-TDN, SR-TDN, RR-TDN, and RS-TDN. Unfortunately, triadimenol also exhibits toxic potency to organisms. A study of the stereo-selectivity of triadimenol metabolism indicated that triadimenol can be enriched in lizards and cause reproductive toxicity, hepatotoxicity and oxidative stress [35,52]. Also, triadimenol can cause neurological toxicity through interference with normal functioning of the acetylcholinesterase (AchE) [35]. Triadimenol exhibited greater inhibition of Scenedesmus obliquus than did triadimefon [36].
Stereo-selectivity in the absorption distribution and metabolic conversion of triadimefon in organisms was taking on. Stereo-selective behavior refers to that chiral drugs entering the environmental system can change the enantiomer ratio due to various chiral factors. This process of changing the enantiomer ratio is referred to as the stereo-selective behavior of chiral molecules. Absorption, distribution, metabolism, and elimination of triadimefon and triadimenol enantiomers in tissues, including liver, brain, fat, kidney, and blood plasma, of the Eremias argus were studied after orally giving lizards with R-TDF or S-TDF [50]. Traits of rapid distribution and slower elimination of triadimefon enantiomers in the tissue have been observed. Biotransformation, degradation, and dynamic metabolic process varied among tissues. The main metabolites of each triadimefon enantiomer were also different. For instance, the main metabolite of R-TDF was RR-TDN while that of S-TDF was SR-TDN. The metabolic rate of these metabolite isomers was also different. Selective metabolism and toxic effect of triadimenol have been observed [35]. Metabolic rates of RS-TDN and RR-TDN were more rapid than that of SR-TDN and SS-TDN, respectively. S-TDF was absorbed faster than R-TDF, and R-TDF was easily metabolism to TDN in lizards [51]. However, in vitro in Rainbow trout (Oncorhynchus mykiss), S-TDF was more easily transformed to triadimenol than R-TDF [53], which illustrated that species differences in stereo-selective metabolism of triadimefon. Therefore, different species and different tissues even different media can exhibit different compositions and distributions of metabolite stereo-isomers [50-52, 54, 55]. Biological activity of triadimenol probably depends on the relative abundances of its particular stereo-isomers. Each isomer is a separate entity, and their behavior in the environment and toxicity to organisms might be different. For example, enantiomers of triadimenol had different toxic effects on fungi and rats [56]. Signi cant differences in metabolism and toxic effects between enantiomers were found in selective metabolism of triadimefon by vegetables [35]. Signi cant differences among isomers also were observed during acute studies of toxicity to the Water ea (Daphnia magna) [55]. Toxic potencies of both triadimefon and triadimenol are complicated. The liver is the main place for metabolism of pollutants in the body, so research on metabolic kinetics of pollutants were carried out by in vitro, by use of liver microsomes. In the early years, some researchers also carried out in vitro liver microsome experiments on triadimefon [57] to study the gender and species differences of triadimefon metabolism in rodents in vitro [58] and to explore the metabolic mechanisms by which triadimefon differed from other conazole fungicides, such as steroidogenesis and carbohydrate metabolism [59].
In summary, although triadimefon does not persist in the body for long time, its metabolite, triadimenol remains for a longer time and may be more toxic. Moreover, its metabolism has stereo-selectivity, which leads to the uncertainty of its metabolite isomer composition and further leads to the complexity of its toxicity. Therefore, these characteristics should be fully taken into account in the risk assessment of triadimefon.

