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Acute and chronic toxic effects of fluoxastrobin on zebrafish (Danio rerio)
Science of the Total Environment 610–611 (2018) 769–775
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Acute and chronic toxic effects of fluoxastrobin on zebrafish (Danio rerio) Cheng Zhang 1, Tongtong Zhou 1, Jun Wang, Shuai Zhang, Lusheng Zhu ⁎, Zhongkun Du, Jinhua Wang College of Resources and Environment, Key Laboratory of Agricultural Environment in Universities of Shandong, Shandong Agricultural University, Taian 271018, China
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Acute and chronic toxic effects of fluoxastrobin on zebrafish were investigated. • Fluoxastrobin can cause oxidative stress and oxidative damage in zebrafish. • The comet assay was the most sensitive of all biomarkers used in the present study.
a r t i c l e
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Article history: Received 30 June 2017 Received in revised form 4 August 2017 Accepted 5 August 2017 Available online xxxx Editor: Jay Gan Keywords: 96 h LC50 Genetic toxicity DNA damage Antioxidant enzyme High-performance liquid chromatography Reactive oxygen species
a b s t r a c t Fluoxastrobin is a new strobilurin fungicide, similar to azoxystrobin and pyraclostrobin. Before the wide application of fluoxastrobin, the present study was performed to assay the acute and chronic toxicity of fluoxastrobin on zebrafish (Danio rerio). The 96-hour median lethal concentration (96 h LC50) after initiation of zebrafish exposure to fluoxastrobin was 0.51 mg/L with a 95% confidence interval of 0.45 to 0.57 mg/L, indicating that fluoxastrobin was highly toxic to zebrafish. As endpoints, we assayed the levels of reactive oxygen species (ROS), malondialdehyde (MDA), the activities of superoxide dismutase (SOD), catalase (CAT), glutathione Stransferase (GST), and the degree of DNA damage at three different doses, 0.001, 0.01, and 0.1 mg/L on days 7, 14, 21, and 28. The antioxidant enzymes partially ameliorated the ROS induced by fluoxastrobin t and were in turn inhibited by excess ROS, especially at 0.1 mg/L. Lipid peroxidation and DNA damage were stimulated by ROS. The fluoxastrobin contents of the tested solutions were also determined; at the fluoxastrobin doses of 0.001, 0.01, and 0.1 mg/L, the contents on day 28 were 3.9, 5.0, and 0.64% greater than those on day 0. Thus, fluoxastrobin was relatively stable in an aquatic environment. In addition, the present study provided more information regarding the toxic effects of fluoxastrobin and the scientific methods for selection and evaluation of fungicides in the future. © 2017 Elsevier B.V. All rights reserved.
1. Introduction
⁎ Corresponding author at: College of Resources and Environment, Shandong Agricultural University, Taian 271018, China. E-mail addresses: [email protected] (J. Wang), [email protected] (L. Zhu), [email protected] (J. Wang). 1 Cheng Zhang and Tongtong Zhou contributed equally to this work.
http://dx.doi.org/10.1016/j.scitotenv.2017.08.052 0048-9697/© 2017 Elsevier B.V. All rights reserved.
Fluoxastrobin, (E)-{2-[6-(2-chlorophenoxy)-5-fluoropyrimidin-4yloxy]phenyl} (5,6-dihydro-1,4,2-dioxazin-3-yl) methanone Omethyloxime, is used on cereal crops, potatoes, greens, coffee, and other crops (Yin et al., 2003). Fluoxastrobin is a new broad-spectrum strobilurin fungicide, similar to azoxystrobin and pyraclostrobin, whose mode of action is to inhibit mitochondrial respiration of the
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Fig. 1. The structural formula of fluoxastrobin.
adult zebrafish (Danio rerio) used in the present study. The fish were groups of half males and half females, with average body weights and lengths of 0.13 ± 0.02 g and 2.44 ± 0.03 cm, respectively. Fish handling was performed as previously described by Zhang et al. (2017b). Chlorine-free tap water was aerated for one week. The pH, temperature and oxygen saturation of the tap water used in the present study were 6.5 to 7.5, 26 ± 1 °C and N70%, respectively, which were also the values of the test water to be applied in the acute and chronic toxicity tests. The photoperiod was 12 h light:12 h dark. The tested fish were acclimated for two weeks under conditions with the aforementioned parameters. The fish were fed commercially available dry flakes every day during the acclimation period. The cumulative mortality did not exceed 5%, and the dead fish were removed in a timely manner. 2.3. Determinations of fluoxastrobin concentrations
target fungus by transferring electrons between cytochrome b and C1 (Bartlett et al., 2002). The structure of fluoxastrobin is shown in Fig. 1. Fluoxastrobin is a white crystal, with a vapor pressure 6 × 10−10 Pa, a log P (octanol/water) of 2.86, and a solubility in water of 2.43 mg/L (pH = 4), 2.29 mg/L (pH = 7), or 2.27 mg/L (pH = 9) at 20 °C (Yin et al., 2003). Thus, it is difficult for fluoxastrobin to remain in the air due to its low vapor pressure. However, it may remain in soil and water because of rain-out or storms due to its preferential high solubility. The Organization for Economic Cooperation and Development (OECD, 1992) selected zebrafish (Danio rerio) as the biological indicator for evaluating the toxicity of external contamination of aquatic environments. Numerous studies have also focused on zebrafish (Chen et al., 2017; Duan et al., 2017; Han et al., 2016; Kais et al., 2017; Keiter et al., 2016; Scholz, 2013; Tu et al., 2016; Wang et al., 2017; Zhang et al., 2017b). Studies on the toxicity of azoxystrobin have focused on acute toxicity with 96-h of exposure (Lin et al., 2014; Zhang et al., 2014), and Han et al. (2016) studied the genetic toxicity and oxidative stress of another strobilurin fungicide, azoxystrobin, in zebrafish livers. They found that azoxystrobin can induce ROS and oxidative stress which inhibited the activity of SOD and induced the activities of CAT and GST. Thus, fluoxastrobin may have toxicity similar to that of azoxystrobin, or the different kinds of strobilurin fungicides may lead to different toxicities in zebrafish. However, information is still scarce of both the acute and chronic toxicity of fluoxastrobin on zebrafish. Thus, it is vital to study the toxic effects of fluoxastrobin on zebrafish. In addition to the acute toxicity using a static test, chronic toxicity using semistatic tests on days 7, 14, 21, and 28 was also studied, including the levels of ROS, the activities of SOD, CAT, and GST, the levels of MDA and the degree of DNA damage. The present study also aimed to find the most sensitive indicator and the relationships among the diverse indicators. 2. Materials and methods 2.1. Chemicals and reagents Fluoxastrobin (99.3% purity, CAS No. 361377-29-9) was provided by Dr. Ehrenstorfer GmbH (Augsburg, Germany). The stock solutions used in the present study were the appropriate doses of fluoxastrobin dissolved in chromatographically pure acetone. The other chemicals and solvents used in the present study were all analytically pure and were obtained from the Solarbio Science &Technology Company (Beijing, China) and Sigma Chemical (St. Louis, Missouri, USA).
To ensure the reliability of the present study, the fluoxastrobin contents of the tested solutions were determined at 1 h, days 7, 14, 21, and 28 after the initiation of contamination as reported by Liu et al. (2015a). High-performance liquid chromatography (HPLC, Agilent 1260, Agilent Technologies Inc., USA) was used to measure the concentrations of the tested strobilurin fungicide for zebrafish in the tested water. The mobile phase in the present study was composed of ultrapure water and chromatographically pure acetonitrile at the ratio of 35:65. Specifically, a C18 column (Eclipse XDB–C18, 4.6 × 250 mm, 5 μm) was utilized to separate the experimental samples at 40 °C, and the ultraviolet-visible (UV/vis) variable wavelength detector (VWD G1314A, Agilent Technologies Inc., USA) was utilized to analyze the experimental samples at 254 nm. The injection volume and flow rate were 10 μL and 1 mL/min, respectively. Triplicate samples were applied to each control and exposure treatment. According to the procedure OECD 203 (1992), the concentrations of the tested fluoxastrobin should range from 80% to 120% of the initial concentrations. However, if the results of the determination were outside that range (N120% or b80%), the experimentally obtained values were considered the experimental exposure concentrations for evaluating the acute toxic, biochemical toxic and genotoxic effects of fluoxastrobin on zebrafish. 2.4. Experimental design of the acute toxicity test for zebrafish The static test to determine the acute toxicity toward the fish was performed as previously described by Zhang et al. (2017b). The pH, temperature and oxygen saturation of the test water were the same as for the fish handling water. The photoperiod was 12 h light:12 h dark. To avoid interference from the solvent, there were solvent control groups using 1 mL in 1500 mL of chlorine-free tap water. Fluoxastrobin dissolved in 1 mL of acetone and the aforementioned chlorine-free tap water were added to 2 L glass beakers such that each beaker contained 1.5 L of the solution, and final doses of 0.01, 0.1, 0.25, 0.5, 0.6, 0.7, and 0.8 mg/L were used in the formal acute toxicity tests, based on the results of preliminary experiments including 10% and 90% mortality. Ten fish were tested in each beaker, and the previously described measurements were taken for 96 h under the various conditions. Triplicate samples were applied to each control and exposure treatment. The fish were fed with commercially available dry flakes every day during the acclimation period until 24 h prior to the acute toxicity tests. The food residues, feces and dead fish were removed in a timely manner to avoid interference. According to Cai (2004) and Lin et al. (2014), 96 h LC50 was used to evaluate the acute toxicity of the pesticides to zebrafish (mg/L): b0.1, rank poison; 0.1 to 1, highly toxic; 1 to 10, moderately toxic; and N10, low toxicity.
