Developmental toxicity and potential mechanisms of

Ecotoxicology and Environmental Safety 151 (2018) 1–9

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Developmental toxicity and potential mechanisms of pyraoxystrobin to zebrafish (Danio rerio) Hui Li, Song Yu, Fangjie Cao, Chengju Wang, Mingqi Zheng, Xuefeng Li, Lihong Qiu

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College of Sciences, China Agricultural University, Beijing 100193, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Zebrafish Pyraoxystrobin Acute toxicity Developmental toxicity Gene expression

As a newly developed, highly efficient strobilurin fungicide, pyraoxystrobin has been reported to be highly toxic to some aquatic organisms. However, the toxicity of pyraoxystrobin to different life stages of fish and the potential underlying mechanisms are still unknown. Hence, in the present study, the acute toxicity of pyraoxystrobin to different life stages of zebrafish (embryo, larva, and adult) was assessed. The developmental toxicity of pyraoxystrobin to zebrafish embryos and its effects on gene transcription in the embryo were also investigated. The results showed that the 96-h LC50 values of pyraoxystrobin to embryos [2 h post-fertilization (hpf)], 12 h post-hatching (hph) larvae (84 hpf), 72 hph larvae (144 hpf), and adult zebrafish were 4.099, 1.069, 3.236, and 5.970 µg/L, respectively. This suggests that pyraoxystrobin has very high toxicity to different life stages of zebrafish, while the newly hatched larvae constitute the most sensitive period of zebrafish to pyraoxystrobin. Decreased heart rate, hatching inhibition, growth regression, and morphological deformities were observed in zebrafish embryos after acute exposure to different concentrations of pyraoxystrobin. The rate of malformation increased in a time- and concentration-dependent manner in embryos, and the most pronounced abnormality was pericardial edema and yolk sac edema. Pyraoxystrobin (2 and 4 μg/L) significantly altered the mRNA levels of genes related to mitochondrial respiratory chain and ATP synthesis (NDI, uqcrc, and ATPo6), oxidative stress (Mn-Sod, Cat, and Gpx), apoptosis (p53, Bcl2, Bax, and Cas3), and immune system (TNFα, IFN, and IL-1b) in zebrafish embryos. This result indicates that the alteration of these genes is a potential mechanism underlying the toxic effects of pyraoxystrobin on zebrafish.

1. Introduction Pyraoxystrobin is a novel strobilurin fungicide that was developed by Shenyang Research Institute of Chemical Industry (SRICI) and has been granted patents in China and America (Chen et al., 2015; Yang et al., 2014). Pyraoxystrobin was demonstrated to have a short degradation half time (DT50; 2.6–2.7 days) in soil, but was stable in water at pH 4 and pH 7, with a half-life of 577.5 days at pH 9 (25 °C) (Chen et al., 2015; Liu et al., 2010). The molecular mechanism of pyraoxystrobin in fungi involves the inhibition of mitochondrial respiration by blocking electron transfer between cytochrome b and cytochrome c1, at the ubiquinol oxidation site (Qo) of cytochrome c reductase (Bartlett et al., 2002; Hnatova et al., 2003). Pyraoxystrobin exerts highly efficient activities against various fungal pathogens, such as those belonging to the ascomycotina, deuteromycotina, and mastigomycotina classes (Li et al., 2011). The acute oral median lethal dose (LD50) of pyraoxystrobin is 1000 mg/kg in male rats and 1022 mg/kg in female rats, which proves that pyraoxystrobin has low toxicity to mammals (Liu et al., 2011).

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To date, the studies on pyraoxystrobin mainly focused on its environmental behavior (Liu et al., 2012), fungicidal activity (Chen et al., 2011), and analytical method (Lin et al., 2015), with very limited attention on its toxicity to environmental organisms. Previous studies have demonstrated that many strobilurin fungicides were highly toxic to fish, for instance, the 96-h LC50 values of azoxystrobin, pyraclostrobin, and picoxystrobin to zebrafish were 0.393, 0.0635, and 0.212 mg/L, respectively (Liu and Zhu, 2015), and the 48-h LC50 values of trifloxystrobin, azoxystrobin, and kresoxim-methyl to grass carp juveniles were 0.051, 0.338, and 0.549 mg/L, respectively (Liu et al., 2013). Chen et al. (2015) reported that pyraoxystrobin exhibits high toxicity to non-target aquatic organisms, for example, the 96-h LC50 values of pyraoxystrobin to Penaeus monodon and Oryzias latipes were 35.10 and 1.22 μg/L, respectively (Chen et al., 2015). However, the toxicity of pyraoxystrobin to different life stages of fish has not yet been investigated. Some previous studies have reported the potential mechanisms underlying the toxicity of strobilurins to fish. Han et al. (2016) reported

Corresponding author. E-mail address: [email protected] (L. Qiu).

https://doi.org/10.1016/j.ecoenv.2017.12.061 Received 25 May 2017; Received in revised form 28 December 2017; Accepted 29 December 2017 Available online 02 January 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.