Bioaccumulation and Biomagni cation
The factors bioconcentration (BCF), bioaccumulation (BAF), and biomagni cation (BMF) are regarded as the main coe cient to describe the potential for compounds to be accumulated into biota. BCF refers to the ratio of the concentration of a pollutant in an aquatic organism to the concentration of this substance in the water. Unlike BCF, BAF represents the net bioaccumulation of pollutants absorbed by organisms from all environmental sources. BMF represents the ability of pollutants to transfer along with trophic levels. BCF can be estimated by use of the octanol-water partitioning coe cient (log K OW ) and metabolism. Triadimefon (log K OW 3.1) [53] is characterized as having moderate lipophilicity and longterm stability [60], almost 95% of triadimefon remained after 28 weeks in water at pH of 3.0, 6.0, or 9.0 [32]. According to the Reregistration Eligibility Decision of US EPA, triadimefon can bioaccumulate into tissues of shes [4]. The elimination half-life has been calculated to be 1.1 days via dietary exposure of juvenile rainbow trout [53]. In addition, there is some information on bioaccumulation of triadimefon in the black-spotted frog tadpoles, Tubifex tubifex, Bellamya aeruginosa, and Scenedesmus obliquus. Bioaccumulation of triadimefon by S. obliquus reached its maximum on the 7th day, and accompanied by metabolism of triadimefon. BCFs of R-TDF and S-TDF were 67.32 L/kg and 67.09 L/kg, respectively [36]. This result indicated that bioaccumulation of triadimefon by S. obliquus was not enantio-selective. However, bioaccumulation in the frog was stereo-selective, and S-TDF was absorbed preferentially. This result was consistent with bioaccumulation of triadimefon by T. tubifex [34]. After exposure to triadimefon, the maximum BCF of B. aeruginosa was 19.4 [61]. Comparing with BCF, the bioaccumulation ability of triadimefon in S. obliquus was signi cantly stronger than the other three. Triadimefon has little enrichment potential. However, the metabolite, triadimenol, might have stronger enrichment capacity. The isomers SS-TDN with BCF of 1.44, are greater than that of S-TDF. Triadimenol could also bioaccumulate in the lizard, especially RR-TDN (produced by R-TDF) [35]. However, there were species differences in this condition, and the opposite result appeared in algae. Bioaccumulation capacity for triadimefon was signi cantly less than that of triadimenol [36].
Concentrations of some pollutants can be increased along trophic levels, which is de ned as biomagni cation. A study related to the biomagni cation of triadimefon demonstrated stereo-selective behavior of triadimefon and triadimenol in a simple food chain of algae-tadpole. Concentration of triadimefon in algae-eating tadpoles was less than that of algae. The biomagni cation factor of the enantiomer for the algae-tadpole system did not result in biomagni cation but rather diminution or a decrease in concentration with trophic level [36]. But the tadpoles that ate algae containing triadimefon were still exposed to the triadimefon enantiomer, which caused chronic toxicity including sub-lethal effects and death. Because of the long-term low doses of triadimefon, more research about bioaccumulation and biomagni cation are needed to better understand the behavior of triadimefon in aquatic environments and aquatic organisms.
Effects and mechanisms of toxicity of triadimefon to aquatic organisms According to the harmonized classi cation and labeling (CLP00) approved by the European Union, triadimefon was toxic to aquatic organisms with long lasting effects. Many relevant studies on effects of triadimefon have been conducted. Since adverse effects of triadimefon on human health were of most concern, most of the studies have focused on animal models, including mammals, such as rats and mice. Toxic effects of triadimefon on aquatic organisms have focused mainly on amphibians, such as the African clawed frog (Xenopus laevis) and Black-spotted frog (Rana nigromaculata), sh such as Zebra sh (Danio rerio), Rare minnow (Gobiocypris rarus), Rainbow trout (Oncorhynchus mykiss), and Japanese medaka (Oryzias latipes), as well as zooplankton water ea (D. magna), and algae. A review of the last 20 years of research found that triadimefon can affect species by causing multiple toxic effects on the thyroid, reproduction system, liver, nervous system as well as carcinogenicity and teratogenicity [12,16,20,62]. Furthermore, synergistic effects with other pesticides were also observed [13,63,64]. In this study, effects and mechanisms of action and toxic potencies of triadimefon have been summarized.