2.2. Fish handling 2.5. Experimental design of the chronic toxicity test for zebrafish The Guiding Principles outlined for the use of animals in toxicology, adopted by the Society of Toxicology in 1989, were applied to the present study (Van, 2002). Kaixin Aquarium (Taian, China) supplied the
The semistatic tests to determine the chronic toxicity toward fish were performed as described by Zhang et al. (2017a). The chlorine-
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free tap water was aerated for one week. The pH, temperature and oxygen saturation of the test water were the same as the fish handling water. The photoperiod was 12 h light:12 h dark. To avoid interference from the solvent, solvent control groups were also established by adding 1 mL of acetone to 20 L chlorine-free tap water. Fluoxastrobin dissolved in 1 mL of acetone and the aforementioned chlorine-free tap water were added to a 25-L aquarium to ensure that each aquarium held 20 L of the solution and to reach final doses of 0.001, 0.01, and 0.1 mg/L, based on the 96 h LC50 value. For each aquarium, 120 fish were acclimatized for 28 days under conditions including the aforementioned parameters. On day 7, day 14, day 21 and day 28, twenty zebrafish were randomly selected for evaluation of the levels of reactive oxygen species (ROS), lipid peroxidation, enzyme activities (SOD, CAT, GST) and DNA damage in livers. Triplicate samples were applied to each control and exposure treatment. The fish were fed with commercially available dry flakes every day during the 28 d of testing until 24 h prior to the chronic toxicity tests. The chronic toxicity test is a semistatic test, and half of the tested water was replaced every two days to maintain constant doses of fluoxastrobin. The food residues, feces and dead fish were removed in a timely manner to avoid interference.
2.6. Determination of ROS levels Reactive oxygen species include hydrogen peroxide (H2O2), super\OH) and singlet oxygen (1O2) oxide anions (O− 2 ), hydroxyl radicals (\ (Han et al., 2016). The present study assayed ROS levels by detecting H2O2 content using the DCFH-DA method (Bothe and Valet, 1990). The method for preparing the suspension was described by Liu et al. (2015a). For each control and experimental group, ten fish were randomly selected to be dissected in 0.1 M ice-cold potassium phosphate buffer at pH 7.4, after which the homogenate was centrifuged (Eppendorf, 5804) at 1000g and 4 °C for 10 min. Subsequently, the resultant supernatant was re-centrifuged at 20,000g and 4 °C for 20 min, after which the precipitate was re-suspended in 500 μL of 0.1 M phosphate buffer to prepare the liver suspension at pH 7.4. Triplicate samples
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were applied to each control and exposure treatment. The ROS content was assayed by using a fluorescent spectrophotometer (Shimadzu, RF5301PC) with an excitation wavelength of 488 nm and emission wavelength of 522 nm. The unit fluorescence-intensity/mg protein (pr) was used to express the ROS levels.
2.7. Determination of enzyme activities (SOD, CAT, and GST) and MDA levels According to the methods performed by Shao et al. (2012), the liver suspension was reserved to measure the content of enzyme proteins, the activities of enzymes and MDA levels. For each control and experimental group, six fish were randomly selected for dissection in icecold 50 mM phosphate buffer, pH 7.0 to prepare the liver suspension, after which the homogenate was centrifuged (Eppendorf, 5804) at 10,000 rpm and 4 °C for 10 min. The enzyme activities and MDA levels were determined by using an ultraviolet-visible spectrophotometer (U-V spectrophotometer, Shimadzu, UV-2550). Triplicate samples were applied to each control and exposure treatment. Bovine serum albumin was utilized as the standard to determine the contents of protein in the fluoxastrobinexposed groups at 595 nm by using the Sigma Bradford method (Bradford, 1976). The methods for assaying the activities of SOD and CAT were both performed according to Song et al. (2009). The photochemical reduction of nitroblue tetrazolium (NBT) after illumination was utilized to assay the activity of SOD at 560 nm. The decomposition of H2O2 was utilized to assay the activity of CAT at 240 nm for 1 min. The amount of enzyme, that caused the reduction of half of the NBT photochemical, was defined as one unit of SOD activity (U). The amount of enzyme that caused 50% of the H2O2 decomposition in 60 s at 25 °C was defined as one unit of CAT activity (U). The unit U/mg pr was used to express the results of both SOD activity and CAT activity. The GST activity was assayed according to the method of Zhu et al. (2011). The changes in absorbance were utilized to evaluate the activity of GST at 340 nm for 3 min, and the unit nmol/min/mg pr was used to express the level of GST activity.
Fig. 2. The ROS levels in zebrafish (Danio rerio) exposed to three different doses of fluoxastrobin. Triplicate samples were applied to each control and exposure treatment. Each column represents the average value of a treatment group, and the error bars represent the standard deviation (SD). The least significant difference (LSD) test was used to analyze the statistical significance of the results among different experimental groups at p b 0.05.
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The method for assaying the levels of MDA, which was considered an indicator of lipid peroxidation, was that reported by Zhang et al. (2013). The unit nmol/mg pr was used to express the MDA levels.
2.8. Determination of the degree of DNA damage The methods for suspension preparation, slide preparation and measurement of the degree of DNA damage using single cell gel electrophoresis (SCGE) were those reported by Shao et al. (2012). For each control and experimental group, four fish were randomly selected for dissection in ice-cold 0.1 M phosphate buffer and were cut up, after which a 200mesh sieve was used to filter the resultant tissue to prepare the homogenate. Then, the homogenate was centrifuged (Eppendorf, 5804) at 200g and 4 °C for 10 min, after which the precipitate with 500 μL of 0.1 M phosphate buffer was re-suspended to prepare the liver suspension at pH 7.4. Triplicate samples were applied to each control and exposure treatment. The comet images were observed using an inverted fluorescence microscope (Olympus, BX51) and analyzed using Comet Assay Software Project (CASP). The distance between the center of the tail and the head, defined as the olive tail moment (OTM), was used to express the results of the degree of DNA damage caused by fluoxastrobin in the livers of the tested zebrafish (Singh et al., 1988).
2.9. Statistical analysis Triplicate samples were applied to each control and exposure treatment. In the present study, we used the Statistical Package for Social Sciences (SPSS) (Standard Version 19.0, SPSS Inc., USA) to evaluate the 96 h LC50 value via probit analysis and to analyze the results of chronic toxicity test via a one-way analysis of variance (ANOVA). We also used a least significant difference (LSD) testing to analyze the statistical significance of the results among different experimental groups at the same treatment time at p b 0.05, and the standard deviation (SD) is presented using the error bars.
3. Results and discussion 3.1. Dynamic change in the fluoxastrobin concentration in water In the controls, no fluoxastrobin was detected. In the present study, the instrument detection limit was 2.33 × 10−9 g, the standard curve was y = 26.618x − 0.0445, and the square of the correlation coefficients was 0.9996. Based on the above data, the recovery rate of fluoxastrobin ranged from 92.9% to 105.3%, which met the guideline OECD 203 (1992). On day 28, the doses of fluoxastrobin in the 0.001,
Fig. 3. The activities of SOD (a), CAT (b) and GST (c) in zebrafish (Danio rerio) exposed to three different doses of fluoxastrobin. Triplicate samples were applied to each control and exposure treatment. Each column represents the average value of three samples, and the standard deviation (SD) is represented with an error bar. The least significant difference (LSD) test was used to analyze the statistical significance of the differences among the experimental groups at p b 0.05.