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incubator (27 ± 1 °C; 14:10 h light/dark photoperiod) to obtain larvae at 12 and 72 h post-hatching (hph). In our laboratory conditions, most embryos (approximately 70%) hatched at around 72 hpf, and the larvae that hatched before 71 hpf and embryos that did not hatch after 73 hpf were removed. The larvae that hatched between 71 and 73 hpf were cultured for another 12 and 72 h, and then used for the larvae of 12 and 72 hph toxicity test, respectively. The hatching time was 72 ± 1 hpf, hence, larvae at 12 and 72 hph were 84 and 144 hpf, respectively.

that chronic exposure to azoxystrobin resulted in the accumulation of reactive oxygen species (ROS), changes in antioxidase activities, generation of lipid peroxide, and DNA damage in zebrafish livers. Liu et al. (2013) demonstrated that trifloxystrobin, azoxystrobin, and kresoximmethyl induced oxidative damage and altered the expression of growthand energy-related genes in grass carp juveniles. Previous studies also indicated that some strobilurin fungicides induced endocrine disruption in fish (Cao et al., 2016b; Zhu et al., 2013). Although mitochondrial respiratory chain might be the potential target of strobilurin fungicides in fish, no information on the toxic effects of strobilurin fungicides on the function of mitochondria in non-target organisms is available yet. Moreover, mitochondrion has been demonstrated to play an important role in the occurrence of oxidative stress and cell apoptosis (Cadenas and Davies, 2000; Green and Reed, 1998), which in turn might affect the components of immune system (Livingstone, 2001). Currently, increasing researches have paid attention to the expression of genes related to oxidative stress, cell apoptosis, and immunotoxicity induced by environmental chemicals. For instance, cyhalofop-butyl has the potential to induce oxidative stress and apoptosis in zebrafish embryo cells (Zhu et al., 2015). Cis-bifenthrin induced the transcription of genes related to oxidative stress, apoptosis, and immunotoxicity in zebrafish (Jin et al., 2013). In the present study, the acute toxicity of pyraoxystrobin to different life stages of zebrafish, as well as the effect of pyraoxystrobin on the expression of genes involved in mitochondrial respiratory chain and adenosine triphosphate (ATP) synthesis, oxidative stress, cell apoptosis, and innate immune response in zebrafish embryos were investigated. The results of this study will help understand the toxicity of pyraoxystrobin to different life stages of fish and provide an insight into the potential toxic mechanisms on embryonic development.

2.3. Acute toxicity assays of pyraoxystrobin to zebrafish Acute-toxicity test of zebrafish embryo was conducted according to the OECD Draft Proposal-Fish Embryo Toxicity (FET) Test (OECD, 2013) and a previously proposed method (Fraysse et al., 2006). The nominal concentrations of pyraoxystrobin were 2.03, 2.44, 2.90, 3.51, 4.22, and 5.08 µg/L, selected based on pre-experiment data. Reconstituted water was used to prepare all test solutions, and it served as blank control (0 mg/L). Solvent control was prepared using 0.005% AT (dissolved in reconstituted water, v/v), which contained the same solvent contents as that in the test solution with the highest concentration of pyraoxystrobin. Normally developed embryos (approximately 2 hpf) were randomly transferred into test solutions in 24-well plates (2 mL solution and 1 embryo per well) for 96 h. Twenty wells contained pyraoxystrobin test solution and four wells contained reconstituted water as the internal control in each plate. For each test concentration and control, three 24-well plates (replicates) were included (60 embryos per concentration). All tested 24-well plates were placed in an incubator (27 ± 1 °C; 14:10 h light/dark photoperiod). The plates were covered with transparent lids to prevent evaporation. The exposure solution was renewed every 24 h to maintain the appropriate concentration of pyraoxystrobin and water quality. The number of dead individuals and the state of embryonic development were examined daily. Spontaneous movements of embryos (spontaneous lashing movements of the tail) at 24 hpf, the heart rate of embryos at 48 and 72 hpf, and hatching rate and body length of the hatched larvae at 96 hpf were tested. In addition, embryonic malformations were checked daily. Morphological development and abnormalities were observed using a light microscope (Olympus BH-2). The body length of larvae was measured from top of head to end of tail by drawing a straight line along the margin of the back axis using GE-5 (Aigo Corp, Beijing) based on the method of Fraysse et al. (2006) with some modifications. Acute toxicity assessment in larvae and adult zebrafish were conducted according to Mu et al. (2013). Two hundred and forty larvae of 12 hph were randomly exposed to 200 mL reconstituted water (blank control), 0.0024% AT (solvent control, dissolved in reconstituted water, v/v), and 1.50, 1.65, 1.83, 2.00, 2.20, and 2.43 µg/L pyraoxystrobin, respectively. Two hundred and ten larvae of 72 hph were randomly exposed to 200 mL reconstituted water (blank control), 0.0037% AT (solvent control, dissolved in reconstituted water, v/v), and 2.50, 2.75, 3.00, 3.30, and 3.65 µg/L pyraoxystrobin, respectively. Two hundred and forty adult zebrafish were exposed to 4 L dechlorinated tap water (blank control), 0.007% AT (solvent control, dissolved in dechlorinated tap water, v/v), and 5.75, 6.00, 6.25, 6.50, 6.75, and 7.00 µg/L pyraoxystrobin for 96 h, respectively. The test concentrations were selected based on pre-experiment data. Reconstituted water and dechlorinated tap water were used to prepare test solutions for larvae and adults, respectively. The solvent control contained the same acetone and Tween-80 contents as that in the highest dosage of solutions of each test. Three replicates for each concentration (30 larvae or adults) were used and each beaker contained 10 larvae or adults as a repetition. The exposure solution was renewed every 24 h to maintain the appropriate concentration of pyraoxystrobin and water quality. The number of dead individuals was recorded daily during the tests. The standard of judging death was the absence of a heartbeat under the microscope (for larvae) (OECD, 1998) and no visible breathing or no moving when the tail was touched (for adult fish) (OECD, 1992). During the acute toxicity