Development and Reproductive toxicity
The developmental and reproductive toxicity of triadimefon have been reported and demonstrated in various organisms and their various life stages, including adult African clawed frog, black-spotted frog tadpoles, zebra sh, rare minnow larvae, and water ea. Triadimefon can adversely affect sexual development and reproductive success through a variety of endocrine processes. Exposure to triadimefon can impair reproductive function in various animals, associating with an imbalance of sex hormones [65] and dysfunction of receptor signaling pathways [16].
Studies of effects of triadimefon and it' s transformation product on adult African clawed frog demonstrated that triadimefon and its metabolite (triadimenol) potentially affected African clawed frog through endocrine-disrupting [14] and inhibiting growth of frogs [15]. Similarly, triadimefon can in uence the development of black-spotted frog tadpoles [66]. Disruption of hormones involved in metamorphosis contributed to toxicity to tadpoles. Toxic potency of triadimefon to tadpoles was greater than that of the metabolite, triadimenol, at both greater and lesser concentrations, which should be taken into account when assessing hazard or risks of triadimefon. There were sex-speci c differences in expressions of genes related to thyroid hormone and reproduction. Thus, triadimefon might contribute to the global decline of amphibians, but as of now, there is no direct evidence of this supposition. But, triadimefon might contribute to population-level effects in wild amphibians, so to ensure better protection of natural communities of amphibians and maintain healthy ecosystem function, some eld-level studied might be warranted. A similar conclusion on developmental toxicity of triadimefon on African clawed frog tadpoles was also suggested [12]. Triadimefon altered expression of genes in the HPT axis, down-regulation of thyroglobulin and up-regulation of genes related to thyroid hormone metabolism (ugt1ab) and disrupted synthesis, regulation and action of thyroid hormones in African clawed frog tadpoles. Thus, disruption of thyroid hormone function in African clawed frog tadpoles exposed to triadimefon consequently delayed thyroid hormone-dependent metamorphic development [12,66,67]. That result was consistent with results of previous studies of the endocrine disrupting effects of triadimefon on the zebra sh [67,68]. Incomplete metamorphosis can cause a complete loss of one generation of pond-breeding frogs, ultimately resulting in a decline of populations [66].
In sh, toxic effects of triadimefon on development and reproduction are signi cant. Triadimefon has negative effects on development of each life stage (embryo [69], larvae [11], adult [70]) of sh such as zebra sh and rare minnow. Incubation, which is the process of breaking out of the chorion from the embryo to the individual, is the transition point of the larva and is sensitive to effects of chemicals. This sensitive developmental stage is often used to assess effects of different factors on early development and reproductive capacity of aquatic animals. Exposure to triadimefon signi cantly reduced hatching success of zebra sh embryos, with exposure to 35.6 mg/L [70]. This effect might have been due to inhibition of secretion of hatching gland cells (HGC) [16]. These results indicated that triadimefon can impair critical events during early life-stages of shes through a speci c pathway. Also, triadimefon disrupted endocrine functions including causing anti-estrogenic effects and oxidative stress [11,71] in larvae of rare minnow. Triadimefon caused anti-estrogenic effects by decreasing vitellogenin (VTG) and CYP19a mRNA level. Because CYP19a can catalyze conversion from testosterone to estradiol (E 2 ), the synthesis of E 2 was restrained due to this effect [11,71,72]. Triadimefon also caused reproductive toxicity to sh and even humans via key receptor signaling pathways. Exposure of triadimefon caused reproductive toxicity to zebra sh, which was expressed in the form of disorder of cell maturation, decrease spawning behavior and production of eggs [23]. These results were consistent with results of a previous study where exposure of 0.25 µg/mL triadimefon reduced reproductive success, based on numbers of eggs produced and spawning behavior [24]. During 21-day oral exposure of triadimefon disrupted endocrine homeostasis in Eremias argus via altering concentrations of sex steroid hormones testosterone and E 2 , and inhibiting activities of the cytochrome P450 enzyme (CYP). Relative expressions of steroidogenic-related genes (Erα and Ar, cyp17 and cyp19, and hsd17β) were also affected after 21days of exposure to triadimefon, leading to abnormal reproductive behavior [100]. These results argue that triadimefon is a potential endocrine disruptive chemical, as de ned by the European Union and World Wide Fund for Nature in 2006. In invertebrates, triadimefon can delay molting and development and cause developmental abnormalities among offspring, eventually reducing reproductive tness of the cladoceran, D. magna [17].