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0.01, and 0.1 mg/L treatments were 3.9, 5.0, and 0.64% greater than those on day 0. Thus, the doses of each fluoxastrobin-exposed group of zebrafish (Danio rerio) in the tested water were considered relatively stable and further demonstrated the dependability of the present study.
3.2. Acute toxic effects of fluoxastrobin on zebrafish (Danio rerio) There were no deaths among the controls. The cumulative mortality increased with the increasing doses of fluoxastrobin. The 96 h LC50 value of fluoxastrobin for exposure to zebrafish was 0.51 mg/L, and the 95% confidence interval extended from 0.45 to 0.57 mg/L. Based on the toxicity grading standards, fluoxastrobin is considered highly toxic to zebrafish (Cai, 2004; Lin et al., 2014). The results were similar to Zhang et al. (2014), in which they evaluated the acute toxicity of azoxystrobin on zebrafish and found that the 96 h LC50 value was 0.67 mg/L with a 95% confidence interval of 0.64 mg/L to 0.72 mg/L. The 96 h LC50 value of fluoxastrobin toward rainbow trout was N0.44 mg/L (Yin et al., 2003). The minor discrepancy between our results and those from aforementioned previous studies may be caused by differences in the strobilurin fungicides or in the fish. Different species can have different susceptibilities and thus different LC50 values.
3.3. Effects of fluoxastrobin on ROS levels The ROS levels in zebrafish exposed to three different doses of fluoxastrobin are depicted in Fig. 2. On days 7, 14, 21, and 28, the ROS levels in each treatment group were all significantly enhanced with increasing test doses compared to the controls. As stated by Liu et al. (2014), there is a dynamic equilibrium between the production and elimination of ROS in general. However, the production and elimination of ROS is disturbed even at the lowest dose (0.001 mg/L). When Han et al. (2016) studied the toxicity of azoxystrobin on female zebrafish, they also observed similar results. Excess ROS can induce oxidative stress, thus the enzyme activities (SOD, CAT, GST) and MDA levels were also evaluated in the present study.
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3.4. Effects of fluoxastrobin on enzyme activities (SOD, CAT and GST) Yonar (2013) has stated that SOD and CAT belong to the antioxidant enzymes, which can partially clean up ROS induced by the external contamination and can be inhibited by excess ROS in turn (Ge et al., 2015; Han et al., 2016; Yan et al., 2015). The activities of SOD (a), CAT (b) and GST(c) in zebrafish after exposure to three different doses of fluoxastrobin are depicted in Fig. 3. Both SOD and CAT belong to the first line of defense for eliminating ROS (Yan et al., 2015). Superoxide dismutase can promote the transformation of O− 2 to H2O2, and CAT can promote the transformation of H2O2 to H2O in a timely manner (Liu et al., 2015a). As illustrated in Fig. 3a, the values for the treatments with fluoxastrobin at 0.001 and 0.01 mg/L are greater than those for the control groups on days 7, 14, 21, and 28. The values for treatment with 0.1 mg/L are all lower than those for the controls on days 7, 14 and 21, while the value for 0.1 mg/L is slightly higher than that for the controls on day 28. The results demonstrated that ROS caused an increase of the SOD activity at low doses of fluoxastrobin (0.001 and 0.01 mg/L), and at the highest dose (0.1 mg/L), SOD activity was inhibited by a disruption of the function of the protein (Du et al., 2014). On day 28, notable differences were found in each fluoxastrobin-treatment group except for the treatment with 0.1 mg/L. As illustrated in Fig. 3b, CAT activity showed a trend similar to that of SOD activity. Specifically, the values in the treatment groups first increased at 0.001 and 0.01 mg/L and then decreased at 0.1 mg/L compared with the controls for the duration of the experimental period. When Du et al. (2014) focused on the toxicity of the IL 1-octyl-3methylimidazolium bromide in zebrafish livers, they observed a similar phenomenon. Significant differences can be observed at each fluoxastrobin-treatment group with the exception of the 0.01 mg/L dose on day 7 and the 0.001 mg/L dose on day 14. Glutathione S-transferase (GST) is considered a type of detoxifying enzyme for cleaning up excess ROS (Zhu et al., 2011). As illustrated in Fig. 3c, the values for the three different doses were all greater than those of the control groups on days 7, 14, 21, and 28, except for the value at the dose of 0.1 mg/L on day 21. Notable differences were found in each fluoxastrobin-treatment group except for the value for the dose of 0.001 mg/L on day 21. The GST activity with treatment at
Fig. 4. The MDA contents in zebrafish (Danio rerio) exposed to three different doses of fluoxastrobin. Triplicate samples were applied to each control and exposure treatment. Each column represents the average value of a condition, and the error bars indicate the standard deviation (SD). The least significant difference (LSD) test was used to analyze the statistical significance of the differences among the different experimental groups at p b 0.05.
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Fig. 5. The degree of DNA damage in zebrafish (Danio rerio) exposed to three different doses of fluoxastrobin. Triplicate samples were applied to each control and exposure treatment. Each column represents the average value of a condition, and the error bars indicate the standard deviation (SD). The least significant difference (LSD) test was used to analyze the statistical significance of the differences among the different experimental groups at p b 0.05.
0.1 mg/L was lower than that with 0.001 or 0.01 mg/L during the entire experimental period, but it was still higher than that in the controls. The results from a study of the toxic effects of azoxystrobin in zebrafish (Han et al., 2016) are similar to our findings. 3.5. Effect of fluoxastrobin on lipid peroxidation Reactive oxygen species (ROS) can induce lipid peroxidation and antioxidant enzymes are the first line of defense to decrease the progression by eliminating the excess ROS (Yan et al., 2015). Lipid peroxidation is considered to have a complex integrated effect that can produce malondialdehyde (MDA). Thus, MDA levels were utilized to assay the degree of lipid peroxidation (Liu et al., 2015a). Fig. 4 depicts the effects of lipid peroxidation according to MDA levels in zebrafish after exposure to three different doses of fluoxastrobin. Compared to the controls, remarkable growth can be observed for each condition with the exception of fluoxastrobin at 0.001 and 0.01 mg/L on day 7. When Han et al. (2016) focused on the toxicity of the fungicide azoxystrobin for zebrafish females, they obtained similar results. Furthermore, no significant difference was found between the values of 0.001 and 0.01 mg/L during the entire environmental period. This may be caused by antioxidant enzymes eliminating ROS to a certain extent with fluoxastrobin treatments at 0.001 and 0.01 mg/L. However, excess ROS can reduce or even inhibit enzyme activities such that ROS cannot be cleared in time at a high dose of fluoxastrobin (0.1 mg/L), which exacerbated oxidative damage and lipid peroxidation (Liu et al., 2015b).
and 0.01 mg/L, the values were less than two times those of the control groups, while the values were three or even four times those of the control groups at the highest dose (0.1 mg/L) in each trial. This result may be due to SOD and GST eliminating ROS to a certain extent at 0.001 and 0.01 mg/L, while at a high dose (0.1 mg/L), the excess ROS inhibited the activities of antioxidant enzymes so that ROS could not be eliminated in time, thereby increasing the degree of DNA damage (Liu et al., 2015b). Significant differences were observed at each time point during the entire experimental period. This result may be due to the DNA strand breakage, which may be dose-dependent. Ge et al. (2015) also drew a similar conclusion when they studied the effect of imidacloprid in zebrafish livers on days 7, 14, 21, and 28.
4. Conclusions In the present study, we analyzed the acute toxic, biochemical toxic and genotoxic effects of fluoxastrobin on zebrafish (Danio rerio). The four following bullet points are the main conclusions: (1) Fluoxastrobin is highly toxic to zebrafish (Danio rerio). (2) The tested fungicide can cause oxidative stress and oxidative damage in zebrafish livers. (3) The most sensitive biomarker of all the biomarkers tested in the present study is the comet assay. (4) Based on the data illustrated in the present study, fluoxastrobin is relatively stable in an aquatic environment for the entire duration of the experimental period.