2. Materials and methods 2.1. Chemicals and reagents Pyraoxystrobin (CAS: 862588-11-2, 95% purity) was supplied by Shenyang Research Institute of Chemical Industry (SRICI). A stock solution of 100 mg/L pyraoxystrobin was prepared with acetone (Sinopharm Chemical Reagent Co., Ltd, analytical reagent), and 0.5% Tween-80 (v/v) (Sinopharm Chemical Reagent Co., Ltd, analytical reagent) was added as a cosolvent. Acetone containing 0.5% Tween-80 (v/v) (abbreviated as AT) was used to prepare solvent control in exposure tests. Acetonitrile (MREDA Technology Inc), which was used for the determination of pyraoxystrobin concentrations in exposure solutions, was HPLC grade. Reconstituted water containing 2 mmol/L Ca2+, 0.5 mmol/L Mg2+, 0.75 mmol/L Na+, and 0.074 mmol/L K+ was prepared in the laboratory according to ISO-7346-3 (ISO, 1996). 2.2. Zebrafish cultivation and egg production Wild type AB-strain zebrafish was purchased from a local aquarium and maintained according to Cao et al. (2016a). Parental zebrafish for egg production (length 3.50 ± 0.50 cm; weight 0.40 ± 0.10 g) and adult zebrafish for chemical exposure (length 2.50 ± 0.50 cm; weight 0.15 ± 0.05 g) were cultured in reconstituted water under laboratory conditions in flow-through feeding equipment (Esen Corp., China) and aquarium, respectively, for at least 2 weeks before the toxicity test. All adult zebrafish were fed live brine shrimp twice a day. Male and female parental zebrafish (male/female ratio was 1/2) were separated in spawning boxes (Esen Corp, Beijing) overnight. On the following morning, spawning was triggered once the light was turned on, and eggs were collected 30 min later and rinsed with reconstituted water. Fertilized and normally developed eggs were selected for embryonic acute toxicity test at approximately 2 h post-fertilization (hpf) using a dissecting microscope. Approximately 1000 eggs of 2 hpf were selected and then cultured in reconstituted water in an 2


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experiment, adult fish and larvae were not fed, and dead individuals were removed immediately from the beakers.

was set to 10% phase A within 0.1 min and stopped at 4.0 min. The injection volume was 10 μL, and the column oven was at 40 °C. Mass spectrometric analysis was performed in the positive ion MRM (multiple reactions monitoring) mode. The source temperature was set at 150 °C, ion spray voltage at 3.0 kV, desolvation temperature at 350 °C, desolvation gas flow at 650 L/h, and cone gas flow at 50 L/h. The optimized cone voltage was 26 V and collision energy was 10 V. The MRM analysis was conducted by monitoring the precursor ion to product ion transition from m/z 412.9–205.4 (qualitative ion) and m/z 412.9–145 (quantitative ion). The UPLC-MS/MS data were collected and analyzed using MassLynx version 4.1 software.

2.4. Gene expression analysis Zebrafish embryos at 2 hpf were exposed to 0.004% AT (solvent control, dissolved in reconstituted water, v/v), 2 and 4 μg/L pyraoxystrobin for 96 h. The dosage was selected based on the no-observedeffect concentration (2.03 µg/L) and the 96-h LC50 value of embryo acute toxicity test. Total RNA was isolated from 30 embryos or newly hatched zebrafish larvae at the end of exposure using the spin column method according to the manufacturer's protocols (Tiangen Biotech, Beijing, China). The RNA concentration and quality were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc, USA) and in 1% agarose gels through electrophoresis. cDNA was synthesized via reverse transcription using Quant RTase Kit (Tiangen Biotech, Beijing, China) following the manufacturer's protocols. Quantitative real-time PCR amplifications were carried out on ABI 7500 q-PCR system (Applied Biosystems, Foster City, CA) using the SYBR Green PCR Master Mix reagent kits (Tiangen Biotech, Beijing, China). The thermal cycle was as follows: denaturation for 10 min at 95 °C, followed by 40 cycles at 95 °C for 15 s, annealing at 60 °C for 20 s, and extension at 72 °C for 32 s. Concentration-related transcriptional changes in genes of interest, including manganese superoxide dismutase gene (Mn-sod), catalase gene (Cat), and glutathione peroxidase gene (Gpx), which encodes antioxidant proteins; tumor suppressor gene (p53), B-cell lymphoma/ leukemia-2 gene (Bcl2), Bcl-2 associated X protein gene (Bax), and Caspase 3 gene (Cas3), which are involved in apoptosis pathway; tumor necrosis factor α gene (TNFα), interferon gene (IFN), and interleukin-1 beta gene (IL-1b), which are associated with the innate immune system; NADH dehydrogenase subunit gene (NDI), ubiquinol-cytochrome c reductase core protein gene (uqcrc), cytochrome c oxidase subunit I gene (COXI), and ATP synthase F0 subunit 6 gene (ATPo6), which are involved in the mitochondrial respiratory chain and ATP synthesis, were determined. The corresponding primer sequences are listed in Table S1. β-actin was used as a house-keeping gene. Melt curve analysis was used to confirm primer specificity. The relative expression levels of genes were calculated using the 2-ΔΔCt method (Livak and Schmittgen, 2001). Three biological replicates and three technical replicates were performed for each sample.

