Effects of penthiopyrad on the development and behaviour of zebrafish in early-life stages

Chemosphere 214 (2019) 184e194

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Effects of penthiopyrad on the development and behaviour of zebrafish in early-life stages Le Qian a, Suzhen Qi b, Fangjie Cao a, Jie Zhang a, Changping Li c, Min Song d, Chengju Wang a, * a

College of Sciences, China Agricultural University, Beijing, People's Republic of China Risk Assessment Laboratory for Bee Product Quality and Safety of Ministry of Agriculture, Institute of Agricultural Research, Chinese Academy of Agricultural Sciences, Beijing, 100093, People's Republic of China c Plant Protection Station, Beijing, People's Republic of China d Institute of Agricultural Research, Taian, Shandong, People's Republic of China b

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


Penthiopyrad has been widely used, but its toxicological assessment is lacking.
Penthiopyrad had an acute toxicity to zebrafish in the early-life stages.
Penthiopyrad induced a series of malformations in embryos and larvae.
Penthiopyrad inhibited behaviours of larvae under sub-lethal exposure.
Penthiopyrad caused the disorder of lipid metabolism and melanin deposition.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 July 2018 Received in revised form 17 September 2018 Accepted 18 September 2018 Available online 19 September 2018

The agricultural use of succinate dehydrogenase inhibitor (SDHI) fungicides has increased dramatically in the US and Europe. As the SDHI fungicides, boscalid, flutolanil and thifluzamide had been reported to induce a series of toxic effects on zebrafish. However, the toxic effects of penthiopyrad on zebrafish have not been reported yet. This study aimed to assess the acute toxicity of penthiopyrad to zebrafish in earlylife stages and investigate behavioural response of larvae and the effects on lipid metabolism and pigmentation under sub-lethal exposure of penthiopyrad. Based on results of the acute toxicity tests of zebrafish embryo and larvae, penthiopyrad had an acute toxicity to early-life stages of zebrafish and induced a series of deformities during development. Based on the results of sub-lethal exposure for 8 days, penthiopyrad resulted in significant decreases in swimming velocity, acceleration speed, distance moved and inactive time of larvae at 0.3, 0.6 and 1.2 mg/L. Penthiopyrad induced the disorders of lipid metabolism via affecting fatty acid synthesis and b-oxidation, in accordance with remarkable changes in the content of triglycerides and cholesterol and the expression of key genes (hmgcra, ppara1, srebf1, cyp51 and acca1) at 1.2 mg/L. In addition, the disorder of melanin synthesis and distribution was caused by penthiopyrad in larvae in accordance with changes in body colour and related gene expression at 8 dpe. © 2018 Elsevier Ltd. All rights reserved.

Handling Editor: David Volz Keywords: Penthiopyrad Toxicity Behaviour Lipid Melanin

* Corresponding author. College of Science, China Agricultural University, No. 2 Yuan mingyuan West Road, Haidian District, Beijing, 100193, People's Republic of China. E-mail address: [email protected] (C. Wang). https://doi.org/10.1016/j.chemosphere.2018.09.117 0045-6535/© 2018 Elsevier Ltd. All rights reserved.

L. Qian et al. / Chemosphere 214 (2019) 184e194

Abbreviations SDHI EFSA EPA DT90 LD50/LC50 LOEC ISO HPLC Hpf OECD Dpe TG TC qPCR ANOVA SD Pe Yse

Succinate dehydrogenase inhibitor European Food Safety Authority U.S. Environmental Protection Agency Time of 90% degradation Half lethal dose/concentration Lowest observed effect concentrations International Organisation for Standardisation High-performance liquid chromatography Hours post-fertilisation Organisation for Economic Co-operation and Development Days post-exposure Total of triglycerides Total of cholesterol Quantitative real-time polymerase chain reaction Analysis of variance Standard deviation Pericardial oedema Yolk sac oedema