3.6. Effect of fluoxastrobin on DNA damage DNA damage, including DNA broken strands, is considered an indicator of oxidative damage induced by external contamination (Liu et al., 2015a). Fig. 5 depicts the degree of DNA damage based on OTM levels in zebrafish after exposure to three different doses of fluoxastrobin. On days 7, 14, 21and 28, the OTM levels were significantly enhanced in all of the treatment groups compared to the controls. With the increasing tested doses, the degree of DNA damage was enhanced at each exposure time, with a trend similar to that of ROS levels. At 0.001
Informed consent Informed consent was obtained from all individual participants included in the study.
Conflicts of interest The authors declare that they have no conflict of interest.
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Acknowledgements This work was supported by the National Key Research and Development Plan [Nos. 2017YFD0200307, 2016YFD0800202 and 2016YFD0201203]; the National Natural Science Foundation of China [Nos. 41771282, 41671320]; the Natural Science Foundation of Shandong Province, China [ZR2017MD005] and the Special Funds of Taishan Scholar of Shandong Province, China. References Bartlett, D.W., Clough, J.M., Godwin, J.R., Hall, A.A., Hamer, M., Parr-Dobrzanski, B., 2002. The strobilurin fungicides. Pest Manag. Sci. 58, 649–662. Bothe, G., Valet, G., 1990. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2′,7′-dichlorofluorescin. J. Leukoc. Biol. 47, 440–448. Bradford, M.M., 1976. A rapid sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Cai, D.J., 2004. Testing Guidelines of Assessing Environmental Safety of Chemical Pesticides. State Environmental Protection Administration of China, Beijing. Chen, Q.Q., Gundlach, M., Yang, S.Y., Jiang, J., Velki, M., Yin, D.Q., Hollert, H., 2017. Quantitative investigation of the mechanisms of microplastics and nanoplastics toward zebrafish larvae locomotor activity. Sci. Total Environ. 584–585, 1022–1031. Du, Z.K., Zhu, L.S., Dong, M., Wang, J.H., Wang, J., Xie, H., Liu, T., Guo, Y.Y., 2014. Oxidative stress and genotoxicity of the ionic liquid 1-octyl-3-methylimidazolium bromide in zebrafish (Danio rerio). Arch. Environ. Contam. Toxicol. 67, 261–269. Duan, J.C., Yu, Y., Li, Y., Jing, L., Yang, M., Wang, J., Li, Y.B., Zhou, X.Q., Miller, M.R., Sun, Z.W., 2017. Comprehensive understanding of PM2.5 on gene and microRNA expression patterns in zebrafish (Danio rerio). Sci. Total Environ. 586, 666–674. Ge, W.L., Yan, S.H., Wang, J.H., Zhu, L.S., Chen, A.M., Wang, J., 2015. Oxidative stress and DNA damage induced by imidacloprid in zebrafish (Danio rerio). J. Agric. Food Chem. 63, 1856–1862. Han, Y.N., Liu, T., Wang, J.H., Wang, J., Zhang, C., Zhu, L.S., 2016. Genotoxicity and oxidative stress induced by the fungicide azoxystrobin in zebrafish (Danio rerio) livers. Pestic. Biochem. Physiol. 133, 13–19. Kais, B., Schiwy, S., Hollert, H., Keiter, S.H., Braunbeck, T., 2017. In vivo EROD assays with the zebrafish (Danio rerio) as rapid screening tools for the detection of dioxin-like activity. Sci. Total Environ. 590–591, 269–280. Keiter, S., Burkhardt-Medicke, K., Wellner, P., Kais, B., Färber, H., Skutlarek, D., Engwall, M., Braunbeck, T., Keiter, S.H., Luckenbach, T., 2016. Does perfluorooctane sulfonate (PFOS) act as chemosensitizer in zebrafish embryos? Sci. Total Environ. 548–549, 317–324. Lin, J., Wang, K.Y., Xu, H., Tang, J.F., Liu, J., 2014. Acute toxicity and safety evaluation of five new fungicides to four kinds of fishes. World Pesticides 36, 34–38. Liu, T., Zhu, L.S., Han, Y.N., Wang, J.H., Wang, J., Zhao, Y., 2014. The cytotoxic and genotoxic effects of metalaxy-M on earthworms (Eisenia fetida). Environ. Toxicol. Chem. 33, 2344–2350.
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Liu, T., Guo, Y.Y., Wang, J.H., Wang, J., Zhu, L.S., Zhang, J., Zhang, C., 2015a. Assessing toxic effects of [Omim]Cl and [Omim]BF4 in zebrafish adults using a biomarker approach. Environ. Sci. Pollut. Res. 23, 7360–7368. Liu, T., Zhu, L.S., Wang, J.H., Wang, J., Xie, H., 2015b. The genotoxic and cytotoxic effects of 1-butyl-3-methylimidazolium chloride in soil on Vicia faba seedlings. J. Hazard. Mater. 285, 27–36. OECD, 1992. Guidelines for the Testing of Chemicals. Test Guideline 203, Fish, Acute Toxicity Test. Organization for Economic Cooperation and Development, Paris. Scholz, S., 2013. Zebrafish embryos as an alternative model for screening of drug-induced organ toxicity. Arch. Toxicol. 87 (5), 767–769. Shao, B., Zhu, L.S., Dong, M., Wang, J., Wang, J.H., Xie, H., Zhang, Q.M., Du, Z.K., Zhu, S.Y., 2012. DNA damage and oxidative stress induced by endosulfan exposure in zebrafish (Danio rerio). Ecotoxicology 21, 1533–1540. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191. Song, Y., Zhu, L.S., Wang, J., Wang, J.H., Liu, W., Xie, H., 2009. DNA damage and effects on antioxidative enzymes in earthworm (Eisenia foetida) induced by atrazine. Soil Biol. Biochem. 41, 905–909. Tu, W.Q., Xu, C., Lu, B., Lin, C.M., Wu, Y.M., Liu, W.P., 2016. Acute exposure to synthetic pyrethroids causes bioconcentration and disruption of the hypothalamus–pituitary– thyroid axis in zebrafish embryos. Sci. Total Environ. 542, 876–885. Van, P.J., 2002. Guiding principles in the use of animals in toxicology. Adopted by the Society of Toxicology in July 1989. Toxicol. Appl. Pharmacol. 2, 6–7. Wang, G.W., Chen, H.Y., Du, Z.K., Li, J.H., Wang, Z.Y., Gao, S.X., 2017. In vivo metabolism of organophosphate flame retardants and distribution of their main metabolites in adult zebrafish. Sci. Total Environ. 590–591, 50–59. Yan, S.H., Wang, J.H., Zhu, L.S., Chen, A.M., Wang, J., 2015. Thiamethoxam induces oxidative stress and antioxidant response in zebrafish (Danio Rerio) livers. Environ. Toxicol. 31, 1–10. Yin, J.J., Ma, H.Y., Guan, A.Y., Liu, C.L., 2003. High-effect fungicide: fluoxastrobin. Pesticides 42, 36–40. Yonar, M.E., 2013. Protective effect of lycopene on oxidative stress and antioxidant status in Cyprinus carpio during cypermethrin exposure. Environ. Toxicol. 28, 609–616. Zhang, Q.M., Zhu, L.S., Wang, J.H., Xie, H., Wang, J., Han, Y.N., Yang, J.H., 2013. Oxidative stress and lipid peroxidation in the earthworm Eisenia fetida induced by low doses of fomesafen. Environ. Sci. Pollut. Res. 20, 201–208. Zhang, G.F., Li, B.J., Wang, J.H., Xia, X.M., 2014. Acute toxicity of technical material and different formulations of difenoconazole and azoxystrobin to zebrafish. J. Agro-Environ. Sci. 33, 2125–2130. Zhang, C., Shao, Y.T., Zhu, L.S., Wang, J.H., Wang, J., Guo, Y.Y., 2017a. Acute toxicity, biochemical toxicity and genotoxicity caused by 1-butyl-3-methylimidazolium chloride and 1-butyl-3-methylimidazolium tetrafluoroborate in zebrafish (Danio rerio) livers. Environ. Toxicol. Pharmacol. 51, 131–137. Zhang, C., Zhu, L.S., Wang, J.H., Wang, J., Zhou, T.T., Xu, Y.Q., Cheng, C., 2017b. The acute toxic effects of imidazolium-based ionic liquids with different alkyl-chain lengths and anions on zebrafish (Danio rerio). Ecotoxicol. Environ. Saf. 140, 235–240. Zhu, L.S., Dong, X.L., Xie, H., Wang, J., Wang, J.H., Su, J., Yu, C.W., 2011. DNA damage and effects on glutathione-S-transferase activity induced by atrazine exposure in zebrafish (Danio rerio). Environ. Toxicol. 26, 480–488.
Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Acute and chronic toxic effects of fluoxastrobin on zebrafish (Danio rerio) Cheng Zhang 1, Tongtong Zhou 1, Jun Wang, Shuai Zhang, Lusheng Zhu ⁎, Zhongkun Du, Jinhua Wang College of Resources and Environment, Key Laboratory of Agricultural Environment in Universities of Shandong, Shandong Agricultural University, Taian 271018, China
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Acute and chronic toxic effects of fluoxastrobin on zebrafish were investigated. • Fluoxastrobin can cause oxidative stress and oxidative damage in zebrafish. • The comet assay was the most sensitive of all biomarkers used in the present study.
a r t i c l e
i n f o
Article history: Received 30 June 2017 Received in revised form 4 August 2017 Accepted 5 August 2017 Available online xxxx Editor: Jay Gan Keywords: 96 h LC50 Genetic toxicity DNA damage Antioxidant enzyme High-performance liquid chromatography Reactive oxygen species
a b s t r a c t Fluoxastrobin is a new strobilurin fungicide, similar to azoxystrobin and pyraclostrobin. Before the wide application of fluoxastrobin, the present study was performed to assay the acute and chronic toxicity of fluoxastrobin on zebrafish (Danio rerio). The 96-hour median lethal concentration (96 h LC50) after initiation of zebrafish exposure to fluoxastrobin was 0.51 mg/L with a 95% confidence interval of 0.45 to 0.57 mg/L, indicating that fluoxastrobin was highly toxic to zebrafish. As endpoints, we assayed the levels of reactive oxygen species (ROS), malondialdehyde (MDA), the activities of superoxide dismutase (SOD), catalase (CAT), glutathione Stransferase (GST), and the degree of DNA damage at three different doses, 0.001, 0.01, and 0.1 mg/L on days 7, 14, 21, and 28. The antioxidant enzymes partially ameliorated the ROS induced by fluoxastrobin t and were in turn inhibited by excess ROS, especially at 0.1 mg/L. Lipid peroxidation and DNA damage were stimulated by ROS. The fluoxastrobin contents of the tested solutions were also determined; at the fluoxastrobin doses of 0.001, 0.01, and 0.1 mg/L, the contents on day 28 were 3.9, 5.0, and 0.64% greater than those on day 0. Thus, fluoxastrobin was relatively stable in an aquatic environment. In addition, the present study provided more information regarding the toxic effects of fluoxastrobin and the scientific methods for selection and evaluation of fungicides in the future. © 2017 Elsevier B.V. All rights reserved.
1. Introduction
⁎ Corresponding author at: College of Resources and Environment, Shandong Agricultural University, Taian 271018, China. E-mail addresses: [email protected] (J. Wang), [email protected] (L. Zhu), [email protected] (J. Wang). 1 Cheng Zhang and Tongtong Zhou contributed equally to this work.
http://dx.doi.org/10.1016/j.scitotenv.2017.08.052 0048-9697/© 2017 Elsevier B.V. All rights reserved.
Fluoxastrobin, (E)-{2-[6-(2-chlorophenoxy)-5-fluoropyrimidin-4yloxy]phenyl} (5,6-dihydro-1,4,2-dioxazin-3-yl) methanone Omethyloxime, is used on cereal crops, potatoes, greens, coffee, and other crops (Yin et al., 2003). Fluoxastrobin is a new broad-spectrum strobilurin fungicide, similar to azoxystrobin and pyraclostrobin, whose mode of action is to inhibit mitochondrial respiration of the
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Fig. 1. The structural formula of fluoxastrobin.
adult zebrafish (Danio rerio) used in the present study. The fish were groups of half males and half females, with average body weights and lengths of 0.13 ± 0.02 g and 2.44 ± 0.03 cm, respectively. Fish handling was performed as previously described by Zhang et al. (2017b). Chlorine-free tap water was aerated for one week. The pH, temperature and oxygen saturation of the tap water used in the present study were 6.5 to 7.5, 26 ± 1 °C and N70%, respectively, which were also the values of the test water to be applied in the acute and chronic toxicity tests. The photoperiod was 12 h light:12 h dark. The tested fish were acclimated for two weeks under conditions with the aforementioned parameters. The fish were fed commercially available dry flakes every day during the acclimation period. The cumulative mortality did not exceed 5%, and the dead fish were removed in a timely manner. 2.3. Determinations of fluoxastrobin concentrations
target fungus by transferring electrons between cytochrome b and C1 (Bartlett et al., 2002). The structure of fluoxastrobin is shown in Fig. 1. Fluoxastrobin is a white crystal, with a vapor pressure 6 × 10−10 Pa, a log P (octanol/water) of 2.86, and a solubility in water of 2.43 mg/L (pH = 4), 2.29 mg/L (pH = 7), or 2.27 mg/L (pH = 9) at 20 °C (Yin et al., 2003). Thus, it is difficult for fluoxastrobin to remain in the air due to its low vapor pressure. However, it may remain in soil and water because of rain-out or storms due to its preferential high solubility. The Organization for Economic Cooperation and Development (OECD, 1992) selected zebrafish (Danio rerio) as the biological indicator for evaluating the toxicity of external contamination of aquatic environments. Numerous studies have also focused on zebrafish (Chen et al., 2017; Duan et al., 2017; Han et al., 2016; Kais et al., 2017; Keiter et al., 2016; Scholz, 2013; Tu et al., 2016; Wang et al., 2017; Zhang et al., 2017b). Studies on the toxicity of azoxystrobin have focused on acute toxicity with 96-h of exposure (Lin et al., 2014; Zhang et al., 2014), and Han et al. (2016) studied the genetic toxicity and oxidative stress of another strobilurin fungicide, azoxystrobin, in zebrafish livers. They found that azoxystrobin can induce ROS and oxidative stress which inhibited the activity of SOD and induced the activities of CAT and GST. Thus, fluoxastrobin may have toxicity similar to that of azoxystrobin, or the different kinds of strobilurin fungicides may lead to different toxicities in zebrafish. However, information is still scarce of both the acute and chronic toxicity of fluoxastrobin on zebrafish. Thus, it is vital to study the toxic effects of fluoxastrobin on zebrafish. In addition to the acute toxicity using a static test, chronic toxicity using semistatic tests on days 7, 14, 21, and 28 was also studied, including the levels of ROS, the activities of SOD, CAT, and GST, the levels of MDA and the degree of DNA damage. The present study also aimed to find the most sensitive indicator and the relationships among the diverse indicators. 2. Materials and methods 2.1. Chemicals and reagents Fluoxastrobin (99.3% purity, CAS No. 361377-29-9) was provided by Dr. Ehrenstorfer GmbH (Augsburg, Germany). The stock solutions used in the present study were the appropriate doses of fluoxastrobin dissolved in chromatographically pure acetone. The other chemicals and solvents used in the present study were all analytically pure and were obtained from the Solarbio Science &Technology Company (Beijing, China) and Sigma Chemical (St. Louis, Missouri, USA).
To ensure the reliability of the present study, the fluoxastrobin contents of the tested solutions were determined at 1 h, days 7, 14, 21, and 28 after the initiation of contamination as reported by Liu et al. (2015a). High-performance liquid chromatography (HPLC, Agilent 1260, Agilent Technologies Inc., USA) was used to measure the concentrations of the tested strobilurin fungicide for zebrafish in the tested water. The mobile phase in the present study was composed of ultrapure water and chromatographically pure acetonitrile at the ratio of 35:65. Specifically, a C18 column (Eclipse XDB–C18, 4.6 × 250 mm, 5 μm) was utilized to separate the experimental samples at 40 °C, and the ultraviolet-visible (UV/vis) variable wavelength detector (VWD G1314A, Agilent Technologies Inc., USA) was utilized to analyze the experimental samples at 254 nm. The injection volume and flow rate were 10 μL and 1 mL/min, respectively. Triplicate samples were applied to each control and exposure treatment. According to the procedure OECD 203 (1992), the concentrations of the tested fluoxastrobin should range from 80% to 120% of the initial concentrations. However, if the results of the determination were outside that range (N120% or b80%), the experimentally obtained values were considered the experimental exposure concentrations for evaluating the acute toxic, biochemical toxic and genotoxic effects of fluoxastrobin on zebrafish. 2.4. Experimental design of the acute toxicity test for zebrafish The static test to determine the acute toxicity toward the fish was performed as previously described by Zhang et al. (2017b). The pH, temperature and oxygen saturation of the test water were the same as for the fish handling water. The photoperiod was 12 h light:12 h dark. To avoid interference from the solvent, there were solvent control groups using 1 mL in 1500 mL of chlorine-free tap water. Fluoxastrobin dissolved in 1 mL of acetone and the aforementioned chlorine-free tap water were added to 2 L glass beakers such that each beaker contained 1.5 L of the solution, and final doses of 0.01, 0.1, 0.25, 0.5, 0.6, 0.7, and 0.8 mg/L were used in the formal acute toxicity tests, based on the results of preliminary experiments including 10% and 90% mortality. Ten fish were tested in each beaker, and the previously described measurements were taken for 96 h under the various conditions. Triplicate samples were applied to each control and exposure treatment. The fish were fed with commercially available dry flakes every day during the acclimation period until 24 h prior to the acute toxicity tests. The food residues, feces and dead fish were removed in a timely manner to avoid interference. According to Cai (2004) and Lin et al. (2014), 96 h LC50 was used to evaluate the acute toxicity of the pesticides to zebrafish (mg/L): b0.1, rank poison; 0.1 to 1, highly toxic; 1 to 10, moderately toxic; and N10, low toxicity.