2.6. Statistical analysis The data presented in this study were checked for normality and homogeneity of variances with Shapiro-Wilk's test and Levene's test, respectively. The differences between control and treatment groups were determined by one-way ANOVA, followed by Dunnett post-hoc comparison using SPSS 16.0 software (SPSS, Chicago, IL, USA). The criterion for statistical significance was p < 0.05. 3. Results 3.1. Solvent effect No significant difference was found between the solvent control and blank control for all indicators in this study. Therefore, the experiment data from the solvent control were used as control for statistical analysis. 3.2. Chemical analysis The calibration curves showed good linearity in the range of 0.5–10 μg/L (R2 = 0.9997) and the level of detection (LOD) was 0.5 μg/L (Fig. S1). At fortification levels of 1, 5, and 10 μg/L for pyraoxystrobin in water samples, the average recoveries of pyraoxystrobin ranged from 88.50% to 107.07% with a relative standard deviation (RSD) of 2.35–3.54% (n = 3) (Table S2). The results indicated that this method provides satisfactory precision and accuracy and can be used for the determination of pyraoxystrobin. Chemical analysis results indicated that the deviations between nominal and actual pyraoxystrobin concentrations were less than 20% in 24 h (Table S3). Since all test solutions were renewed daily, the nominal dosage represents the actual concentration of pyraoxystrobin in the study, according to OECD guidelines (OECD, 1992, 2006).

2.5. Determination of pyraoxystrobin in water samples The concentrations of pyraoxystrobin in exposure solutions of all treatments were analyzed at the beginning of exposure and before water renewal after 24 h of exposure. The samples (10 mL) were transferred into a 50 mL centrifuge tube, followed by the addition of 10 mL of acetonitrile and 3.0 g of sodium chloride. Then, the mixture was vortexed for 1 min and centrifuged for 5 min at 5000×g. The supernatant acetonitrile layer was filtered through a 0.22 µm filter membrane and transferred into an auto sampler vial for analysis. A recovery experiment was conducted to determine the accuracy and precision of the analytical method. The concentrations of pyraoxystrobin (1, 5, and 10 μg/L) were designed by adding the quantitative standard substance to blank water samples, after which the samples were analyzed according to the steps above. The analysis was performed on a Waters Acquity UPLC system coupled to a triple-quadrupole Xevo-TQD equipped with an electrospray ionization source (ESI) (Waters Corp., Milford, MA, USA). The chromatographic separation was achieved on an Acquity UPLC BEH Shield RP18 column (2.1 × 100 mm, 1.7 µm particle size, Milford, MA, USA) with a gradient of acetonitrile (A)/water (B) at a flow rate of 0.4 mL/min. The gradient elution program was as follows: mobile phase A started at 10% and was held for 0.5 min, then increased linearly to 90% from 0.5 to 1.25 min and held for 1.75 min; finally, the gradient

3.3. Acute toxicity of pyraoxystrobin to zebrafish The results of acute toxicity test showed that pyraoxystrobin is highly toxic to zebrafish, with 96-h LC50 values of 4.099, 1.697, 3.236, and 5.970 μg/L to zebrafish embryos, 12 hph larvae, 72 hph larvae, and adults, respectively (Table 1). The lethal sensitivity of different life stages of zebrafish to pyraoxystrobin was as follows: 12 hph larvae > 72 hph larvae > embryos > adults. 3.4. Effects of pyraoxystrobin on embryonic development There was no significant difference between embryos of control and treatment groups on the number of spontaneous movements in 20 s (Fig. 1a), and no spontaneous movements of embryos were observed in 4.22 and 5.08 μg/L treatments. The heartbeat rates of embryos decreased with increase in pyraoxystrobin concentrations. Pyraoxystrobin (2.90 μg/L) significantly inhibited the number of heartbeats in 20 s at 48 hpf. At 72 hpf, the heartbeat rate in 20 s was 60.33 ± 0.41 beats in the control group, but only 25.6 ± 3.2 beats in 5.08 μg/L pyraoxystrobin exposure group (Fig. 1b). Meanwhile, arrhythmia was detected 3


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Table 1 Acute toxicity of pyraoxystrobin to different life stages of zebrafish. Stages

Regression equation

Embryos 12hph larvae 72hph larvae Adults

Y Y Y Y

= = = =

4.484X − 6.325 6.652X − 1.527 16.044X − 8.130 12.730X − 9.810

R2

LC50 value (μg/L)

95% confidence limit (μg/L)

0.990 0.992 0.984 0.986

4.099 1.697 3.236 5.970

3.880–4.289 1.553–1.830 3.132–3.346 5.523–6.190

hph: hours post-hatching.

in groups exposed to pyraoxystrobin at concentrations of 2.90 μg/L and above. Different levels of hatching inhibition were detected following exposure to various concentrations of pyraoxystrobin for 96 h. The hatching rate of 2.03 μg/L pyraoxystrobin-treated group was similar to that of the control; however, a 20% decline was observed in 2.44 μg/L pyraoxystrobin-treated group when compared to the control group. Simultaneously, no hatching was observed in the 5.08 μg/L pyraoxystrobin-treated group (Fig. 1c). Furthermore, treatments with 2.90 μg/ L or higher concentrations of pyraoxystrobin significantly reduced the body length of hatched larvae at 96 hpf (Fig. 1d). The body length of hatched larvae in 4.22 and 5.08 μg/L pyraoxystrobin-treated groups was not illustrated because embryos in these two concentrations hatched rarely or with a hatching rate of zero.

edema, yolk sac edema, growth retardation, pigmentation defect, and spine deformation. The most pronounced malformation caused by pyraoxystrobin was pericardial edema, which appeared visibly at a concentration of 2.90 μg/L of pyraoxystrobin at 72 hpf. Notably, a slight decrease in pericardial edema rate was observed at 96 hpf at lower concentrations (2.03–3.51 μg/L) compared with that at 72 hpf. However, the percentage of pericardial edema in 4.22 and 5.08 μg/L pyraoxystrobin-treated groups increased in a time-dependent manner, which reached approximately 60% and 70%, respectively, when compared with the control at 96 hpf (Fig. 3a). Another obvious malformation caused by pyraoxystrobin was yolk sac edema, which apparently occurred at a concentration of 3.51 μg/L at 96 hpf. In addition, yolk sac edema rate at 96 hpf reduced when compared with that at 72 hpf at higher concentrations of pyraoxystrobin. For instance, the percentage of yolk sac edema reduced from 90% at 72 hpf to 57% at 96 hpf at a concentration of 5.08 μg/L of pyraoxystrobin (Fig. 3b).

