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Toxicity effects of captan on different life stages of zebrafish (Danio rerio)
Environmental Toxicology and Pharmacology 69 (2019) 80–85
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Toxicity effects of captan on different life stages of zebrafish (Danio rerio) ⁎
T
Yimeng Zhou, Xiangguang Chen, Miaomiao Teng, Jie Zhang, Chengju Wang China Agricultural University, Beijing, 100193, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Captan Zebrafish Acute toxicity Embryonic development
The objective of this study was to evaluate the toxicity and developmental effects of captan on different life stages (embryo and adult) of zebrafish (Danio rerio). The results showed that the 96-h lethal concentration 50 (LC50) values of embryo and adult zebrafish (exposed to captan) were 0.81(0.75−0.87) mg/L and 0.65(0.62−0.68) mg/L, respectively. The results of developmental effect experiment showed that captan can significantly decrease the heartbeats and inhibit the hatching rate and growth of zebrafish embryos. Moreover, captan exposure can induce a series of deformities, including pericardial edema, yolk sac edema, spine curvature, and tail bending, in zebrafish embryos during the developmental period. Among these, the most significant were tail bending and spine curvature.
1. Introduction Captan [N-(trichloromethylthio)-cyclohex-4-ene-1,2-dicarboximide], as a broad-spectrum nonsystematic phthalimide fungicide, is used for the treatment of foliar, soil-borne, and seed-borne diseases. The action mechanism of captan in fungal death involves inhibiting the respiration and metabolic processes of fungi by reacting with sulfhydryl groups to generate nonspecific sulfhydryl reactants (Barreda et al., 2006). Ultimately, captan reduces fungal spore germination, growth, and oxygen uptake (Owens and Novotny, 1959). Captan has significant effects in controlling a variety of diseases in fruits, vegetables, rice, and other crops (Xu et al., 2008; Gopalakrishnan et al., 2012). Some adverse effects associated with the wide use of captan include the presence of captan residues in the aquatic environment, which have been already reported in some places. For example, captan was detected at 0.78 μg/L in the surface water of an irrigated farming area in the region of Guaira county (Sao Paulo, Brazil) (Filizola et al., 2005) and also in the river water of Greece (Loudias, Axios, Nestos, Evrotas, and Louros rivers) with the maximum concentrations of 24, 40, 80, 260, and 32 ng/L (Konstantinou et al., 2006). Previous studies have reported that captan has adverse effects in humans and other nontarget organisms. On October 27, 2017, the World Health Organization's International Agency for Research on Cancer published a preliminary list of carcinogens, and captan was included in the list of three types of carcinogens. As early as the 1980s, it was found that captan had mutagenicity and was teratogenic to rodents (Legator, 1981; Waters et al., 1982). A recent study showed that captan can induce chromosome damage on human bronchial epithelial
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cells, and the main manifestations were structural aberrations (Xu et al., 2010). In addition, 90-d oral toxicity tests conducted on rats found that the forestomach and liver were the toxic target organs of 95% captan, and the maximum no-effect dose to rats was estimated to be 4 000 ppm (Liu et al., 2013). It is reported that the exposure of captan can cause inflammation and necrosis in the liver, trunk kidney, and spleen of juvenile rainbow trout (Oncorhynchus mykiss) (Boran et al., 2012). Zebrafish (Danio rerio) is a typical small tropical aquarium fish, which has a long history of application in toxicology research, considering its low cost, small size, fast breeding cycle, easy mass rearing, and the ability of fast propagation (Lele and Krone, 1996). Zebrafish embryos are also characterized by in vitro fertilization, rapid embryonic development, and optical transparency, which allow to easily detect their morphological endpoints or observe their development in the early stages of life (Hill et al., 2005). Zebrafish has become a common model organism in assessing the negative effects of toxic substances in the aquatic environment (Chow et al., 2013). Recently, zebrafish embryos have been used to evaluate the developmental neurotoxicity of 7 well-characterized compounds, which suggested that zebrafish is useful in determining whether a compound causes specific neurotoxicity or general developmental toxicity (Ton et al., 2006). Teng et al. found that difenoconazole exposure at environmentally relevant concentrations elicited estrogenic endocrine disruption effects via altering the homeostasis of sex steroid hormones in the HPGL axis, and the adverse effects can be transferred to the offspring (Teng et al., 2017). Although the toxicity of captan on humans and rats (Que et al., 2009; Liu et al., 2013) has been widely reported, it is very rarely reported on aquatic organisms, especially in fish. And the study of acute
Corresponding author. E-mail address: [email protected] (C. Wang).
https://doi.org/10.1016/j.etap.2019.04.003 Received 31 October 2018; Received in revised form 28 March 2019; Accepted 2 April 2019 Available online 03 April 2019 1382-6689/ © 2019 Elsevier B.V. All rights reserved.
Environmental Toxicology and Pharmacology 69 (2019) 80–85
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glass rod to touch the tail of the fish without visible motion.
and developmental toxicity of captan on zebrafish has hardly been reported. To reflect the potential threat of captan on aquatic organisms and to lay the foundation for further study, the acute and developmental toxicity of captan on adult and embryo zebrafish was evaluated. Moreover, this study will provide a scientific basis for the proper application and risk assessment of captan in environments.
2.4. Drug content monitoring Exposure solutions in each replicate were taken at the beginning of the test, added 4.0 g sodium chloride, 10 mL acetonitrile, vortex extraction, and centrifuged at 5000 rpm for 5 min. The supernatant was collected, evaporated with a rotary evaporator, blow dried, brought to volume with 1 mL acetonitrile, passed the 0.22 μm organic filter, and injected into the injection bottle. High performance liquid chromatography (Agilent 1260 Infinit, Agilent) conditions were as follows: column was Eclipse Plus C18 (size 4.6 × 100 mm × 3.5 μm), column temperature was 30℃, detector was diode array detector(DAD), detection wavelength was 210 nm, column flow rate was 0.6 mL/min, the mobile phase was acetonitrile/water = 55/45 (v/v), and the amount of each injection was 10 μL.