2.2. Fish handling 2.5. Experimental design of the chronic toxicity test for zebrafish The Guiding Principles outlined for the use of animals in toxicology, adopted by the Society of Toxicology in 1989, were applied to the present study (Van, 2002). Kaixin Aquarium (Taian, China) supplied the
The semistatic tests to determine the chronic toxicity toward fish were performed as described by Zhang et al. (2017a). The chlorine-
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free tap water was aerated for one week. The pH, temperature and oxygen saturation of the test water were the same as the fish handling water. The photoperiod was 12 h light:12 h dark. To avoid interference from the solvent, solvent control groups were also established by adding 1 mL of acetone to 20 L chlorine-free tap water. Fluoxastrobin dissolved in 1 mL of acetone and the aforementioned chlorine-free tap water were added to a 25-L aquarium to ensure that each aquarium held 20 L of the solution and to reach final doses of 0.001, 0.01, and 0.1 mg/L, based on the 96 h LC50 value. For each aquarium, 120 fish were acclimatized for 28 days under conditions including the aforementioned parameters. On day 7, day 14, day 21 and day 28, twenty zebrafish were randomly selected for evaluation of the levels of reactive oxygen species (ROS), lipid peroxidation, enzyme activities (SOD, CAT, GST) and DNA damage in livers. Triplicate samples were applied to each control and exposure treatment. The fish were fed with commercially available dry flakes every day during the 28 d of testing until 24 h prior to the chronic toxicity tests. The chronic toxicity test is a semistatic test, and half of the tested water was replaced every two days to maintain constant doses of fluoxastrobin. The food residues, feces and dead fish were removed in a timely manner to avoid interference.
2.6. Determination of ROS levels Reactive oxygen species include hydrogen peroxide (H2O2), super\OH) and singlet oxygen (1O2) oxide anions (O− 2 ), hydroxyl radicals (\ (Han et al., 2016). The present study assayed ROS levels by detecting H2O2 content using the DCFH-DA method (Bothe and Valet, 1990). The method for preparing the suspension was described by Liu et al. (2015a). For each control and experimental group, ten fish were randomly selected to be dissected in 0.1 M ice-cold potassium phosphate buffer at pH 7.4, after which the homogenate was centrifuged (Eppendorf, 5804) at 1000g and 4 °C for 10 min. Subsequently, the resultant supernatant was re-centrifuged at 20,000g and 4 °C for 20 min, after which the precipitate was re-suspended in 500 μL of 0.1 M phosphate buffer to prepare the liver suspension at pH 7.4. Triplicate samples
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were applied to each control and exposure treatment. The ROS content was assayed by using a fluorescent spectrophotometer (Shimadzu, RF5301PC) with an excitation wavelength of 488 nm and emission wavelength of 522 nm. The unit fluorescence-intensity/mg protein (pr) was used to express the ROS levels.
2.7. Determination of enzyme activities (SOD, CAT, and GST) and MDA levels According to the methods performed by Shao et al. (2012), the liver suspension was reserved to measure the content of enzyme proteins, the activities of enzymes and MDA levels. For each control and experimental group, six fish were randomly selected for dissection in icecold 50 mM phosphate buffer, pH 7.0 to prepare the liver suspension, after which the homogenate was centrifuged (Eppendorf, 5804) at 10,000 rpm and 4 °C for 10 min. The enzyme activities and MDA levels were determined by using an ultraviolet-visible spectrophotometer (U-V spectrophotometer, Shimadzu, UV-2550). Triplicate samples were applied to each control and exposure treatment. Bovine serum albumin was utilized as the standard to determine the contents of protein in the fluoxastrobinexposed groups at 595 nm by using the Sigma Bradford method (Bradford, 1976). The methods for assaying the activities of SOD and CAT were both performed according to Song et al. (2009). The photochemical reduction of nitroblue tetrazolium (NBT) after illumination was utilized to assay the activity of SOD at 560 nm. The decomposition of H2O2 was utilized to assay the activity of CAT at 240 nm for 1 min. The amount of enzyme, that caused the reduction of half of the NBT photochemical, was defined as one unit of SOD activity (U). The amount of enzyme that caused 50% of the H2O2 decomposition in 60 s at 25 °C was defined as one unit of CAT activity (U). The unit U/mg pr was used to express the results of both SOD activity and CAT activity. The GST activity was assayed according to the method of Zhu et al. (2011). The changes in absorbance were utilized to evaluate the activity of GST at 340 nm for 3 min, and the unit nmol/min/mg pr was used to express the level of GST activity.
Fig. 2. The ROS levels in zebrafish (Danio rerio) exposed to three different doses of fluoxastrobin. Triplicate samples were applied to each control and exposure treatment. Each column represents the average value of a treatment group, and the error bars represent the standard deviation (SD). The least significant difference (LSD) test was used to analyze the statistical significance of the results among different experimental groups at p b 0.05.
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The method for assaying the levels of MDA, which was considered an indicator of lipid peroxidation, was that reported by Zhang et al. (2013). The unit nmol/mg pr was used to express the MDA levels.
2.8. Determination of the degree of DNA damage The methods for suspension preparation, slide preparation and measurement of the degree of DNA damage using single cell gel electrophoresis (SCGE) were those reported by Shao et al. (2012). For each control and experimental group, four fish were randomly selected for dissection in ice-cold 0.1 M phosphate buffer and were cut up, after which a 200mesh sieve was used to filter the resultant tissue to prepare the homogenate. Then, the homogenate was centrifuged (Eppendorf, 5804) at 200g and 4 °C for 10 min, after which the precipitate with 500 μL of 0.1 M phosphate buffer was re-suspended to prepare the liver suspension at pH 7.4. Triplicate samples were applied to each control and exposure treatment. The comet images were observed using an inverted fluorescence microscope (Olympus, BX51) and analyzed using Comet Assay Software Project (CASP). The distance between the center of the tail and the head, defined as the olive tail moment (OTM), was used to express the results of the degree of DNA damage caused by fluoxastrobin in the livers of the tested zebrafish (Singh et al., 1988).
2.9. Statistical analysis Triplicate samples were applied to each control and exposure treatment. In the present study, we used the Statistical Package for Social Sciences (SPSS) (Standard Version 19.0, SPSS Inc., USA) to evaluate the 96 h LC50 value via probit analysis and to analyze the results of chronic toxicity test via a one-way analysis of variance (ANOVA). We also used a least significant difference (LSD) testing to analyze the statistical significance of the results among different experimental groups at the same treatment time at p b 0.05, and the standard deviation (SD) is presented using the error bars.
3. Results and discussion 3.1. Dynamic change in the fluoxastrobin concentration in water In the controls, no fluoxastrobin was detected. In the present study, the instrument detection limit was 2.33 × 10−9 g, the standard curve was y = 26.618x − 0.0445, and the square of the correlation coefficients was 0.9996. Based on the above data, the recovery rate of fluoxastrobin ranged from 92.9% to 105.3%, which met the guideline OECD 203 (1992). On day 28, the doses of fluoxastrobin in the 0.001,
Fig. 3. The activities of SOD (a), CAT (b) and GST (c) in zebrafish (Danio rerio) exposed to three different doses of fluoxastrobin. Triplicate samples were applied to each control and exposure treatment. Each column represents the average value of three samples, and the standard deviation (SD) is represented with an error bar. The least significant difference (LSD) test was used to analyze the statistical significance of the differences among the experimental groups at p b 0.05.