Teratogenic and Carcinogenic effect
Studies on teratogenic effects of triadimefon have focused on malformations of development, primarily of branchial arches in amphibians and zebra sh or mammalian embryos. After being exposed to triadimefon during the neurula stage, teratogenic effects were observed in cartilages and muscles of the 1st and 2nd branchial arches in X. laevis [73]. Exposure to 4 µg/ml triadimefon caused teratogenic effects during development of zebra sh embryos [69]. In mammals, relatively small doses of triadimefon or triadimenol can affect branchial development in rat embryos and exhibit teratogenic effects after being exposed during neurulation [21]. Exposure of mouse embryos to triadimefon during early embryo development caused craniofacial malformations like reducing and fusing the 1st and 2nd branchial arches [74][75][76], axial deformities, and maxillary ectopic cartilage [76]. When early development embryos of the ascidian Phallusia mammilla (Chordata Ascidiacea) were exposed to triadimefon, malformations were observed in a dose-dependent manner [77]. Both triadimefon and triadimenol have been classi ed as "possible human carcinogens" [4]. Triadimefon can induce tumorigenesis in rodents, including thyroid tumors and liver tumors [78]. In sh, triadimefon enhanced CYP3A and CYP1A activities, which were associated with tumorigenesis in adult medaka [79]. But there are fewer studies of how triadimefon and/or its metabolite(s) cause these effects.

Neurotoxicity, Hepatotoxicity and Cardiovascular toxicity
Some studies have indicated that triadimefon and triadimenol can cause neurotoxicity, hepatotoxicity, and cardiovascular toxicity in mammals and sh. These ndings suggest potential effects might occur not only in aquatic ecosystems, but in humans as well. Triadimefon and triadimenol cause effects on mammalian central nervous systems [80] of rats, mice and rabbits [4,59,81]. Exposure of mammals to triadimefon resulted in increases in concentrations of dopamine at synapses. The mechanism of action for this effect was similar to that caused by cocaine, which was to bind to the dopamine transporter, thus inhibiting uptake of monoamine [82]. Neurotoxic syndrome in rats, is characterized by increasing motor activity [82,83] can be related to aggressiveness when adult zebra sh were exposed to 5 mg/L triadimefon. This result suggests that triadimefon could also cause neurotoxicity in aquatic ecosystems [18]. The least concentration altered neither locomotor activity nor concentrations of dopamine, but caused changes in expressions of two genes, tyrosine hydroxylase 1 (th1) and dopamine transporter (dat). Triadimefon also reduced extracellular serotonin and had an anxiolytic-like effect, accompanied by lesser production of cortisol. Greater concentrations of triadimefon resulted in a dose-dependent reduction in locomotion, which was reversed or enhanced by SCH-23390 (D1) or Haloperidol (D2) dopamine receptor antagonists, respectively [18].
Liver tissue is rich in metabolic detoxi cation enzymes, such as CYP [84] and is important for detoxi cation, but subject to cause damage of tissues due to oxidative stress. Exposure to triadimefon might inhibit CYP enzyme activity and can damage the liver, which increased duration of magnitude of exposure. During a 21-day orally exposure of triadimefon and its enantiomers to Eremias argus, triadimefon caused hepatocellular damage and affected normal physiological function of the liver. The two enantiomers exhibited different degrees of damage [100]. Damage to liver cells may in turn lead to reproductive and developmental effects. When toxic effects of triadimefon and triadimenol on human hepatocytes were explored in vitro, effects were dose-dependent. Exposure to lesser concentrations (< 20.998 µg/mL) signi cantly (P < 0.05) promoted cell proliferation, while at high concentrations  µg/mL) signi cantly inhibited cell proliferation (P < 0.05). All cells died at concentrations of triadimefon greater than 190.203 µg/mL [85]. In rodents, triadimefon also caused hepatocyte hypertrophy in a dosedependent manner [19]. When studying toxic effects on liver, it is necessary to consider toxicity of chiral enantiomers and metabolites of triadimefon as well as their combined toxicity.
Triadimefon can adversely affect the cardiovascular system, but less information is available. Triadimefon negatively affected morphology and functions of the zebra sh cardiovascular system by down-regulating genes related to the calcium signaling pathway and cardiac muscle contraction [20]. It was speci cally shown in pericardial edema, circulation abnormalities, serious venous thrombosis, greater distance between the sinus venosus, bradycardia, and a signi cantly reduced of cardiac output. More attention should be paid to the harm of triadimefon to human health, especially the occupational population, including farmers, retailers, and pharmaceutical workers. But the risk of triadimefon to the cardiovascular system of humans has been rarely studied, which might should considered in future studies and assessments of effects of triadimefon on health of humans.