2. Materials and methods 2.1. Reagents The recombined water was formulated according to ISO-7346-3 (ISO, 1996), containing 2 mmol/L Ca2+, 0.5 mmol/L Mg2+, 0.075 mmol/L Na+, and 0.074 mmol/L K+. Captan (80% water dispersible granules) (CAS:133-06-2) was provided by the Chinese Agriculture University. It was dissolved in recombined water and stored at 4℃. All reagents used in this study were of analytical grade.
2.5. Data processing 2.2. Zebrafish culture and embryo collection Statistical software SPSS 16.0 was used to calculate LC50, coefficient of determination, and confidence interval. All the data were expressed as the mean ± standard deviation (SD). Significance analysis was performed using one-way ANOVA followed by Dunnett post hoc comparison. P < 0.05 was considered significant, P < 0.01 was considered highly significant.
Wild-type zebrafish (4 months old) were purchased from Beijing Hongdagaofeng Aquarium Department. Male and female fish were maintained in Esen equipment on a 14-h light/10-h dark cycle at 26℃ and fed with hatched Artemia nauplii twice a day. Adult zebrafish were acclimatized for two weeks before experiment, and mortality was less than 5% during this period (Mu et al., 2013). Before the test, male and female zebrafish were placed in a special spawning box at a ratio of 1:2 overnight, separated by a transparent partition, covered with a lid and then with a black cloth completely at night. Next morning, the black cloth and the partitions were removed. About half an hour later, the embryos were collected and washed with recombined water. Then, the embryos were observed under the microscope, and those embryos in a division period and had no obvious abnormalities were selected for exposure experiments (Mu et al., 2013).
3. Results 3.1. Captan analysis The analytical results showed that the measured and theoretical concentration deviations of the water samples of all treatment groups were less than 20% (Table 1). According to the OECD test guideline, the theoretical concentration of this test can represent the measured concentration for testing (OECD, 1992).
2.3. Acute toxicity of captan
3.2. Lethal effect of captan
2.3.1. Embryonic acute toxicity test The acute toxicity test of embryo was performed according to the OECD test guideline No. 236 (Busquet et al., 2013) and Mu (Mu et al., 2013). According to the pretest, embryos about 1 h post-fertilization (hpf) were randomly placed in a 5-cm diameter petri dish containing 20-mL captan exposure solution (0, 0.58, 0.66, 0.75, 0.86, 1.00 and 1.16 mg/L, n = 3 replicates). The final content of acetone was 0.005% in all the six groups. Each replicate had 10 embryos, and the exposure media was renewed every 24 h. The external conditions during exposure were the same as the culture environment. The number of dead embryos was recorded daily and promptly removed. Embryos with egg coagulation, developmental stagnation, and no heartbeat were considered dead (Nagel, 2002). To study the developmental toxicity of captan to embryo, spontaneous movements (in 20 s) at 24 hpf, heartbeats (in 20 s) at 48, 72 hpf, and the body length of hatched individuals at 96 hpf were tested. Moreover, the hatched and malformations of embryos were recorded daily. Spontaneous movements, heartbeats, and malformations were observed under microscope, and body length was measured under Aigo GE-5 digital microscope (Aigo, Beijing, China).
From Table 2, we can observe that captan had high toxicity to embryo and adult zebrafish. The acute toxicity/lethality showed a time effect and increased with the exposure time. The sensitivity of embryonic and adult fish to captan was different, as the 96 h-LC50 values of embryo and adult zebrafish exposed to captan were 0.81 (0.75−0.87) mg/L and 0.65(0.62−0.68) mg/L, respectively. The toxicity of captan to the two different stages of zebrafish was as follows: adult fish > embryos. 3.3. Embryonic developmental effects of captan 3.3.1. Effect of captan on the spontaneous movements of embryos The zebrafish embryos exposed to 24 hpf have regular spontaneous movement. As shown in Fig. 1A, compared with the control group, Table 1 Chemical analysis results of acute toxicity test.
2.3.2. Adult fish acute toxicity test The acute toxicity test of adult fish was performed according to the OECD test guideline No.203 (OECD, 1992) and Mu (Mu et al., 2013). According to the pretest, adult fish were randomly placed in 5 L tanks containing 4 L captan exposure solution (0, 0.56, 0.61, 0.66, 0.72, and 0.78 mg/L) for 4 days. Three replicates per treatment with 10 healthy fish in each replicate were used and renewed the solution daily. The number of dead fish was recorded at 24 h, 48 h, 72 h, and 96 h and removed them timely. The standard of judging the dead fish is using a
Test stage
Nominal con.(mg/L)
Actual con.(mg/L)
Deviationb(%)
Embryo
Control 0.58 0.66 0.76 0.87 1.00 1.16
n.d.a 0.482 0.634 0.718 1.038 0.924 1.057
n.d. −0.169 −0.039 −0.055 0.193 −0.076 −0.089
a b
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n.d.= not detect. deviation= (actual content-nominal content) / nominal content.
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Table 2 96 h-LC50 values of two different stages of zebrafish (exposed to captan). Life stage
Exposure time/h
LC50(95% CLa)mg/L
X2b
R²
embryo
24 48 72 96 24 48 72 96
0.81(0.76-0.88) 0.81(0.75-0.87) 0.81(0.75-0.87) 0.81(0.75-0.87) 0.78(0.73-0.93) 0.69(0.66-0.72) 0.66(0.63-0.69) 0.65(0.62-0.68)
y = 7.50x+1.25 y = 7.50x+1.25 y = 7.50x+1.25 y = 7.50x+1.25 y = 7.00x+0.90 y = 10.00x+1.75 y = 10.00x+2.00 y = 10.00x+2.00
0.91 0.93 0.93 0.93 0.92 0.95 0.98 0.99
adult
a b
Table 3 Hatching rate of zebrafish embryos at 72 hpf and 96 hpf.
CL = Confidence limit. X2 = Chi-square.