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0.01, and 0.1 mg/L treatments were 3.9, 5.0, and 0.64% greater than those on day 0. Thus, the doses of each fluoxastrobin-exposed group of zebrafish (Danio rerio) in the tested water were considered relatively stable and further demonstrated the dependability of the present study.
3.2. Acute toxic effects of fluoxastrobin on zebrafish (Danio rerio) There were no deaths among the controls. The cumulative mortality increased with the increasing doses of fluoxastrobin. The 96 h LC50 value of fluoxastrobin for exposure to zebrafish was 0.51 mg/L, and the 95% confidence interval extended from 0.45 to 0.57 mg/L. Based on the toxicity grading standards, fluoxastrobin is considered highly toxic to zebrafish (Cai, 2004; Lin et al., 2014). The results were similar to Zhang et al. (2014), in which they evaluated the acute toxicity of azoxystrobin on zebrafish and found that the 96 h LC50 value was 0.67 mg/L with a 95% confidence interval of 0.64 mg/L to 0.72 mg/L. The 96 h LC50 value of fluoxastrobin toward rainbow trout was N0.44 mg/L (Yin et al., 2003). The minor discrepancy between our results and those from aforementioned previous studies may be caused by differences in the strobilurin fungicides or in the fish. Different species can have different susceptibilities and thus different LC50 values.
3.3. Effects of fluoxastrobin on ROS levels The ROS levels in zebrafish exposed to three different doses of fluoxastrobin are depicted in Fig. 2. On days 7, 14, 21, and 28, the ROS levels in each treatment group were all significantly enhanced with increasing test doses compared to the controls. As stated by Liu et al. (2014), there is a dynamic equilibrium between the production and elimination of ROS in general. However, the production and elimination of ROS is disturbed even at the lowest dose (0.001 mg/L). When Han et al. (2016) studied the toxicity of azoxystrobin on female zebrafish, they also observed similar results. Excess ROS can induce oxidative stress, thus the enzyme activities (SOD, CAT, GST) and MDA levels were also evaluated in the present study.
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3.4. Effects of fluoxastrobin on enzyme activities (SOD, CAT and GST) Yonar (2013) has stated that SOD and CAT belong to the antioxidant enzymes, which can partially clean up ROS induced by the external contamination and can be inhibited by excess ROS in turn (Ge et al., 2015; Han et al., 2016; Yan et al., 2015). The activities of SOD (a), CAT (b) and GST(c) in zebrafish after exposure to three different doses of fluoxastrobin are depicted in Fig. 3. Both SOD and CAT belong to the first line of defense for eliminating ROS (Yan et al., 2015). Superoxide dismutase can promote the transformation of O− 2 to H2O2, and CAT can promote the transformation of H2O2 to H2O in a timely manner (Liu et al., 2015a). As illustrated in Fig. 3a, the values for the treatments with fluoxastrobin at 0.001 and 0.01 mg/L are greater than those for the control groups on days 7, 14, 21, and 28. The values for treatment with 0.1 mg/L are all lower than those for the controls on days 7, 14 and 21, while the value for 0.1 mg/L is slightly higher than that for the controls on day 28. The results demonstrated that ROS caused an increase of the SOD activity at low doses of fluoxastrobin (0.001 and 0.01 mg/L), and at the highest dose (0.1 mg/L), SOD activity was inhibited by a disruption of the function of the protein (Du et al., 2014). On day 28, notable differences were found in each fluoxastrobin-treatment group except for the treatment with 0.1 mg/L. As illustrated in Fig. 3b, CAT activity showed a trend similar to that of SOD activity. Specifically, the values in the treatment groups first increased at 0.001 and 0.01 mg/L and then decreased at 0.1 mg/L compared with the controls for the duration of the experimental period. When Du et al. (2014) focused on the toxicity of the IL 1-octyl-3methylimidazolium bromide in zebrafish livers, they observed a similar phenomenon. Significant differences can be observed at each fluoxastrobin-treatment group with the exception of the 0.01 mg/L dose on day 7 and the 0.001 mg/L dose on day 14. Glutathione S-transferase (GST) is considered a type of detoxifying enzyme for cleaning up excess ROS (Zhu et al., 2011). As illustrated in Fig. 3c, the values for the three different doses were all greater than those of the control groups on days 7, 14, 21, and 28, except for the value at the dose of 0.1 mg/L on day 21. Notable differences were found in each fluoxastrobin-treatment group except for the value for the dose of 0.001 mg/L on day 21. The GST activity with treatment at
Fig. 4. The MDA contents in zebrafish (Danio rerio) exposed to three different doses of fluoxastrobin. Triplicate samples were applied to each control and exposure treatment. Each column represents the average value of a condition, and the error bars indicate the standard deviation (SD). The least significant difference (LSD) test was used to analyze the statistical significance of the differences among the different experimental groups at p b 0.05.
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Fig. 5. The degree of DNA damage in zebrafish (Danio rerio) exposed to three different doses of fluoxastrobin. Triplicate samples were applied to each control and exposure treatment. Each column represents the average value of a condition, and the error bars indicate the standard deviation (SD). The least significant difference (LSD) test was used to analyze the statistical significance of the differences among the different experimental groups at p b 0.05.
0.1 mg/L was lower than that with 0.001 or 0.01 mg/L during the entire experimental period, but it was still higher than that in the controls. The results from a study of the toxic effects of azoxystrobin in zebrafish (Han et al., 2016) are similar to our findings. 3.5. Effect of fluoxastrobin on lipid peroxidation Reactive oxygen species (ROS) can induce lipid peroxidation and antioxidant enzymes are the first line of defense to decrease the progression by eliminating the excess ROS (Yan et al., 2015). Lipid peroxidation is considered to have a complex integrated effect that can produce malondialdehyde (MDA). Thus, MDA levels were utilized to assay the degree of lipid peroxidation (Liu et al., 2015a). Fig. 4 depicts the effects of lipid peroxidation according to MDA levels in zebrafish after exposure to three different doses of fluoxastrobin. Compared to the controls, remarkable growth can be observed for each condition with the exception of fluoxastrobin at 0.001 and 0.01 mg/L on day 7. When Han et al. (2016) focused on the toxicity of the fungicide azoxystrobin for zebrafish females, they obtained similar results. Furthermore, no significant difference was found between the values of 0.001 and 0.01 mg/L during the entire environmental period. This may be caused by antioxidant enzymes eliminating ROS to a certain extent with fluoxastrobin treatments at 0.001 and 0.01 mg/L. However, excess ROS can reduce or even inhibit enzyme activities such that ROS cannot be cleared in time at a high dose of fluoxastrobin (0.1 mg/L), which exacerbated oxidative damage and lipid peroxidation (Liu et al., 2015b).
and 0.01 mg/L, the values were less than two times those of the control groups, while the values were three or even four times those of the control groups at the highest dose (0.1 mg/L) in each trial. This result may be due to SOD and GST eliminating ROS to a certain extent at 0.001 and 0.01 mg/L, while at a high dose (0.1 mg/L), the excess ROS inhibited the activities of antioxidant enzymes so that ROS could not be eliminated in time, thereby increasing the degree of DNA damage (Liu et al., 2015b). Significant differences were observed at each time point during the entire experimental period. This result may be due to the DNA strand breakage, which may be dose-dependent. Ge et al. (2015) also drew a similar conclusion when they studied the effect of imidacloprid in zebrafish livers on days 7, 14, 21, and 28.
4. Conclusions In the present study, we analyzed the acute toxic, biochemical toxic and genotoxic effects of fluoxastrobin on zebrafish (Danio rerio). The four following bullet points are the main conclusions: (1) Fluoxastrobin is highly toxic to zebrafish (Danio rerio). (2) The tested fungicide can cause oxidative stress and oxidative damage in zebrafish livers. (3) The most sensitive biomarker of all the biomarkers tested in the present study is the comet assay. (4) Based on the data illustrated in the present study, fluoxastrobin is relatively stable in an aquatic environment for the entire duration of the experimental period.
3.6. Effect of fluoxastrobin on DNA damage DNA damage, including DNA broken strands, is considered an indicator of oxidative damage induced by external contamination (Liu et al., 2015a). Fig. 5 depicts the degree of DNA damage based on OTM levels in zebrafish after exposure to three different doses of fluoxastrobin. On days 7, 14, 21and 28, the OTM levels were significantly enhanced in all of the treatment groups compared to the controls. With the increasing tested doses, the degree of DNA damage was enhanced at each exposure time, with a trend similar to that of ROS levels. At 0.001
Informed consent Informed consent was obtained from all individual participants included in the study.