Toxic Mechanisms and Pathways
Toxic mechanisms and pathways of triadimefon on aquatic organisms, including sh, amphibians, and invertebrates, were developed and summarized in this section (Figs. 2-4).
Toxic mechanisms and pathways of triadimefon on shes and amphibians include embryo hatching, fecundity, the ratio of male to female, neurotoxicity, and cardiovascular toxicity (Figs. 2 and 3).

Metamorphic development was a predominant effect of triadimefon on amphibians. Triadimefon can
interfere with thyroid hormones via altering expression of genes related to the HPT axis, eventually affecting rates of hatching of embryos and reducing reproductive capacity in sh [16]. This pathway also applies to amphibians [12]. Triadimefon also regulated expressions of mRNA coding for VTG, the precursor of the lipoproteins and phosphoproteins that make up most of the protein content of egg yolk, and CPY19a, thereby inhibiting the aromatase activity and concentrations of VTG to cause an antiestrogenic effect and reduce fecundity [86]. Accelerating conversion of androstenedione to testosterone and forestalling transformation from testosterone to E 2 were other pathways that could reduce fecundity.
The signaling pathways of reproductive toxicity were related to the decrease in the secretory function of the HGCs, inhibition of sperm and egg production and maturation [56], reduction of sperm density and motility, and immature gonad development exposed of sh to triadimefon. Dopamine-related neurotoxicity might be associated with secretion of chitinolytic enzyme, which would link neurotoxicity to reproductive toxicity. Oxidative stress and peroxidation of the membrane lipids were detected whether in sh [11,23] or amphibians [14]. Besides, changes in expressions of genes related to ATPase, including atp2a1l, atp1b2b and atp1a3b, calcium channel-related genes, cacna1ab and cacna1da and cardiac troponin C (tnnc1a) might also be molecular initiating events involved in the mechanism of triadimefon in cardiovascular toxicity [20].
Toxic mechanisms and pathways of triadimefon on aquatic invertebrates (Fig. 4) is relatively simple since, currently information on mechanisms of toxic effects of triadimefon to invertebrates are relatively limited. Triadimefon reduces synthesis of ecdysis hormone in crustaceans by inhibiting activity of P450 enzymes [65,59]. Results of these studies demonstrated that exposure to triadimefon (0.05 mg/L) signi cantly reduced numbers of molts and prolonged durations of inter-molt periods times of D. magna, thus affecting populations [87]. Malformations of eyes of among exposed D. magna offspring were also observed. Therefore, it can be speculated that triadimefon can adversely affect ecological tness of invertebrates, which could result in population-level decreases in tness.
As a fungicide, triadimefon is seldom applied alone, but often together with other agricultural chemicals including herbicides and insecticides in aquatic ecosystems, thus, its combined toxicity cannot be ignored. Per uorooctanoic Acid, which can be present in some formulations, signi cantly increased toxic potency of triadimefon [64]. The 96-h exposure of rare minnow to a mixture of fenvalerate (0.288 mg/L) and triadimefon (6.44 mg/L) illustrated that the two chemicals can interact to cause acute toxicity, endocrine disruption, and oxidative stress during rare minnow embryo development at lesser doses [13].
Co-exposure of zebra sh to triadimefon and butachlor [63] or udioxonil [88] also exhibited a synergistic effect on the embryonic development and thyroid endocrine disruption. Combined toxic effects of triadimefon and other pollutants in the ecological risk assessment of triadimefon in aquatic ecosystems deserves more attention.