3.5. Teratogenic effects caused by pyraoxystrobin A series of morphological abnormalities (Fig. 2) were induced by pyraoxystrobin during embryonic development, including pericardial

Fig. 1. Developmental effects of pyraoxystrobin on zebrafish embryos. a. Number of spontaneous movements in 20 s at 24 hpf (n = 18 per concentration). b. Number of heartbeats in 20 s at 48 and 72 hpf (n = 18 per concentration). c. Hatching rate of embryos at 96 hpf (n = 60 per concentration). d. Body length of larvae at 96 hpf (n = 18 per concentration). Asterisks denote significant differences between treatment and control groups (determined by Dunnett post hoc comparison, *p < 0.05, **p < 0.01, ***p < 0.001). Error bars indicate standard deviation (SD).

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Fig. 2. Morphological effects of pyraoxystrobin on zebrafish embryos. a. Normal embryo at 24 hpf. b. c. Embryo with growth retardation(GR) at 24 hpf. d. Normal embryo at 48 hpf. e. Embryo with pericardial edema(PE) and yolk sac edema (YSE) at 48 hpf. f. Embryo with yolk sac edema (YSE) and pigmentation defect (PD) at 48 hpf. g. Normal embryo at 72 hpf. h. Embryo with yolk sac edema (YSE) at 72 hpf. i. Embryo with pericardial edema (PE) at 72 hpf. j. Normal hatched larvae at 96 hpf. k. Larvae with pericardial edema (PE) and spine deformation (SD) at 96 hpf. l. Larvae with pericardial edema (PE) at 96 hpf.

Fig. 3. Rate of pericardial edema (Pe) and yolk sac edema (Yse) caused by pyraoxystrobin (n = 3 replicates, with 20 embryos per replicate). a. Pericardial edema rate of zebrafish at 72 and 96 hpf. b. Yolk sac edema rate of zebrafish at 72 and 96 hpf. Asterisks denote significant differences between treatment and control groups (determined by Dunnett post hoc comparison, *p < 0.05, **p < 0.01, ***p < 0.001). Error bars indicate standard deviation (SD).

treated with 2 μg/L pyraoxystrobin but downregulated in the group treated with 4 μg/L pyraoxystrobin. However, the mRNA expression of Cat was significantly downregulated after exposure to 2 and 4 μg/L pyraoxystrobin, which was 40% and 80% lower than that in the control group, respectively. The mRNA levels of p53, Cas3, Bax, and Bcl2, which are involved in apoptotic signaling processes, decreased significantly after 96 h of exposure to pyraoxystrobin. Moreover, downregulation of p53 and Bax was observed in both 2 and 4 μg/L pyraoxystrobin-treated groups; however, the expression of Cas3 and Bcl2 was inhibited only in the 4 μg/L pyraoxystrobin-treated group (Fig. 4c). Pyraoxystrobin exposure also changed the expression of genes related to innate immune system (Fig. 4d). Pyraoxystrobin treatment at a concentration of 4 μg/L upregulated the mRNA levels of IL-1b and IFN significantly, with 2.2- and 3.8-fold increase, respectively, when compared with the control group. Meanwhile, the transcription level of TNFа was upregulated 1.5-fold in the 2 μg/L pyraoxystrobin-treated group, but decreased by 40% in 4 μg/L pyraoxystrobin-treated group when compared with that in the control.

3.6. Effects of pyraoxystrobin on gene expression To explore the toxic mechanisms of pyraoxystrobin on zebrafish embryos further, we examined the transcription of genes related to mitochondrial respiratory chain and ATP synthesis, oxidative stress, apoptosis, and innate immune system. The results showed that exposure to pyraoxystrobin significantly altered the mRNA expression of genes involved in mitochondrial respiratory chain and ATP synthesis. The transcription level of NDI and ATPo6 was markedly upregulated, with 2.2- and 1.6-fold increase, after exposure to 4 μg/L pyraoxystrobin, while the mRNA level of uqcrc in 2 and 4 μg/L pyraoxystrobin-treated groups decreased by 55% and 85% respectively, when compared with that in the control group. For COXI, no significant difference was observed after exposure to pyraoxystrobin (Fig. 4a). The expression of genes related to oxidative stress was significantly altered after exposure to pyraoxystrobin. As shown in Fig. 4b, a concentration-dependent upregulation of Mn-sod gene expression was observed upon exposure to 2 and 4 μg/L pyraoxystrobin, with 2.1- and 3.0-fold increase, respectively, relative to the control group. In addition, the mRNA level of Gpx was upregulated markedly in the group 5


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Fig. 4. Expression of mRNA of mitochondrial respiratory chain-, oxidative stress-, apoptosis- and immune-related genes in zebrafish embryos after exposure to pyraoxystrobin (n = 3 replicates). a. Relative mRNA level of NDI, uqcrc, COXI, ATPo6. b. Relative mRNA level of Mn-sod, Cat, Gpx. c. Relative mRNA level of p53, Cas3, Bax, Bcl2. d. Relative mRNA level of IL1b, IFN, TNFa. Asterisks denote significant differences between treatment and control groups (determined by Dunnett post hoc comparison, *p < 0.05, **p < 0.01, ***p < 0.001). Error bars indicate standard deviation (SD).