1. Introduction Succinate dehydrogenase inhibitor (mitochondrial complex II or succinate-ubiquinone oxidoreductase, SDHI) fungicides play an important role in the prevention of many plant diseases, which inhibit fungal respiration by specifically blocking the ubiquinonebinding sites in mitochondrial complex II. Over the past decade, the agricultural use of SDHI fungicides has increased dramatically in the US and Europe (Wu et al., 2018), especially boscalid, fluxapyroxad, flutolanil, thifluzamide and penthiopyrad (Qiu and Bai, 2015). Increased authorisations of SDHI fungicides on national and European Union levels will be accompanied by increased usage of these compounds and their applications on different crops. Consequently, these fungicides are likely to be frequently detected in aquatic ecosystem and pose a potential risk to aquatic organisms in the future. Previous reports have shown that flutolanil and boscalid were frequently detected in the environment, especially in ~ asco et al., 2008; Tanabe and Kawata, 2009; Tsuda et al., water (An 2009). According to existing reports, flutolanil was frequently detected in Lake Kojima and Drainage canal (Japan) with the ~ asco et al., maximum concentrations of 30.3 and 1.64 mg/L (An 2009). Boscalid was one of the most frequently detected pesticides (in greater than 90% of the samples) in 3 main coastal estuaries in California, USA, with the maximum detection concentration of 36 mg/L (Vu et al., 2016). As one of the most popular SDHI fungicides, penthiopyrad [(RS) eN-[2-(1,3-dimethylbutyl)-3-thienyl]-1-methyl-3-(trifluoromethyl) pyrazole-4-carboxamide] was developed by Mitsui Chemicals (Japan) and has been authorised for use in the US and Canada (EFSA, 2012). Penthiopyrad is a carboxamide fungicide that is active against grey mold, powdery mildew and apple scab and its physico-chemical properties are shown in Table S1 (http://sitem. herts.ac.uk/aeru/iupac/Reports/12-10.htm). Based on the environmental fate and ecological risk assessment of penthiopyrad from Environmental Protection Agency (EPA) (EPA, 2011), penthiopyrad is characterized as a non-volatile, lipophilic compound that is moderately mobile in soil with the maximum DT90 values of 169 days in the field studies and over 1000 days in the laboratory studies (EFSA, 2012). Penthiopyrad, as a soil drench, effectively controlled Rhizoctonia solani on sugar beet when applied at 210,

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280, 420, or 550 g a.i./ha in-furrow at planting, which might increase the residue of penthiopyrad in the soil and the risk of entering surface and ground water via leaching and runoff of dissolved or sorbed residues (Meyer and Hausbeck, 2013; Liu and Khan, 2016). Penthiopyrad was detected four times in brood (reaching 115.3 mg/kg of brood), which indicated that it might occur in significant quantities in pollen as pollen is fed to the brood (Piechowicz et al., 2018). In addition, penthiopyrad has potentially direct chronic effects on mammals and direct adverse effects on listed freshwater fish, estuarine/marine fish and invertebrates via spray drift (EPA, 2011). Penthiopyrad is highly toxic for freshwater fish, with 96 h-LC50 value of 290 mg/L for fathead minnow (Pimephales promelas), 386 mg/L for rainbow trout (Oncorhynchus mykiss) and 572 mg/L for common Carp (Cyprinus carpio). However, the toxic mechanism of penthiopyrad on fish has not been reported yet, especially zebrafish embryo and larvae. Moreover, boscalid, flutolanil and thifluzamide had been reported to induce a series of toxic effects on the development of zebrafish (Yang et al., 2016a, 2016b; Qian et al., 2018). Penthiopyrad could have the similar toxic effects on zebrafish due to the similar mode of action. Behavioural changes provide a unique toxicological perspective to study environmental pollutant effects, as it links both biochemical and ecological consequences (Scott and Sloman, 2004), which are widely used biomarkers at the individual level and have been proposed as a sensitive index of sublethal toxicity of environmental pollutants (Warner, 1967). Considering that many ecologically relevant behaviours of fish are easily observed and quantified in a controlled setting, fish is an excellent model to study behavioural changes (Atchison et al., 1987). Changes in fish behaviour have been applied as indicators for ecologically relevant monitoring of environmental pollutants in some studies (Little and Finger, 1990; Gerlai et al., 2006; Nusser et al., 2016; Cambier et al., 2018). Zebrafish are one of the most favoured and representative vertebrate model organisms due to their higher sensitivity in earlylife stages and ability to swim in 4e5 dpf (Kimmel et al., 1995; Robertson et al., 2007). The impact of pollutants on the swimming behaviour of zebrafish in early-life stages has become the focus of environmental toxicology research (Nusser et al., 2016; Cambier et al., 2018). In addition, zebrafish have been also applied in genetics, developmental biology, neurophysiology and biomedicine due to their small size, high fecundity and rapid external development and toxicant-sensitivity (Ali et al., 2011). The purpose of this study was to investigate the toxic effects of penthiopyrad on the early-life stage of zebrafish. In the acute toxicity tests, the LC50 values of embryo and larvae were determined. The developmental indicators of embryo and larvae were recorded, including spontaneous movement, heartbeat, the development of yolk sac and pericardium, hatching rate, swim bladders and pigmentation. According to the apparent abnormities in the acute toxicity tests, an 8-d sub-lethal developmental toxicity test was carried out to further explore the developmental toxicity of zebrafish in early-life stages. The lowest observed effect concentration (LOEC) values of penthiopyrad to zebrafish embryo and sacfry stages were calculated. The locomotor behaviours of hatched larvae were recorded after embryos exposed to 5, 6, 7 and 8 days. In addition, lipid metabolism and melanin synthesis and distribution were also studied. Our findings reflect the potential risk of penthiopyrad on aquatic organisms and provide a foundation for further study of its toxic modes of action. 2. Materials and methods 2.1. Chemicals and reagents Penthiopyrad