Assessment Of Risk Posed By Triadimefon To Aquatic Organisms
Predicted no effect concentration of triadimefon based on various measurement endpoints Screening for potential endocrine disrupting effects and in particular effects on estrogen, suggested that triadimefon resulted in endocrine disrupting effects, and particularly estrogenic effects [9]. Although the concentration of triadimefon in surface waters is generally not very great, triadimefon can persist in aquatic environments for relatively long durations and can cause reproductive damage to aquatic organisms even at small concentrations. Various species or different life stages of the same species might exhibit different responses and mechanisms, and there is gender-speci c effects. As a result of the existence of stereo-selectivity, toxic effects interpretation of this chiral compound is further complicated.
Also, triadimenol, a degradation product of triadimefon, can exhibit greater toxic potency than triadimefon. Triadimefon poses ecological risks to populations of aquatic animals that can result in dysfunction of ecosystem processes and minimize ecological services. US EPA established a screening value of 550 µg/L for protection of underground drinking water sources [4]. The OPP Aquatic Criterion also has de ned an acute toxicity value of 2050 µg/L for sh, a chronic toxicity value of 170 µg/L and an acute toxicity value of 800 µg/L for invertebrates. However, it has been demonstrated that triadimefon affects reproductive systems of aquatic organisms at concentrations as small as 5 µg/L. It is necessary to derive more scienti c safety thresholds based on the endocrine disruptive potential effect, in order to reduce uncertainty and provide the basis for developing criteria for protection of aquatic ecosystems.
In this study, data for toxic potencies of triadimefon (Table S4) were collected from the ECOTOX Knowledgebase (https://cfpub.epa.gov/ecotox/search.cfm) developed by US EPA, following the principles of accuracy, relevance and reliability [29]. A total of 28 toxicity values based on various endpoints for aquatic species met the criteria for data quality and were thus selected for further use. The numbers of toxicity values for triadimefon, based on endpoints including mortality, growth, reproduction and biochemical-cellular responses were 10, 6, 7 and 5, respectively. Toxicity data used to derive PNECs were selected using a hierarchical method. No observed effect concentrations (NOEC) or EC 10 were preferred for growth and development, reproduction and biochemical-cellular endpoints [89]. In the absence of a NOEC or EC 10 , the lowest observed effect concentration (LOEC) or the median effect concentration (EC 50 ) divided by an assessment factor (AF) of 2 or 10 was used [89,90]. Median lethal concentration (LC 50 ) was preferred for mortality. The assumption of a log-normal distribution was tested for each data set by use of the Anderson-Darling (A-D) test by ETX 2.0 software packages [91]. Species sensitivity distributions (SSDs) based on various assessment endpoints were tted (Fig. 5) and the HC 5 (hazardous concentration for 5% species affected) values were then derived by SigmaPlot 14 software packages. PNECs were calculated as the derived HC 5 divided by a safety factor of 5, which depended on the amount of supporting toxicity data [92]. The obtained PNECs based on assessment endpoints of mortality, growth and development, reproduction, and biochemical-cellular were 604.97, 3.95, 3.28 and 0.51 µg triadimefon/L, respectively (Table S5). Based on SSDs curves, the most sensitive endpoint was biochemical-cellular. Sensitivity distributions were compared, by use of the two-sample Kolmogorov-Smirnov test, using the SPSS software version 26 (Table S6). Differences in sensitivity among the endpoints of growth and development, reproduction, and biochemical-cellular were not statistically signi cant. While the sensitivity distribution of mortality was signi cantly different from those of the other three. Second screening for growth and development, reproduction and biochemical was conducted to get the nal PNEC (1.30 µg/L), which was chosen for use in calculating risk quotients. This value was slightly greater than the PNEC of 0.34 µg/L that was calculated by dividing the chronic toxicity value of two trophic levels of aquatic organisms by the safety factor 50 according to the method recommended in the technical guidelines for risk assessment of the European Union [28]. Alternatively, the PNEC based on the most sensitive test endpoint of 9 aquatic organisms, was 3.66 µg/L [24], which was consistent with the PNEC based on growth/development (3.95 µg/L) and reproduction (3.28 µg/L) derived in this study.