Exposure concentration (mg/L)
Hatching rate at 72 hpf (%)
Hatching rate at 96 hpf (%)
0.00 0.58 0.66 0.76 0.87 1.00 1.16
96. 7 ± 5.8 83.3 ± 15.3 53.3 ± 15.3 50.0 ± 17.3 40.0 ± 10.0 23.3 ± 10.0 3.0 ± 5.8
100.0 ± 0.0 86.7 ± 15.3 63.3 ± 11.5 56.7 ± 20.8 43.3 ± 15.3 30.0 ± 17.3 6.7 ± 11.5
3.3.3. Effect of captan on embryonic hatching Under normal conditions, zebrafish embryos start hatching at 48 hpf. All the embryos in the control group hatched at 72 hpf, while 0.58 mg/L and other higher concentration groups of captan had significant inhibition effect on the hatching rate of zebrafish embryos at 72 hpf and 96 hpf (Fig. 1D). Furthermore, compared with the control group, the hatching rate of the 1.00 mg/L group was 30.0% ± 17.3% at 96 hpf, and the hatching rate of the highest concentration group was only 6.7% ± 11.5% at 96 hpf (Table 3).
captan exposure had no significant effect on the spontaneous movements of zebrafish embryos in all captan treatments, including in 1.16 mg/L of the highest concentration.
3.3.2. Effect of captan on embryonic heartbeat As shown in Fig. 1B, compared with the control group, captan exposure inhibited the heartbeats of zebrafish embryos in a concentrationdependent manner. The inhibition effect of captan on embryonic heartbeat at 48 hpf and 72 hpf was not significant in low-dose treatment groups, but significant in 0.76 mg/L and other higher concentration groups. Because of the high concentration of captan, almost all the embryos in the 1.16 mg/L concentration group were dead, and hence the data were not representative.
3.3.4. Effect of captan on embryonic body length Compared with the control group, high concentration of captan (concentrations of 0.76 mg/L and above) could significantly reduce the body length of hatched larvae at 96 hpf (Fig. 1C). And the inhibition rate increased with the increase of captan concentration, and the
Fig. 1. A. Spontaneous movements of embryos at 24 hpf. B. Heartbeats of embryos at 48 hpf and 72 hpf. C. Body length of hatched larvae at 96 hpf. D. Hatching rate of embryos at 72 hpf and 96 hpf. Asterisks denote significant difference between the treatment and control groups (determined by Dunnett post hoc comparison, P < 0.05*; P < 0.01**). Error bars indicate standard deviation. 82
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flutolanil. However, many studies have found that the early life stages of aquatic organisms, in comparison with adult stages, were more sensitive to toxic substances. According to Lele (Lele and Krone, 1996) and Westemhagen (Westernhagen, 1988), the embryo and larvae stages were the most sensitive life stages of Osteichthyes fish because metabolic detoxification is very weak in the embryonic stage compared with adult individuals (Embry et al., 2010). Therefore, further studies are required to identify the factors that cause differences in the susceptibility of zebrafish embryos and adults. And a previous study found that the ability of test substances to penetrate the chorion of embryo may be a factor (Embry et al., 2010). In addition, zebrafish embryonic toxicity of silver nanoparticles (AgNPs) in the absence of chorion was greater than when it was present (Kim and Tanguay, 2014). The function of zebrafish embryo chorion to captan might be tested through experiments on zebrafish larva. Determining the most sensitive phase of environmental organisms to toxic substances can reduce the level of toxicity to environmental organisms and thus reduce the incorrect use of pesticides. Furthermore, our results showed that captan can induce a series of adverse effects in the development of zebrafish embryos, including decreased heart rate, growth inhibition, and various malformations. The results showed that high concentration of captan (0.76 mg/L and other higher concentration groups) significantly inhibited the heartbeats of zebrafish embryos at 48 hpf and 72 hpf, accompanied by yolk sac deformities and pericardial deformities, suggesting that captan can affect the heart development of zebrafish embryos. Heart is a very important functional organ. Yu et al. found that an organosulfur fungicide—thiram can decrease the heartbeats of zebrafish embryos and cause pericardial edema (Yu et al., 2011) because the exposure of thiram induced renal (Hill et al., 2004) dysfunction, water barrier damage, and drainage dysfunction in the body of zebrafish larvae (Cao et al., 2003). Detailed examination by Raldúa et al. (2008) suggested that during the embryonic development of oviparous fish, nutrients were mainly derived from endogenous yolk nutrients (Raldúa et al. 2008); therefore, the abnormal development of yolk sac is likely to result in impaired nutritional supply of zebrafish embryos. Sufficient energy supply is required for the proper functioning of heart, and insufficient energy supply results in symptoms such as decreased heart rate (Kodde et al., 2007). It is reported that flutolanil exposure can cause slower heartbeat in zebrafish embryos, and the pericardial area of zebrafish larvae in the treated groups is significantly larger than that of the control group. And metabolomics analysis showed that energy metabolism-related pathways were also changed in zebrafish embryos exposed to flutolanil (Teng et al., 2018). In addition to the decrease in the heartbeats of zebrafish embryos, the body length of zebrafish larvae was also inhibited when exposed to
Table 4 Inhibition rate of captan on the body length of zebrafish hatched larvae. Exposure concentration (mg/L)
Body length (mm)
Inhibition rate (%)
0.00 0.58 0.66 0.76 0.87 1.00
3.68 3.42 3.29 2.90 2.79 2.56
0 5.26 8.86 19.67 22.71 29.09
± ± ± ± ± ±
0.06 0.06 0.03 0.16 0.10 0.17