Conflicts of interest The authors declare that they have no conflict of interest.
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Acknowledgements This work was supported by the National Key Research and Development Plan [Nos. 2017YFD0200307, 2016YFD0800202 and 2016YFD0201203]; the National Natural Science Foundation of China [Nos. 41771282, 41671320]; the Natural Science Foundation of Shandong Province, China [ZR2017MD005] and the Special Funds of Taishan Scholar of Shandong Province, China. References Bartlett, D.W., Clough, J.M., Godwin, J.R., Hall, A.A., Hamer, M., Parr-Dobrzanski, B., 2002. The strobilurin fungicides. Pest Manag. Sci. 58, 649–662. Bothe, G., Valet, G., 1990. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2′,7′-dichlorofluorescin. J. Leukoc. Biol. 47, 440–448. Bradford, M.M., 1976. A rapid sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Cai, D.J., 2004. Testing Guidelines of Assessing Environmental Safety of Chemical Pesticides. State Environmental Protection Administration of China, Beijing. Chen, Q.Q., Gundlach, M., Yang, S.Y., Jiang, J., Velki, M., Yin, D.Q., Hollert, H., 2017. Quantitative investigation of the mechanisms of microplastics and nanoplastics toward zebrafish larvae locomotor activity. Sci. Total Environ. 584–585, 1022–1031. Du, Z.K., Zhu, L.S., Dong, M., Wang, J.H., Wang, J., Xie, H., Liu, T., Guo, Y.Y., 2014. Oxidative stress and genotoxicity of the ionic liquid 1-octyl-3-methylimidazolium bromide in zebrafish (Danio rerio). Arch. Environ. Contam. Toxicol. 67, 261–269. Duan, J.C., Yu, Y., Li, Y., Jing, L., Yang, M., Wang, J., Li, Y.B., Zhou, X.Q., Miller, M.R., Sun, Z.W., 2017. Comprehensive understanding of PM2.5 on gene and microRNA expression patterns in zebrafish (Danio rerio). Sci. Total Environ. 586, 666–674. Ge, W.L., Yan, S.H., Wang, J.H., Zhu, L.S., Chen, A.M., Wang, J., 2015. Oxidative stress and DNA damage induced by imidacloprid in zebrafish (Danio rerio). J. Agric. Food Chem. 63, 1856–1862. Han, Y.N., Liu, T., Wang, J.H., Wang, J., Zhang, C., Zhu, L.S., 2016. Genotoxicity and oxidative stress induced by the fungicide azoxystrobin in zebrafish (Danio rerio) livers. Pestic. Biochem. Physiol. 133, 13–19. Kais, B., Schiwy, S., Hollert, H., Keiter, S.H., Braunbeck, T., 2017. In vivo EROD assays with the zebrafish (Danio rerio) as rapid screening tools for the detection of dioxin-like activity. Sci. Total Environ. 590–591, 269–280. Keiter, S., Burkhardt-Medicke, K., Wellner, P., Kais, B., Färber, H., Skutlarek, D., Engwall, M., Braunbeck, T., Keiter, S.H., Luckenbach, T., 2016. Does perfluorooctane sulfonate (PFOS) act as chemosensitizer in zebrafish embryos? Sci. Total Environ. 548–549, 317–324. Lin, J., Wang, K.Y., Xu, H., Tang, J.F., Liu, J., 2014. Acute toxicity and safety evaluation of five new fungicides to four kinds of fishes. World Pesticides 36, 34–38. Liu, T., Zhu, L.S., Han, Y.N., Wang, J.H., Wang, J., Zhao, Y., 2014. The cytotoxic and genotoxic effects of metalaxy-M on earthworms (Eisenia fetida). Environ. Toxicol. Chem. 33, 2344–2350.
775
Liu, T., Guo, Y.Y., Wang, J.H., Wang, J., Zhu, L.S., Zhang, J., Zhang, C., 2015a. Assessing toxic effects of [Omim]Cl and [Omim]BF4 in zebrafish adults using a biomarker approach. Environ. Sci. Pollut. Res. 23, 7360–7368. Liu, T., Zhu, L.S., Wang, J.H., Wang, J., Xie, H., 2015b. The genotoxic and cytotoxic effects of 1-butyl-3-methylimidazolium chloride in soil on Vicia faba seedlings. J. Hazard. Mater. 285, 27–36. OECD, 1992. Guidelines for the Testing of Chemicals. Test Guideline 203, Fish, Acute Toxicity Test. Organization for Economic Cooperation and Development, Paris. Scholz, S., 2013. Zebrafish embryos as an alternative model for screening of drug-induced organ toxicity. Arch. Toxicol. 87 (5), 767–769. Shao, B., Zhu, L.S., Dong, M., Wang, J., Wang, J.H., Xie, H., Zhang, Q.M., Du, Z.K., Zhu, S.Y., 2012. DNA damage and oxidative stress induced by endosulfan exposure in zebrafish (Danio rerio). Ecotoxicology 21, 1533–1540. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191. Song, Y., Zhu, L.S., Wang, J., Wang, J.H., Liu, W., Xie, H., 2009. DNA damage and effects on antioxidative enzymes in earthworm (Eisenia foetida) induced by atrazine. Soil Biol. Biochem. 41, 905–909. Tu, W.Q., Xu, C., Lu, B., Lin, C.M., Wu, Y.M., Liu, W.P., 2016. Acute exposure to synthetic pyrethroids causes bioconcentration and disruption of the hypothalamus–pituitary– thyroid axis in zebrafish embryos. Sci. Total Environ. 542, 876–885. Van, P.J., 2002. Guiding principles in the use of animals in toxicology. Adopted by the Society of Toxicology in July 1989. Toxicol. Appl. Pharmacol. 2, 6–7. Wang, G.W., Chen, H.Y., Du, Z.K., Li, J.H., Wang, Z.Y., Gao, S.X., 2017. In vivo metabolism of organophosphate flame retardants and distribution of their main metabolites in adult zebrafish. Sci. Total Environ. 590–591, 50–59. Yan, S.H., Wang, J.H., Zhu, L.S., Chen, A.M., Wang, J., 2015. Thiamethoxam induces oxidative stress and antioxidant response in zebrafish (Danio Rerio) livers. Environ. Toxicol. 31, 1–10. Yin, J.J., Ma, H.Y., Guan, A.Y., Liu, C.L., 2003. High-effect fungicide: fluoxastrobin. Pesticides 42, 36–40. Yonar, M.E., 2013. Protective effect of lycopene on oxidative stress and antioxidant status in Cyprinus carpio during cypermethrin exposure. Environ. Toxicol. 28, 609–616. Zhang, Q.M., Zhu, L.S., Wang, J.H., Xie, H., Wang, J., Han, Y.N., Yang, J.H., 2013. Oxidative stress and lipid peroxidation in the earthworm Eisenia fetida induced by low doses of fomesafen. Environ. Sci. Pollut. Res. 20, 201–208. Zhang, G.F., Li, B.J., Wang, J.H., Xia, X.M., 2014. Acute toxicity of technical material and different formulations of difenoconazole and azoxystrobin to zebrafish. J. Agro-Environ. Sci. 33, 2125–2130. Zhang, C., Shao, Y.T., Zhu, L.S., Wang, J.H., Wang, J., Guo, Y.Y., 2017a. Acute toxicity, biochemical toxicity and genotoxicity caused by 1-butyl-3-methylimidazolium chloride and 1-butyl-3-methylimidazolium tetrafluoroborate in zebrafish (Danio rerio) livers. Environ. Toxicol. Pharmacol. 51, 131–137. Zhang, C., Zhu, L.S., Wang, J.H., Wang, J., Zhou, T.T., Xu, Y.Q., Cheng, C., 2017b. The acute toxic effects of imidazolium-based ionic liquids with different alkyl-chain lengths and anions on zebrafish (Danio rerio). Ecotoxicol. Environ. Saf. 140, 235–240. Zhu, L.S., Dong, X.L., Xie, H., Wang, J., Wang, J.H., Su, J., Yu, C.W., 2011. DNA damage and effects on glutathione-S-transferase activity induced by atrazine exposure in zebrafish (Danio rerio). Environ. Toxicol. 26, 480–488.