Ecological risk assessment of triadimefon
In order to provide a more rigorous scienti c basis and technical support for risk management options for triadimefon, two methods of ecological risk assessment were applied. These included both semiprobabilistic and fully probabilistic approaches.

Risk Quotient (RQ)
Ecological risks caused by triadimefon in surface waters of China were assessed rst by use of risk quotients (RQs), which were calculated as quotients of the maximum concentration of triadimefon in waters divided by the predicted no effect concentration (PNEC) (Equation 1). The results are classi ed as insigni cant if RQ < 0.1, low risk if 0.1 ≤ RQ < 1, moderate risk if 1 ≤ RQ < 10, and high risk if RQ ≥10 [29]. This approach was semi-probabilistic in that the PNEC was derived based on a probabilistic determination but compared to a point estimate of exposures (C m ) Where C m is the maximum concentration of triadimefon at each surface water; PNEC is the predicted no effect concentration derived by the second screening chronic toxicity data, with AF of 5.
RQs for Baihua Lake, Aha Reservoir, Red Maple Lake in Guizhou and the water around rice paddy in Zhejiang were 4.02, 3.15, 2.61, and 9.23, respectively, all of which were de ned as moderate risk (Fig. 6).
The RQ for the Jiulongjiang River (0.13) and waters in Hainan (0.1) exceeded 0.1, but were less than 1.0, which were characterized as low risk. Other regions were de ned as insigni cant according to the results of RQ. If the PNEC of the most sensitive endpoints (biochemical-cellular) was chose to calculate the RQ, Baiyangdian (0.25) in Hebei and Liangshui River in Beijing (0.11) would be also rated low risk. RQ can be in uenced by the choice of PNEC based on different endpoints. In addition, environmental exposure data were collected from limited areas, the actual risk of triadimefon in whole China or world is uncertain.
More work needed to do to acquire detected concentrations of other representative areas. We would also further assessment the risk of triadimefon caused to aquatic ecosystem by drawing the joint probability curves (JPCs).

Joint Probability Curve
Joint probability curves (JPC) are a re ned fully probabilistic method of assessing risks, which can protect the complex ecosystems better [93,94]. The JPG represents the probability of exposure as well as the probability of a species being affected, which can be based on both acute and chronic responses of various endpoints, ranging from lethality to growth or reproduction, which are the apical assessment endpoints most often employed in ecological risk assessments. JPCs are charted by the cumulative function of all biological toxicity data and the inverse cumulative function of pollutant exposure concentration, and the conclusion of risk assessment is expressed in the form of a continuous distribution curve while considering the uncertainty and variability of exposure concentration and toxicity data. The X-axis represents the proportion of aquatic species predicted to be affected at a de ned level of response. The Y-axis represents the probability of exceeding the concentration associated with a particular level of effect (proportion of species affected to a de ned level of effect). This approach combines the duration and intensity of effects and expresses it as a probability of exposure and response [95,96]. The relationship indicates that while at greater concentrations the probability of response is greater, the probability of exceeding that concentration would be less. Each point along the JPC curve represents the same product of the probability of exposure and associated probability of effect. Each point in the curve indicates the joint probability that a certain percentage of organisms will be affected in the target water body. The area under the curve is the region that would be expected to be affected up to that joint probability. The closer the joint probability density curve is to the X-axis, the less likely organisms will be affected [94]. By this approach, ecological risks are characterized by for regions of combined exposure and response: de-minimis risk ( ), low risk ( ), intermediate risk ( ) and high risk ( ) [97,29]. The JPCs of triadimefon based on different test endpoints (mortality, growth, reproduction, biochemical-cellular and the second screening of the 3 chronic endpoints) in China (Fig. 7) demonstrate that, risks of toxic effect of triadimefon to aquatic organisms based on endpoints of growth/development, reproduction and G+R+B were characterized as "low", while risks based on biochemical-cellular effects was classi ed as "intermediate", which indicated that current concentrations of triadimefon in surface waters of China might pose risks of reproductive damage to aquatic organisms.
The probability that reproduction of 5% of aquatic organisms in surface water in China would be affected by current concentrations of triadimefon was close to 15%, while the probability of growth being affected was characterized as "low". The results of risk assessment about triadimefon were consistent with conclusions of previous studies [24]. The low risk was represented in the studies of Sun et al. (2020) [98] and Zhou et al. (2020) [99]. Since data of exposure and toxicity effect are limited, risk assessments of triadimefon on aquatic environments determined here were uncertain. triadimefon might have certain risks to aquatic organisms and even aquatic biological population and aquatic ecosystems. Much work about concentration surveys was needed especially water bodies near agriculture land. According to previous surveys, the water bodies near agriculture land might have a higher concentration of triadimefon than water bodies far from land of much triadimefon use.