(99.5%

purity)

was

provided

by

Mitsui

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Chemicals Agro Corporation (Japan). A stock solution of penthiopyrad (50,000 mg/L) was prepared in acetone and kept at 4  C in dark condition and diluted appropriately for each experiment. The reconstituted water considered as a standard solution for embryolarvae tests was prepared in our laboratory with the formula of iso7346-3, which contained 2 mmol/L of Ca2þ, 0.5 mmol/L of Mg2þ, 0.75 mmol/L of Naþ and 0.074 mmol/L of Kþ (ISO, 1996). All other reagents were of analytical grade and were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 2.2. Analysis of exposure solutions All test solutions were renewed every 24 h to maintain the appropriate concentration of penthiopyrad and water quality. Two millilitres of water samples were added into 2 mL of acetonitrile and 1.0 g of sodium chloride, then extracted with a vortex mixer for 10 min and centrifuged at 5000 g at room temperature for 5 min. After this, 1 mL of organic solvent phase was filtered with a 0.22-mm organic membrane filter and analysed by high-performance liquid chromatography (HPLC). Separations were carried out on a column (4.6 mm  250 mm  5 mm, Inert Sustain C18). The mobile phase consisted of 25% purified water/0.1% formic acid aqueous solution (v/v) and 75% acetonitrile (v/v). The detector operating temperature was 35  C, the detection time was 15 min, and the detection wavelength was 230 nm. 2.3. Zebrafish maintenance Healthy juveniles of AB strain zebrafish (Danio rerio) were obtained from Beijing Hongda Gaofeng Aquarium Department (Beijing, China) and cultured in the fish facility (Esen Corp. China) at 28 ± 1  C with a photoperiod of 14/10 (light/dark). The adult zebrafish were fed with live brine shrimp three times a day. Embryos were obtained within 2 h of natural spawning of healthy adults with a ratio of 1:2 (female to male). Healthy developing embryos were identified under a microscope and larvae were selected for the subsequent tests after 72 h post fertilisation (hpf). 2.4. Acute toxicity tests of penthiopyrad to embryo and larvae Acute toxicity tests of penthiopyrad to embryo were carried out according to the Organisation for Economic Co-operation and Development (OECD) guidelines (OECD, 2013). Zebrafish embryos of approximately 2 hpf (during the 16-cell stage) were randomly transferred into 2 mL of test solutions with concentrations of 0, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 and 2.9 mg/L penthiopyrad in 24-well plates. The health status of embryos was recorded during the exposure period, including spontaneous movement, heartbeat, development of yolk sac and pericardium and hatching rate. Morphological development was recorded by fluorescence microscope (Axio Vert.A1 microscopes, Germany) and the area of yolk sac oedema was measured using a dissecting microscope (Olympus BH-2) with a digital viewer (Aigo GE-5, China). Acute toxicity test of penthiopyrad to larvae was conducted following the method mentioned in the previous study (Mu et al., 2013). Seventy-two hours after hatching, larvae were exposed to 100 mL test solutions, which contained concentrations of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 and 2.6 mg/L of penthiopyrad in a 200-mL beaker. Malformation and mortality of larvae were recorded at 24, 48, 72 and 96 h to calculate the values of LC50. Both blank and solvent control (0.01% acetone in water, v/v) were arranged in the embryo and larvae toxicity tests. Each group consisted of three replicates. The exposure time was 4 days. All test solutions were renewed every 24 h, and dead eggs or larvae were immediately removed. The experiments were performed in this