4. Discussion

in them because the adaptive immune system in zebrafish emerges from 4 to 6 weeks after fertilization (Novoa and Figueras, 2012). In this study, exposure to pyraoxystrobin caused several developmental abnormalities, such as hatching inhibition, decreased heart rates, growth inhibition, and morphological deformities. Among the malformations, the most pronounced morphological alterations were pericardial edema and yolk sac edema. Similar results have also been reported in zebrafish embryos exposed to environmental toxicants, such as cyhalofop-butyl (Zhu et al., 2015), difenoconazole (Mu et al., 2013), and azoxystrobin (Cao et al., 2016a). Heart is the first functional organ developed in zebrafish and heart rate is an important toxicology endpoint in fish embryonic test (Glickman and Yelon, 2002; OECD, 2006). The results of the present study showed that the heart rate of zebrafish embryos was significantly inhibited by pyraoxystrobin. Similar results were reported for other strobilurin fungicides (trifloxystrobin, azoxystrobin, and kresoxim-methyl) on grass carp juveniles (Liu et al., 2013), and pesticides, e.g. thifluzamide (Yang et al., 2016b), cyhalofop-butyl (Zhu et al., 2015), and imazalil (Jin et al., 2016), suggesting that alterations in heartbeat rate may be a common response in fish embryos after exposure to toxicants. Moreover, pyraoxystrobin exposure induced obvious pericardial edema in developing embryos, and the pericardial edema rate in the 5.08 μg/L treatment group reached up to approximately 70% at 96 hpf. This result indicates that the developing heart may be an important target for pyraoxystrobin toxicity in zebrafish. Enormous amount of ATP is needed to maintain a constant energy-consuming contractile state of cardiomyocytes, and the primary source of intracellular energy is mitochondria. Previous studies demonstrated that drugs, which directly interact with the electron transport chain, induced myocardial mitochondrial dysfunction and led to subsequent myocardial dysfunction (Kuzmicic et al., 2011; Varga et al., 2015). In the current study, exposure to pyraoxystrobin significantly increased the mRNA levels of NDI and ATPo6 and inhibited the expression of uqcrc, which are essential genes encoding mitochondrial complex I,V, and III, respectively,

In this study, the acute toxicity of pyraoxystrobin to different life stages of zebrafish was evaluated. The results clearly indicated that pyraoxystrobin has high toxicity to all the measured life stages of zebrafish. The 96-h LC50 value of pyraoxystrobin to adult zebrafish was 5.97 μg/L, which was similar to that reported by Chen et al. (2015). By comparing the 96-h LC50 values of pyraclostrobin, azoxystrobin, and picoxystrobin to adult zebrafish, which were 0.0635, 0.393, and 0.212 mg/L respectively, it was observed that pyraoxystrobin demonstrated a higher level of toxicity to zebrafish. Determining the most sensitive period would help protect aquatic organisms from external toxic interferences (Newman and Unger, 2002); thus, it is important to discover the most vulnerable stage via toxicity tests at multiple stages before the development of a scientific protection method. The early life stages of fish are generally more sensitive than adult stage when exposed to chemicals (Lele and Krone, 1996; Westernhagen, 1988), but there are also differences between fish embryos and larvae. Yang et al. (2016b) reported that thifluzamide showed higher toxicity to zebrafish embryos than to larvae, while Cao et al. (2016a) demonstrated that zebrafish larvae were more sensitive to cyhalofop-butyl than the embryos. As shown in the present study, the most sensitive stage of zebrafish to pyraoxystrobin is 12 hph larvae, followed by 72 hph larvae, embryo, and adult fish, suggesting that newly hatched larvae (12 and 72 hph larvae) were more sensitive to pyraoxystrobin than embryos. Similar result has also been reported by Mu et al. (2013), who found that zebrafish larvae were more sensitive to difenoconazole compared to embryos and adult fish. The possible reasons for the sensitivity of zebrafish larvae are as follows: first, unlike embryos, newly hatched larvae lack chorion, which serves as a protective barrier against exposure to waterborne chemicals (Embry et al., 2010); second, the development of detoxification mechanisms is incomplete in 12 and 72 hph larvae (Gomez-Requeni et al., 2010; Parichy et al., 2009); finally, 12 and 72 hph larvae only have innate immunity 6