Conclusions
Environmental exposure, migration and transformation in the environment, bioaccumulation and metabolism in organisms, and effects on and toxic potencies to organisms of triadimefon were reviewed.
Risks posed by triadimefon in various aquatic environments were assessed based on both semiprobabilistic and fully probabilistic approaches, in which the risk quotient and joint probability density curve were used, respectively. The main conclusions are as follows: (1) triadimefon is ubiquitous in surface waters with high rate of detection at relatively low limits of quanti cation; (2) abiotic degradation of triadimefon via hydrolysis and photolysis, is affected by multiple factors, including pH, temperature, solvent, and radiation wavelength; (3) stereo-selectivity during biotic degradation of triadimefon occurs in soil and during transformation by organisms; (4) different enantiomers might be biotransformation and degraded differentially and cause multiple effects via various mechanisms. Although triadimefon has potential for enrichment, its enrichment capacity is relatively small, but enrichment of its metabolite triadimenol might be more signi cant; (5) toxic effects of triadimefon on aquatic organisms, especially on reproduction, is a key assessment endpoint that cannot be ignored. However, studies of effects of triadimefon have focused primarily on amphibians and sh, and then there are relatively few studies on invertebrates, which need to be investigated more comprehensively. The metabolite of triadimefon, triadimenol, might have greater toxic potency than triadimefon. Toxic effects of triadimenol and its stereo-selectivity need to be considered when assessing risks of triadimefon; (6) the risk of triadimefon based on JPCs rated low or intermediate risk based on different effects. This suggests that triadimefon may cause harm at the population level or even at the ecosystem level. Although the toxicity and risk of triadimefon were determined to be low, measurements of exposure in various aquatic environments are limited especially in areas outside of China. And few studies have considered the chiral nature of triadimefon and it' s transformation products. Studies of triadimefon in China have focused primarily in the Beijing-Tianjin-Hebei and Yangtze River Delta regions, while the data of other regions where agricultural production is greater and triadimefon might be more applied such as northwest and northeast China are still limited. Currently, there is surface water environmental quality standard, so it is suggested to strengthen regulations to control potential effects of triadimefon and it' s transformation products.  Availability of data and material

Abbreviations
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.  Schematic diagram of toxic mechanisms and pathway for sh exposed to triadimefon. The green portion of the diagram represents effects on gene-level, while the violet portion of the diagram represents the adverse outcome expressed at the individual. Finally, the cyan portion of the diagram represents response on protein or hormone where links to gene-level response could be linked to individual-level response.

Figure 4
Schematic diagram of toxic mechanisms and pathway for exposure of aquatic invertebrates to triadimefon, which is based on currently available results. The cyan portion of the diagram represents response on protein or hormone, while the violet portion of the diagram represents the adverse outcome expressed at the individual.

Figure 5
Species sensitivity distributions (SSDs) for triadimefon (triadimefon) based on several endpoints for aquatic organisms.

Figure 6
Distribution of maximum risk quotients (RQs) for triadimefon in some Chinese rivers and reservoirs; risk value was represented by different colors. RQ < 0.1 represented insigni cant risk, 0.1 ≤ RQ < 1 represented low risk, 1 ≤ RQ < 10 represented moderate risk.

Supplementary Files
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