study in accordance with current Chinese legislation and were approved by the independent animal ethics committee at China Agricultural University. 2.5. The sub-lethal developmental toxicity test 2.5.1. Short-term developmental toxicity test of penthiopyrad to embryo and sac-fry stages A 4-d developmental toxicity test of penthiopyrad to embryo was carried out with the concentrations of 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8 mg/L. Sub-lethal concentrations were set on the basis of penthiopyrad-LC50, without causing death. Sub-lethal effects on embryo and sac-fry stages induced by penthiopyrad were recorded, including spontaneous movement inhibition, heartbeat reduction, hatching inhibition, and body length reduction. In addition, LOEC values of penthiopyrad to zebrafish embryo and sac-fry stages were calculated. 2.5.2. Exposure experiment for assessment of behaviour Based on the previous experiment with slight modification (OECD, 1998; Nusser et al., 2016; Cambier et al., 2018), healthy embryos (approximately 2 hpf) were randomly transferred into 24well plates with 2 mL of each test solutions. The exposure concentrations were 0, 0.3, 0.6 and 1.2 mg/L of penthiopyrad, which were set on the basis of the 1/8, 1/4, 1/2 of 96-LC50 value for embryo, without causing death. Both blank and solvent control (0.01% acetone in water, v/v) were arranged. The exposure time was 8 days. The test solutions were changed every 24 h. Video recording of larvae was conducted using a USB 3.0 colour video camera with a e2v CMOS sensor (UI-3240CP-C-GL, IDS Imaging Development Systems GmbH, Obersulm, Germany). The locomotor activity was continually measured for 15 min under constant darkness at 5, 6, 7 and 8 days post-exposure (dpe). Video recordings were analysed using LoliTrack Version 4.2.0 software (Loligo Systems, United States). 2.5.3. Determination of body length and sample collection In addition, the body length of the hatched larvae was measured by an Aigo GE-5 digital microscope (Aigo, Beijing, China), and 25 larvae from each beaker were collected for the measurement of total triglyceride (TG) and cholesterol content (TC) at 4 and 8 dpe. Similarly, another 25 larvae from each beaker were collected for RNA extraction. All samples were stored at 80  C for use. 2.6. Gene expression analysis Total RNA was extracted from sampled larvae by an RNAprep pure tissue kit (Tiangen Biotech, China). The purity of the RNA was assessed based on the ratio of A260/A280, and the concentration was determined to be A260 by spectrophotometer (DeNOVIX, DS-11). The synthesis of first-strand complementary DNA (cDNA) and performance of quantitative real-time polymerase chain reaction (qPCR) were based on the previous reports (Zhu et al., 2015; Qian et al., 2018). Relative quantification of the target gene normalised by b-actin was performed by the 2DD Ct method. The stability of bactin after penthiopyrad exposure was validated using Bestkeeper (Figs. S1 and S2). The most specific and efficient primer sequences are cited in other studies (Deng et al., 2009; Jin et al., 2010; Mu et al., 2015; Yin et al., 2016), and the sequences are provided in Table S2. 2.7. Statistical analysis All statistical analyses were undertaken using SPSS 17.0 software (SPSS, USA). Differences were determined by one-way analysis of