maximum inhibition rate was 29.09% at 1.10 mg/L concentration group (Table 4). Almost all the embryos in the 1.16 mg/L concentration group were dead, and hence the data was not representative. 3.3.5. Teratogenic effects of captan on embryos Exposure of captan induced a series of deformities in zebrafish embryos, including pericardial edema (Pe), yolk sac edema (Ysc), tail bending (Tb), and spine curvature (Sc) (Fig. 2). The most significant teratogenic effects were tail bending and spine curvature. As shown in Fig. 3, compared with the control group, captan groups with the concentrations of 0.76 mg/L and above can significantly induce tail bending and spine curvature at 72 hpf. However, captan exposure had no significant effect on yolk sac edema at 48 hpf and 72hpf, and pericardial edema rate was reduced at 72 hpf than at 48 hpf. 4. Discussion Multiple toxic substances have toxic effects on different stages of zebrafish. It has been reported that 96-h LC50 values of embryo and adult zebrafish (exposed to triclosan) were 0.42 mg/L and 0.34 mg/L, respectively. Moreover, embryo toxicity, delay in otolith formation, eye and body pigmentation, and malformations including spine malformations, pericardial edema and undersize, were evident. However, triclosan did not change biomarker levels and did not elicit a micronucleus in adults (Oliveira et al., 2009). Table 5 shows the 96 h-LC50 values of several common fungicides (difenoconazole, thifluzamide, bromothalonil, flutolanil, pyraoxystrobin, penthiopyrad) on different stages of zebrafish and their main teratogenic effects on embryos (Mu et al., 2013; Yang et al., 2016a, 2016b; Li et al., 2018; Qian et al., 2019). Compared with those fungicides, our results suggested that captan had high toxicity to both adult and embryo zebrafish. And it was found that zebrafish embryos, in comparison with adult fish, were more resistant to captan, which is consistent with the results of Mu (Mu et al., 2013) and Yang (Yang et al., 2016a,b). The results revealed that adult zebrafish were more sensitive to difenoconazole, bromothalonil, and
Fig. 2. Teratogenic effects of captan on zebrafish embryos. A. Normal embryo in the control group at 48 hpf. B. Embryo with pericardial edema (Pe) and spine curvature (Sc) at 48 hpf. C. Embryo with pericardial edema (Pe) and yolk sac edema (Yse), and spine curvature (Sc) at 48 hpf. D. Embryo with spine curvature (Sc) at 48 hpf. E. Normal hatched larvae in the control group at 72 hpf. F. Hatched larvae with pericardial edema (Pe) at 72 hpf. G. Hatched larvae with tail bending (Tb) at 72 hpf. H. Hatched larvae with spine curvature (Sc) at 72 hpf. 83
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Fig. 3. Deformity rate of zebrafish embryos at 48 hpf and 72 hpf. Pe: pericardial edema; Ysc: yolk sac edema; Tb: tail bending; Sc: spine curvature. Asterisks denote significant difference between the treatment and control groups (determined by Dunnett post hoc comparison, P < 0.05*; P < 0.01**). Error bars indicate standard deviation.
Table 5 96 h-LC50 values of several common fungicides on different stages of zebrafish and their main teratogenic effects on embryos. Fungicide
96h-LC50(95% CLa)of adult fish (mg/L)
96h-LC50(95%CLa) of embryo (mg/L)
Main deformities in zebrafish embryos
References
captan difenoconazole thifluzamide bromothalonil flutolanil pyraoxystrobin penthiopyrad
0.81(0.75-0.87) 1.45(1.38-1.53) 4.19(4.07-4.29) 2.52 (4.17–4.47) 2.70 (2.58–2.83) 0.0060(0.0052-0.0062)
0.65(0.62-0.68) 2.34(2.27-2.40) 3.08(2.96-3.22) 4.34 (4.25–4.77) 5.47 (5.04–5.84) 0.0041(0.0039-0.0043) 2.77(2.73-2.82)
tail bending spine curvature yolk sac edema yolk sac edema pericardial edema notochord deformation short body length pericardial edema and yolk sac edema yolk sac edema pericardial edema
Mu et al., 2013 Yang et al., 2016a,b Yang et al., 2016a,b Yang et al., 2016a,b Li et al., 2018 Qian et al., 2019
Acknowledgment
captan. The results showed that 0.76 mg/L and other higher concentrations of captan significantly inhibited the body length of zebrafish larvae, the maximum inhibition rate reached 29.09%, and significant tail bending and spine curvature can also be observed. Myristoylated alanine-rich C-kinase substrate (MARCKS) is an actin binding protein substrate of protein kinase C (PKC). Ott, L.E. et.al found that Marcksa and Marcksb, two MARCKS paralogs, play an important role in the tail development of zebrafish, morpholino-based targeting of either MARCKS protein resulted in increasing mortality and a range of gross phenotypic abnormalities, such as a slight curve of a full-length tail, a severe curve or twist of a full-length tail and a truncated tail (Ott et al., 2011). Besides, studies have shown that lysyl oxidase is of great importance in the development of spines in aquatic vertebrates, and inhibition leads to abnormalities in aquatic spines (Zhou et al., 2009). Tail deformity may be related to myristoylated alanine-rich C-kinase or lysyl oxidase or both. While the most significant teratogenic effect induced by captan was spine curvature, other fungicides have different developmental toxicity on zebrafish embryos (Table 5), and this may be due to their specific action mechanism. Therefore, further research is required to identify the exact mechanism of captan on zebrafish embryo tail development.
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5. Conclusion Our study showed that captan had high toxicity to adult and embryo zebrafish, the 96 h-LC50 values of embryo and adult zebrafish (exposed to captan) were 0.81(0.75−0.87) mg/L and 0.65(0.62−0.68) mg/L, respectively. Captan exposure can induce a series of negative effects on embryos, including heart rate decrease, hatching regression, and growth inhibition. Captan can also induce significant teratogenic effects, including spine curvature and tail bending, on zebrafish embryos. Further studies on the action mechanism of captan on zebrafish can provide the scientific basis for the proper application of captan.
Conflict of interest The authors declare that they have no conflict of interest.