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might have been caused by the flux of superoxide radicals that could inhibit CAT activity. Jin et al. (2015) reported that the activity of GPX decreased after exposure to higher concentrations of chlorpyrifos, which might have resulted from the depletion of other antioxidases. Ansari and Ansari (2014) suggested that the increase or decrease in enzyme activity is related to the intensity of cellular damage. Based on the results of the above-mentioned studies, we speculated that exposure to pyraoxystrobin, especially at higher concentrations, might cause severe cellular damage in zebrafish embryos, then resulting in excessive production of superoxide radicals and inhibition of Cat and Gpx. However, the sensitivity of different antioxidant enzymes to pesticides may be different. To confirm this speculation, further study is still needed at enzyme level. Apoptosis, the intrinsic cell death program, can achieve tissue homeostasis through targeted elimination of a single cell without disrupting the biological functionality of the tissue (Schattenberg et al., 2006). Previous researches have demonstrated that mitochondrion plays a vital role in the regulation of apoptosis (Fan et al., 2001). Mitochondrial dysfunction will disrupt cellular homeostasis (Green and Reed, 1998), which in turn would lead to the occurrence of diseases. For instance, apoptosis induction leads to organ insufficiency and apoptosis inhibition causes hyperplasia and cancer (Yazici et al., 2009). Most apoptotic signaling processes are related to alterations in apoptosis-related molecules, such as p53, Bcl-2/Bax, and cytochrome c, which then trigger caspase activation and subsequently induce cell apoptosis (Zhao et al., 2009). Recently, the changes in mRNA expression levels of apoptosis-related genes induced by environmental pollutants in zebrafish have been widely studied (Deng et al., 2009; Shi et al., 2008; Zeng et al., 2014). Zhu et al. (2015) reported that cyhalofop-butyl significantly induced the mRNA expression of p53, Puma, Cas3, and Cas9, and decreased the ratio of Bcl-2/Bax in zebrafish embryos after 96 h of exposure. This suggests that cyhalofop-butyl induced apoptosis in zebrafish embryos via activation of p53, which directly induced the transcription of genes that encode proapoptotic proteins (Puma, Bax), and then triggered the key executor of apoptosis (Cas3) activation and subsequently induced cell apoptosis and embryonic malformation. In the current study, the expression of p53, Cas3, Bax, and Bcl2 decreased by 77%, 47%, 73%, and 50% in zebrafish embryos, respectively, when compared with the control group after exposure to 4 μg/L pyraoxystrobin for 96 h, indicating that the normal process of apoptosis in zebrafish was disturbed by pyraoxystrobin. Innate immune system plays an important role in primary defense during the early life stages of a fish (Lam et al., 2004; Trede et al., 2004). The cytokines secreted by immune cells, including TNFа, IL-1b, and IFN, are important for regulating innate immune response (Sieger et al., 2009). Previous studies have demonstrated that oxidative stress and cell apoptosis triggered by environmental pollutants would affect the components of immune system (Gao et al., 2015; Livingstone, 2001; Yoo et al., 1997). Pesticides would affect the innate immune system by disturbing the mRNA expression of related genes in zebrafish (Jiang et al., 2016; Tu et al., 2013). However, no study has reported about the immunotoxicity of strobilurin fungicides on aquatic organisms to date. The results of the present study demonstrated that the mRNA expression levels of IFN, IL-1b, and TNFа were significantly upregulated by pyraoxystrobin at concentrations of 4, 4, and 2 μg/L, respectively. The mRNA expression levels of IL-1b and TNFа were downregulated by pyraoxystrobin at concentrations of 2 and 4 μg/L, respectively, indicating that pyraoxystrobin interfered with the innate immune responses in zebrafish. Previous studies indicated that the transcriptional alterations in innate immune related-genes caused by different pesticides might be different. For instance, the mRNA levels of TNFα, IFN, IL8, CXCL-C1C, CC-chem, and IL-1b in zebrafish larvae increased significantly after exposure to 100 or 300 μg/L chlorpyrifos for 96 hpf (Jin et al., 2015); however, the transcription of CXCL-C1C, IL-8, and IL-1b in zebrafish embryos decreased significantly after pretilachlor exposure (Jiang et al., 2016). The up/downregulation of genes might be a

suggesting that pyraoxystrobin might interfere with the electron transport chain and ATP synthesis in the mitochondria of zebrafish embryos. Yang et al. (2016b) reported that exposure to thifluzamide, an inhibitor of succinate dehydrogenase (SDH) synthesis, significantly altered the expression of mitochondrial complex-related genes, changed the activity of SDH, and finally led to mitochondrial structure damage and mitochondrial dysfunction. Thus, we speculate that pyraoxystrobin may interfere with the energy synthesis of cardiomyocytes in zebrafish embryos via breaking mitochondrial electron transport chain, which in turn would further induce cardiac dysfunction accompanied by the occurrence of pericardial edema, reduction of heart rate, and arrhythmia. To the best of our knowledge, this is the first study investigating the expression of genes involved in mitochondrial respiratory chain and ATP synthesis as potential mechanisms underlying the toxic effects of strobilurin fungicides in aquatic organisms. It should be noted that yolk sac edema rate of embryos in 4.22 and 5.08 μg/L pyraoxystrobin exposure groups, as well as pericardial edema rate in groups treated with lower concentrations (2.03–3.51 μg/L) decreased after zebrafish embryos hatched to larvae. Similar result has been reported by Fraysse et al. (2006), who found that the edemas of zebrafish embryos detected after treatment with 0.8, 1.5, 3, and 6 μM malathion at 48 hpf were observable only at the highest concentration at 80 hpf. The possible reason could be that some slight non-lethal malformations were alleviated by the enhanced immunity during development of zebrafish embryos. Mitochondrion is the major intracellular source of ROS (Turrens, 2003). Abnormal generation of ROS occurs when the mitochondria is impaired (Murphy, 2009), which can be considered as an important signal of oxidative damage (Barzilai and Yamamoto, 2004; Valko et al., 2007). The elevated levels of ROS can subsequently be eliminated by antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), which convert superoxide anions (O2-) into H2O2 and then into H2O and O2 (Valavanidis et al., 2006; Zhang et al., 2009). Therefore, examining the change in activities of antioxidant enzymes, such as SOD, CAT, and GPx, is considered an effective method for determining oxidative stress. Liu et al. (2013) demonstrated that trifloxystrobin, azoxystrobin, and kresoxim-methyl could weaken the antioxidant defense system and enhance oxidative stress through increasing CAT and peroxidase (POD) activity and decreasing SOD activity in grass carp juveniles. Although alterations in gene expression do not mean that the protein expression has been changed, the expression trend of genes is consistent with that of corresponding proteins in most cases (Jiang et al., 2016; Yang et al., 2016a). The expression of genes encoding antioxidant proteins has also been used to monitor the impact of chemical pollutants (Sheader et al., 2006; Woo et al., 2009). Jiang et al. (2016) reported that pretilachlor exposure could increase the activities of antioxidant proteins as well as the transcription of corresponding genes (e.g., SOD, CAT, GPX), suggesting that pretilachlor induced oxidative stress during zebrafish embryo development. The increase in transcription and accumulation of antioxidant enzymes might contribute to elimination of ROS induced by pretilachlor. In the current study, 2 and 4 μg/L pyraoxystrobin significantly induced the mRNA expression of Mn-sod, with 2.1- and 3.0fold increase, respectively, and 2 μg/L pyraoxystrobin induced the mRNA expression of Gpx with 1.4- fold increase, suggesting the two enzymes might be induced to higher contents or activities to eliminate the superoxide radicals caused by pyraoxystrobin. However, the transcription of Cat was markedly inhibited by both concentrations of pyraoxystrobin, while that of Gpx was inhibited at 4 μg/L of pyraoxystrobin. Similar results have also been reported in some previous studies (Ahmad et al., 2000; Ansari and Ansari, 2014; Crestani et al., 2007; Kubrak et al., 2010; Liu et al., 2008). Ansari and Ansari (2014) indicated that the activity of CAT in zebrafish decreased significantly in a dose-dependent manner after exposure to dimethoate, and they suggested that the reduced CAT activity might stem from the decrease in reaction rates resulting from the excess production of H2O2, which 7


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potential mechanism of zebrafish to adapt to the different stimuli induced by exogenous toxicants. Further study is necessary to investigate the mechanisms underlying the different changes in immune-related genes.

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5. Conclusions In summary, our study confirmed that pyraoxystrobin exhibits high acute toxicity to multiple stages of zebrafish, among which the 12 hph larvae was the most sensitive period. Negative effects were observed during embryonic development, indicating developmental toxicity induced by pyraoxystrobin. These results suggested that the application of pyraoxystrobin even at a low dose might pose a risk to fishes in aquatic environment. The results of gene expression test suggested that alterations in the mRNA levels of genes related to mitochondrial respiratory chain and ATP synthesis, oxidative stress, apoptosis, and immune system might be the potential mechanisms underlying the toxic effects of pyraoxystrobin to zebrafish during embryogenesis. However, further research is warranted to elucidate the underlying toxic mechanisms of pyraoxystrobin to zebrafish. Acknowledgments The present study was financially supported by National Key R&D Program of China (Grant no. 2017YFD0200504). Conflict of interest The authors declare that they have no conflict of interest. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2017.12.061 References Ahmad, I., Hamid, T., Fatima, M., 2000. Induction of hepatic antioxidants in freshwater catfish (Channa punctatus Bloch) is a biomarker of paper mill effluent exposure. Biochim. Biophys. Acta 1523, 37–48. Ansari, S., Ansari, B.A., 2014. Temporal variations of CAT, GSH, and LPO in gills and livers of zebrafish, Danio rerio, exposed to dimethoate. Arch. Pol. Fish. 22, 101–109. Bartlett, D.W., Clough, J.M., Godwin, J.R., et al., 2002. The strobilurin fungicides. Pest Manag. Sci. 58, 649–662. Barzilai, A., Yamamoto, K., 2004. DNA damage responses to oxidative stress. DNA Repair 3, 1109–1115. Cadenas, E., Davies, K.J.A., 2000. Mitochondrial free radical generation, oxidative stress, and aging. Free Radic. Biol. Med. 29, 222–230. Cao, F., Liu, X., Wang, C., et al., 2016a. Acute and short-term developmental toxicity of cyhalofop-butyl to zebrafish (Danio rerio). Environ. Sci. Pollut. Res. Int. 23, 10080–10089. Cao, F., Zhu, L., Li, H., et al., 2016b. Reproductive toxicity of azoxystrobin to adult zebrafish (Danio rerio). Environ. Pollut. 219, 1109–1121. Chen, L., Liu, J., Si, N., 2011. Mixture of pyraoxystrobin and epoxiconazole against rice blast. Agrochemicals 50, 759–772. Chen, L., Song, Y., Tang, B., et al., 2015. Aquatic risk assessment of a novel strobilurin fungicide: a microcosm study compared with the species sensitivity distribution approach. Ecotoxicol. Environ. Saf. 120, 418–427. Crestani, M., Menezes, C., Glusczak, L., et al., 2007. Effect of clomazone herbicide on biochemical and histological aspects of silver catfish (Rhamdia quelen) and recovery pattern. Chemosphere 67, 2305–2311. Deng, J., Yu, L., Liu, C., et al., 2009. Hexabromocyclododecane-induced developmental toxicity and apoptosis in zebrafish embryos. Aquat. Toxicol. 93, 29–36. Embry, M.R., Belanger, S.E., Braunbeck, T.A., et al., 2010. The fish embryo toxicity test as an animal alternative method in hazard and risk assessment and scientific research. Aquat. Toxicol. 97, 79–87. Fan, T.J., Xia, L., Han, Y.R., 2001. Mitochondrion and apoptosis. Acta Biochim. Biophys. Sin. 33, 7–12. Fraysse, B., Mons, R., Garric, J., 2006. Development of a zebrafish 4-day embryo-larval bioassay to assess toxicity of chemicals. Ecotoxicol. Environ. Saf. 63, 253–267. Gao, H., Wang, D., Zhang, S., et al., 2015. Roles of ROS mediated oxidative stress and DNA damage in 3-methyl-2-quinoxalin benzenevinylketo-1, 4-dioxide-induced immunotoxicity of Sprague-Dawley rats. Regul. Toxicol. Pharmacol. 73, 587–594. Glickman, N.S., Yelon, D., 2002. Cardiac development in zebrafish: coordination of form

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