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Environ. Pollut. 141 (3), 555. Legator, M.S., 1981. Mutagenicity of captan. Mutat. Res. Mutagen. Relat. Subj. 85 (4) 220-220. Lele, Z., Krone, P.H., 1996. The zebrafish as a model system in developmental, toxicological and transgenic research. Biotechnol. Adv. 14 (1), 57–72. Li, H., Yu, S., Cao, F., Wang, C., Zheng, M., Li, X., et al., 2018. Developmental toxicity and potential mechanisms of pyraoxystrobin to zebrafish (danio rerio). Ecotoxicol. Environ. Saf. 151, 1–9. Liu, Y.E., Zhang, Y.B., Shi, L., 2013. Toxicity of 95% captan in rats: an oral toxicity test. Chin. J. Public Health. Mu, X., Pang, S., Sun, X., Gao, J., Chen, J., Chen, X., Li, X., Wang, C., 2013. Evaluation of acute and developmental effects of difenoconazole via multiple stage zebrafish assays. Environ. Pollut. 175 (8), 147–157. Nagel, R., 2002. DarT: the embryo test with the zebrafish Danio rerio - a general model in ecotoxicology and toxicology. Altex-Alternativen Zu Tierexperimenten. 19, 38–48. OECD, 1992. ). Test no. 203: fish, acute toxicity test. Oecd Guidelines for the Testing of Chemicals, vol. 1. pp. 1–10 (10). Oliveira, R., Domingues, I., Koppe, G.C., Soares, A.M., 2009. Effects of triclosan on zebrafish early-life stages and adults. Environ. Sci. Pollut. Res. Int. 16 (6), 679. Ott, L.E., Mcdowell, Z.T., Turner, P.M., Law, M.H., Adler, K.B., Yoder, J.A., Jones, S.L., 2011. Two myristoylated alanine-rich C-kinase substrate (MARCKS) paralogs are required for normal development in zebrafish. Anat. Rec. Adv. Integr. Anat. Evol. Biol. 294 (9), 1511–1524. Owens, R., Novotny, H., 1959. Mechanism of Action of the Fungicide Captan. 20. pp. 171–190. Qian, L., Qi, S., Cao, F., Zhang, J., Li, C., Song, M., et al., 2019. (2018). Effects of penthiopyrad on the development and behavior of zebrafish in early-life stages. Chemosphere 214, 184–194. Que, B.L., Ge, Y.C., Liang, L.Y., 2009. Research on the sub-chronic oral toxicity of captan. China Occup. Med. Raldúa, D., André, M., Babin, P.J., 2008. Clofibrate and gemfibrozil induce an embryonic
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Toxicity effects of captan on different life stages of zebrafish (Danio rerio) ⁎
T
Yimeng Zhou, Xiangguang Chen, Miaomiao Teng, Jie Zhang, Chengju Wang China Agricultural University, Beijing, 100193, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Captan Zebrafish Acute toxicity Embryonic development
The objective of this study was to evaluate the toxicity and developmental effects of captan on different life stages (embryo and adult) of zebrafish (Danio rerio). The results showed that the 96-h lethal concentration 50 (LC50) values of embryo and adult zebrafish (exposed to captan) were 0.81(0.75−0.87) mg/L and 0.65(0.62−0.68) mg/L, respectively. The results of developmental effect experiment showed that captan can significantly decrease the heartbeats and inhibit the hatching rate and growth of zebrafish embryos. Moreover, captan exposure can induce a series of deformities, including pericardial edema, yolk sac edema, spine curvature, and tail bending, in zebrafish embryos during the developmental period. Among these, the most significant were tail bending and spine curvature.
1. Introduction Captan [N-(trichloromethylthio)-cyclohex-4-ene-1,2-dicarboximide], as a broad-spectrum nonsystematic phthalimide fungicide, is used for the treatment of foliar, soil-borne, and seed-borne diseases. The action mechanism of captan in fungal death involves inhibiting the respiration and metabolic processes of fungi by reacting with sulfhydryl groups to generate nonspecific sulfhydryl reactants (Barreda et al., 2006). Ultimately, captan reduces fungal spore germination, growth, and oxygen uptake (Owens and Novotny, 1959). Captan has significant effects in controlling a variety of diseases in fruits, vegetables, rice, and other crops (Xu et al., 2008; Gopalakrishnan et al., 2012). Some adverse effects associated with the wide use of captan include the presence of captan residues in the aquatic environment, which have been already reported in some places. For example, captan was detected at 0.78 μg/L in the surface water of an irrigated farming area in the region of Guaira county (Sao Paulo, Brazil) (Filizola et al., 2005) and also in the river water of Greece (Loudias, Axios, Nestos, Evrotas, and Louros rivers) with the maximum concentrations of 24, 40, 80, 260, and 32 ng/L (Konstantinou et al., 2006). Previous studies have reported that captan has adverse effects in humans and other nontarget organisms. On October 27, 2017, the World Health Organization's International Agency for Research on Cancer published a preliminary list of carcinogens, and captan was included in the list of three types of carcinogens. As early as the 1980s, it was found that captan had mutagenicity and was teratogenic to rodents (Legator, 1981; Waters et al., 1982). A recent study showed that captan can induce chromosome damage on human bronchial epithelial
⁎
cells, and the main manifestations were structural aberrations (Xu et al., 2010). In addition, 90-d oral toxicity tests conducted on rats found that the forestomach and liver were the toxic target organs of 95% captan, and the maximum no-effect dose to rats was estimated to be 4 000 ppm (Liu et al., 2013). It is reported that the exposure of captan can cause inflammation and necrosis in the liver, trunk kidney, and spleen of juvenile rainbow trout (Oncorhynchus mykiss) (Boran et al., 2012). Zebrafish (Danio rerio) is a typical small tropical aquarium fish, which has a long history of application in toxicology research, considering its low cost, small size, fast breeding cycle, easy mass rearing, and the ability of fast propagation (Lele and Krone, 1996). Zebrafish embryos are also characterized by in vitro fertilization, rapid embryonic development, and optical transparency, which allow to easily detect their morphological endpoints or observe their development in the early stages of life (Hill et al., 2005). Zebrafish has become a common model organism in assessing the negative effects of toxic substances in the aquatic environment (Chow et al., 2013). Recently, zebrafish embryos have been used to evaluate the developmental neurotoxicity of 7 well-characterized compounds, which suggested that zebrafish is useful in determining whether a compound causes specific neurotoxicity or general developmental toxicity (Ton et al., 2006). Teng et al. found that difenoconazole exposure at environmentally relevant concentrations elicited estrogenic endocrine disruption effects via altering the homeostasis of sex steroid hormones in the HPGL axis, and the adverse effects can be transferred to the offspring (Teng et al., 2017). Although the toxicity of captan on humans and rats (Que et al., 2009; Liu et al., 2013) has been widely reported, it is very rarely reported on aquatic organisms, especially in fish. And the study of acute
Corresponding author. E-mail address: [email protected] (C. Wang).
https://doi.org/10.1016/j.etap.2019.04.003 Received 31 October 2018; Received in revised form 28 March 2019; Accepted 2 April 2019 Available online 03 April 2019 1382-6689/ © 2019 Elsevier B.V. All rights reserved.
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glass rod to touch the tail of the fish without visible motion.
and developmental toxicity of captan on zebrafish has hardly been reported. To reflect the potential threat of captan on aquatic organisms and to lay the foundation for further study, the acute and developmental toxicity of captan on adult and embryo zebrafish was evaluated. Moreover, this study will provide a scientific basis for the proper application and risk assessment of captan in environments.
2.4. Drug content monitoring Exposure solutions in each replicate were taken at the beginning of the test, added 4.0 g sodium chloride, 10 mL acetonitrile, vortex extraction, and centrifuged at 5000 rpm for 5 min. The supernatant was collected, evaporated with a rotary evaporator, blow dried, brought to volume with 1 mL acetonitrile, passed the 0.22 μm organic filter, and injected into the injection bottle. High performance liquid chromatography (Agilent 1260 Infinit, Agilent) conditions were as follows: column was Eclipse Plus C18 (size 4.6 × 100 mm × 3.5 μm), column temperature was 30℃, detector was diode array detector(DAD), detection wavelength was 210 nm, column flow rate was 0.6 mL/min, the mobile phase was acetonitrile/water = 55/45 (v/v), and the amount of each injection was 10 μL.
2. Materials and methods 2.1. Reagents The recombined water was formulated according to ISO-7346-3 (ISO, 1996), containing 2 mmol/L Ca2+, 0.5 mmol/L Mg2+, 0.075 mmol/L Na+, and 0.074 mmol/L K+. Captan (80% water dispersible granules) (CAS:133-06-2) was provided by the Chinese Agriculture University. It was dissolved in recombined water and stored at 4℃. All reagents used in this study were of analytical grade.
2.5. Data processing 2.2. Zebrafish culture and embryo collection Statistical software SPSS 16.0 was used to calculate LC50, coefficient of determination, and confidence interval. All the data were expressed as the mean ± standard deviation (SD). Significance analysis was performed using one-way ANOVA followed by Dunnett post hoc comparison. P < 0.05 was considered significant, P < 0.01 was considered highly significant.
Wild-type zebrafish (4 months old) were purchased from Beijing Hongdagaofeng Aquarium Department. Male and female fish were maintained in Esen equipment on a 14-h light/10-h dark cycle at 26℃ and fed with hatched Artemia nauplii twice a day. Adult zebrafish were acclimatized for two weeks before experiment, and mortality was less than 5% during this period (Mu et al., 2013). Before the test, male and female zebrafish were placed in a special spawning box at a ratio of 1:2 overnight, separated by a transparent partition, covered with a lid and then with a black cloth completely at night. Next morning, the black cloth and the partitions were removed. About half an hour later, the embryos were collected and washed with recombined water. Then, the embryos were observed under the microscope, and those embryos in a division period and had no obvious abnormalities were selected for exposure experiments (Mu et al., 2013).
3. Results 3.1. Captan analysis The analytical results showed that the measured and theoretical concentration deviations of the water samples of all treatment groups were less than 20% (Table 1). According to the OECD test guideline, the theoretical concentration of this test can represent the measured concentration for testing (OECD, 1992).
2.3. Acute toxicity of captan
3.2. Lethal effect of captan
2.3.1. Embryonic acute toxicity test The acute toxicity test of embryo was performed according to the OECD test guideline No. 236 (Busquet et al., 2013) and Mu (Mu et al., 2013). According to the pretest, embryos about 1 h post-fertilization (hpf) were randomly placed in a 5-cm diameter petri dish containing 20-mL captan exposure solution (0, 0.58, 0.66, 0.75, 0.86, 1.00 and 1.16 mg/L, n = 3 replicates). The final content of acetone was 0.005% in all the six groups. Each replicate had 10 embryos, and the exposure media was renewed every 24 h. The external conditions during exposure were the same as the culture environment. The number of dead embryos was recorded daily and promptly removed. Embryos with egg coagulation, developmental stagnation, and no heartbeat were considered dead (Nagel, 2002). To study the developmental toxicity of captan to embryo, spontaneous movements (in 20 s) at 24 hpf, heartbeats (in 20 s) at 48, 72 hpf, and the body length of hatched individuals at 96 hpf were tested. Moreover, the hatched and malformations of embryos were recorded daily. Spontaneous movements, heartbeats, and malformations were observed under microscope, and body length was measured under Aigo GE-5 digital microscope (Aigo, Beijing, China).
From Table 2, we can observe that captan had high toxicity to embryo and adult zebrafish. The acute toxicity/lethality showed a time effect and increased with the exposure time. The sensitivity of embryonic and adult fish to captan was different, as the 96 h-LC50 values of embryo and adult zebrafish exposed to captan were 0.81 (0.75−0.87) mg/L and 0.65(0.62−0.68) mg/L, respectively. The toxicity of captan to the two different stages of zebrafish was as follows: adult fish > embryos. 3.3. Embryonic developmental effects of captan 3.3.1. Effect of captan on the spontaneous movements of embryos The zebrafish embryos exposed to 24 hpf have regular spontaneous movement. As shown in Fig. 1A, compared with the control group, Table 1 Chemical analysis results of acute toxicity test.
2.3.2. Adult fish acute toxicity test The acute toxicity test of adult fish was performed according to the OECD test guideline No.203 (OECD, 1992) and Mu (Mu et al., 2013). According to the pretest, adult fish were randomly placed in 5 L tanks containing 4 L captan exposure solution (0, 0.56, 0.61, 0.66, 0.72, and 0.78 mg/L) for 4 days. Three replicates per treatment with 10 healthy fish in each replicate were used and renewed the solution daily. The number of dead fish was recorded at 24 h, 48 h, 72 h, and 96 h and removed them timely. The standard of judging the dead fish is using a
Test stage
Nominal con.(mg/L)
Actual con.(mg/L)
Deviationb(%)
Embryo
Control 0.58 0.66 0.76 0.87 1.00 1.16
n.d.a 0.482 0.634 0.718 1.038 0.924 1.057
n.d. −0.169 −0.039 −0.055 0.193 −0.076 −0.089
a b
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n.d.= not detect. deviation= (actual content-nominal content) / nominal content.
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Table 2 96 h-LC50 values of two different stages of zebrafish (exposed to captan). Life stage
Exposure time/h
LC50(95% CLa)mg/L
X2b
R²
embryo
24 48 72 96 24 48 72 96
0.81(0.76-0.88) 0.81(0.75-0.87) 0.81(0.75-0.87) 0.81(0.75-0.87) 0.78(0.73-0.93) 0.69(0.66-0.72) 0.66(0.63-0.69) 0.65(0.62-0.68)
y = 7.50x+1.25 y = 7.50x+1.25 y = 7.50x+1.25 y = 7.50x+1.25 y = 7.00x+0.90 y = 10.00x+1.75 y = 10.00x+2.00 y = 10.00x+2.00
0.91 0.93 0.93 0.93 0.92 0.95 0.98 0.99
adult
a b
Table 3 Hatching rate of zebrafish embryos at 72 hpf and 96 hpf.
CL = Confidence limit. X2 = Chi-square.
Exposure concentration (mg/L)
Hatching rate at 72 hpf (%)
Hatching rate at 96 hpf (%)
0.00 0.58 0.66 0.76 0.87 1.00 1.16
96. 7 ± 5.8 83.3 ± 15.3 53.3 ± 15.3 50.0 ± 17.3 40.0 ± 10.0 23.3 ± 10.0 3.0 ± 5.8
100.0 ± 0.0 86.7 ± 15.3 63.3 ± 11.5 56.7 ± 20.8 43.3 ± 15.3 30.0 ± 17.3 6.7 ± 11.5
3.3.3. Effect of captan on embryonic hatching Under normal conditions, zebrafish embryos start hatching at 48 hpf. All the embryos in the control group hatched at 72 hpf, while 0.58 mg/L and other higher concentration groups of captan had significant inhibition effect on the hatching rate of zebrafish embryos at 72 hpf and 96 hpf (Fig. 1D). Furthermore, compared with the control group, the hatching rate of the 1.00 mg/L group was 30.0% ± 17.3% at 96 hpf, and the hatching rate of the highest concentration group was only 6.7% ± 11.5% at 96 hpf (Table 3).
captan exposure had no significant effect on the spontaneous movements of zebrafish embryos in all captan treatments, including in 1.16 mg/L of the highest concentration.
3.3.2. Effect of captan on embryonic heartbeat As shown in Fig. 1B, compared with the control group, captan exposure inhibited the heartbeats of zebrafish embryos in a concentrationdependent manner. The inhibition effect of captan on embryonic heartbeat at 48 hpf and 72 hpf was not significant in low-dose treatment groups, but significant in 0.76 mg/L and other higher concentration groups. Because of the high concentration of captan, almost all the embryos in the 1.16 mg/L concentration group were dead, and hence the data were not representative.
3.3.4. Effect of captan on embryonic body length Compared with the control group, high concentration of captan (concentrations of 0.76 mg/L and above) could significantly reduce the body length of hatched larvae at 96 hpf (Fig. 1C). And the inhibition rate increased with the increase of captan concentration, and the
Fig. 1. A. Spontaneous movements of embryos at 24 hpf. B. Heartbeats of embryos at 48 hpf and 72 hpf. C. Body length of hatched larvae at 96 hpf. D. Hatching rate of embryos at 72 hpf and 96 hpf. Asterisks denote significant difference between the treatment and control groups (determined by Dunnett post hoc comparison, P < 0.05*; P < 0.01**). Error bars indicate standard deviation. 82
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flutolanil. However, many studies have found that the early life stages of aquatic organisms, in comparison with adult stages, were more sensitive to toxic substances. According to Lele (Lele and Krone, 1996) and Westemhagen (Westernhagen, 1988), the embryo and larvae stages were the most sensitive life stages of Osteichthyes fish because metabolic detoxification is very weak in the embryonic stage compared with adult individuals (Embry et al., 2010). Therefore, further studies are required to identify the factors that cause differences in the susceptibility of zebrafish embryos and adults. And a previous study found that the ability of test substances to penetrate the chorion of embryo may be a factor (Embry et al., 2010). In addition, zebrafish embryonic toxicity of silver nanoparticles (AgNPs) in the absence of chorion was greater than when it was present (Kim and Tanguay, 2014). The function of zebrafish embryo chorion to captan might be tested through experiments on zebrafish larva. Determining the most sensitive phase of environmental organisms to toxic substances can reduce the level of toxicity to environmental organisms and thus reduce the incorrect use of pesticides. Furthermore, our results showed that captan can induce a series of adverse effects in the development of zebrafish embryos, including decreased heart rate, growth inhibition, and various malformations. The results showed that high concentration of captan (0.76 mg/L and other higher concentration groups) significantly inhibited the heartbeats of zebrafish embryos at 48 hpf and 72 hpf, accompanied by yolk sac deformities and pericardial deformities, suggesting that captan can affect the heart development of zebrafish embryos. Heart is a very important functional organ. Yu et al. found that an organosulfur fungicide—thiram can decrease the heartbeats of zebrafish embryos and cause pericardial edema (Yu et al., 2011) because the exposure of thiram induced renal (Hill et al., 2004) dysfunction, water barrier damage, and drainage dysfunction in the body of zebrafish larvae (Cao et al., 2003). Detailed examination by Raldúa et al. (2008) suggested that during the embryonic development of oviparous fish, nutrients were mainly derived from endogenous yolk nutrients (Raldúa et al. 2008); therefore, the abnormal development of yolk sac is likely to result in impaired nutritional supply of zebrafish embryos. Sufficient energy supply is required for the proper functioning of heart, and insufficient energy supply results in symptoms such as decreased heart rate (Kodde et al., 2007). It is reported that flutolanil exposure can cause slower heartbeat in zebrafish embryos, and the pericardial area of zebrafish larvae in the treated groups is significantly larger than that of the control group. And metabolomics analysis showed that energy metabolism-related pathways were also changed in zebrafish embryos exposed to flutolanil (Teng et al., 2018). In addition to the decrease in the heartbeats of zebrafish embryos, the body length of zebrafish larvae was also inhibited when exposed to
Table 4 Inhibition rate of captan on the body length of zebrafish hatched larvae. Exposure concentration (mg/L)
Body length (mm)
Inhibition rate (%)
0.00 0.58 0.66 0.76 0.87 1.00
3.68 3.42 3.29 2.90 2.79 2.56
0 5.26 8.86 19.67 22.71 29.09
± ± ± ± ± ±
0.06 0.06 0.03 0.16 0.10 0.17
maximum inhibition rate was 29.09% at 1.10 mg/L concentration group (Table 4). Almost all the embryos in the 1.16 mg/L concentration group were dead, and hence the data was not representative. 3.3.5. Teratogenic effects of captan on embryos Exposure of captan induced a series of deformities in zebrafish embryos, including pericardial edema (Pe), yolk sac edema (Ysc), tail bending (Tb), and spine curvature (Sc) (Fig. 2). The most significant teratogenic effects were tail bending and spine curvature. As shown in Fig. 3, compared with the control group, captan groups with the concentrations of 0.76 mg/L and above can significantly induce tail bending and spine curvature at 72 hpf. However, captan exposure had no significant effect on yolk sac edema at 48 hpf and 72hpf, and pericardial edema rate was reduced at 72 hpf than at 48 hpf. 4. Discussion Multiple toxic substances have toxic effects on different stages of zebrafish. It has been reported that 96-h LC50 values of embryo and adult zebrafish (exposed to triclosan) were 0.42 mg/L and 0.34 mg/L, respectively. Moreover, embryo toxicity, delay in otolith formation, eye and body pigmentation, and malformations including spine malformations, pericardial edema and undersize, were evident. However, triclosan did not change biomarker levels and did not elicit a micronucleus in adults (Oliveira et al., 2009). Table 5 shows the 96 h-LC50 values of several common fungicides (difenoconazole, thifluzamide, bromothalonil, flutolanil, pyraoxystrobin, penthiopyrad) on different stages of zebrafish and their main teratogenic effects on embryos (Mu et al., 2013; Yang et al., 2016a, 2016b; Li et al., 2018; Qian et al., 2019). Compared with those fungicides, our results suggested that captan had high toxicity to both adult and embryo zebrafish. And it was found that zebrafish embryos, in comparison with adult fish, were more resistant to captan, which is consistent with the results of Mu (Mu et al., 2013) and Yang (Yang et al., 2016a,b). The results revealed that adult zebrafish were more sensitive to difenoconazole, bromothalonil, and
Fig. 2. Teratogenic effects of captan on zebrafish embryos. A. Normal embryo in the control group at 48 hpf. B. Embryo with pericardial edema (Pe) and spine curvature (Sc) at 48 hpf. C. Embryo with pericardial edema (Pe) and yolk sac edema (Yse), and spine curvature (Sc) at 48 hpf. D. Embryo with spine curvature (Sc) at 48 hpf. E. Normal hatched larvae in the control group at 72 hpf. F. Hatched larvae with pericardial edema (Pe) at 72 hpf. G. Hatched larvae with tail bending (Tb) at 72 hpf. H. Hatched larvae with spine curvature (Sc) at 72 hpf. 83
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Fig. 3. Deformity rate of zebrafish embryos at 48 hpf and 72 hpf. Pe: pericardial edema; Ysc: yolk sac edema; Tb: tail bending; Sc: spine curvature. Asterisks denote significant difference between the treatment and control groups (determined by Dunnett post hoc comparison, P < 0.05*; P < 0.01**). Error bars indicate standard deviation.
Table 5 96 h-LC50 values of several common fungicides on different stages of zebrafish and their main teratogenic effects on embryos. Fungicide
96h-LC50(95% CLa)of adult fish (mg/L)
96h-LC50(95%CLa) of embryo (mg/L)
Main deformities in zebrafish embryos
References
captan difenoconazole thifluzamide bromothalonil flutolanil pyraoxystrobin penthiopyrad
0.81(0.75-0.87) 1.45(1.38-1.53) 4.19(4.07-4.29) 2.52 (4.17–4.47) 2.70 (2.58–2.83) 0.0060(0.0052-0.0062)
0.65(0.62-0.68) 2.34(2.27-2.40) 3.08(2.96-3.22) 4.34 (4.25–4.77) 5.47 (5.04–5.84) 0.0041(0.0039-0.0043) 2.77(2.73-2.82)
tail bending spine curvature yolk sac edema yolk sac edema pericardial edema notochord deformation short body length pericardial edema and yolk sac edema yolk sac edema pericardial edema
Mu et al., 2013 Yang et al., 2016a,b Yang et al., 2016a,b Yang et al., 2016a,b Li et al., 2018 Qian et al., 2019
Acknowledgment
captan. The results showed that 0.76 mg/L and other higher concentrations of captan significantly inhibited the body length of zebrafish larvae, the maximum inhibition rate reached 29.09%, and significant tail bending and spine curvature can also be observed. Myristoylated alanine-rich C-kinase substrate (MARCKS) is an actin binding protein substrate of protein kinase C (PKC). Ott, L.E. et.al found that Marcksa and Marcksb, two MARCKS paralogs, play an important role in the tail development of zebrafish, morpholino-based targeting of either MARCKS protein resulted in increasing mortality and a range of gross phenotypic abnormalities, such as a slight curve of a full-length tail, a severe curve or twist of a full-length tail and a truncated tail (Ott et al., 2011). Besides, studies have shown that lysyl oxidase is of great importance in the development of spines in aquatic vertebrates, and inhibition leads to abnormalities in aquatic spines (Zhou et al., 2009). Tail deformity may be related to myristoylated alanine-rich C-kinase or lysyl oxidase or both. While the most significant teratogenic effect induced by captan was spine curvature, other fungicides have different developmental toxicity on zebrafish embryos (Table 5), and this may be due to their specific action mechanism. Therefore, further research is required to identify the exact mechanism of captan on zebrafish embryo tail development.
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5. Conclusion Our study showed that captan had high toxicity to adult and embryo zebrafish, the 96 h-LC50 values of embryo and adult zebrafish (exposed to captan) were 0.81(0.75−0.87) mg/L and 0.65(0.62−0.68) mg/L, respectively. Captan exposure can induce a series of negative effects on embryos, including heart rate decrease, hatching regression, and growth inhibition. Captan can also induce significant teratogenic effects, including spine curvature and tail bending, on zebrafish embryos. Further studies on the action mechanism of captan on zebrafish can provide the scientific basis for the proper application of captan.
Conflict of interest The authors declare that they have no conflict of interest.
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