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variance (ANOVA), followed by Dunnett's post hoc comparison, and p < 0.05 was considered a significant difference. Values are shown as the mean ± standard deviation (SD). Behavioural profiles were analysed by LoliTrack Version 4.2.0 software (Loligo Systems, United States). 3. Results 3.1. Solvent effect and chemical analysis There was no significant difference in all test indicators between the solvent and the blank control. Thus, only the solvent control was used for the expression and discussion of results. Analysis results indicated that the deviations between nominal and actual concentrations of penthiopyrad were less than 20% (Table S3), and the theoretical concentrations could be used in the following sections. 3.2. Lethal effects of penthiopyrad on zebrafish early-life stage 3.2.1. LC50 values of penthiopyrad According to Table S4, the 96 h-LC50 values (95% CL, mg/L) was 2.77 (2.73e2.82) for embryo with regression equation y ¼ 55.810x24.690 (R2 ¼ 0.987), and 2.38 (2.32e2.42) for larvae with regression equation y ¼ 27.810x-10.46 (R2 ¼ 0.976). 3.2.2. Developmental toxicity of penthiopyrad to embryo and larvae Under the condition of acute toxicity exposure test, penthiopyrad induced yolk sac oedema (Yse) and pericardial oedema (Pe) (Fig. 1A and B) in embryos. The area of Yse was elevated with the

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increase of penthiopyrad concentration and exposure time (Fig. 1C). The number of spontaneous movements increased remarkably at 2.5, 2.6 and 2.7 mg/L penthiopyrad but obviously decreased at 2.9 mg/L (Fig. 2A). The hatching rate of embryo was recorded from 24 to 96 hpf, and a reduction in the hatching rate of embryo was observed in all the treated groups at 72 and 96 hpf (Fig. 2B). In addition, heart beat was significantly inhibited (Fig. 2C) and the rate of Yse and Pe increased in all treated groups at 48, 72 and 96 hpf (Fig. 2D). In addition to the lethal effect of penthiopyrad on larvae, it induced 4 apparent abnormities in larvae, including Yse, Pe, pigmentation and failure of swim bladder inflation. The most striking malformation was observed in the yolk sac of larvae after 48 and 72 h (Fig. 3) exposure. Obvious Pe was observed in larvae in the 2.0, 2.1, 2.2 and 2.5 mg/L penthiopyrad groups. Additionally, longitudinal microscopic observation on the head and abdomen of larvae showed that normal larvae were transparent with a clearly visible internal structure, while notable pigmentation appeared in the head and abdomen of the larvae in the treated groups. Moreover, penthiopyrad could also affect swim bladder inflation of larvae in the acute toxicity test. Larvae in all the treated groups showed failure of the swim bladder to inflate at 48 and 72 h exposure. At 48 h, failure of swim bladder inflation and notable melanin deposition appeared in the head and abdomen (B-H); Yse (C-H) and Pe (B-E and G-H); At 72 h, failure of swim bladder inflation (C-H) and notable melanin deposition appeared in the head and abdomen (D-H); Yse (C-H) and Pe (B-E and G) (blue arrows for swim bladder; black arrows for pigmentation; red asterisk for Pe; red circles for Yse) (100  ).

Fig. 1. Yolk sacs of embryos at 48 and 72 hpf. A: Embryo in control group (Control-A) with normal yolk sac; embryos exposed to 2.3 (A-1), 2.6 (A-2) and 2.9 (A-3) mg/L penthiopyrad; Embryos were observed 48 h after exposure. B: Embryo in control group (Control-B) with normal yolk sac; embryos exposed to 2.3 (B-1), 2.6 (B-2) and 2.9 (B-3) mg/L penthiopyrad. Embryos were observed 72 h after exposure. C: Area of yolk sac oedema caused by penthiopyrad (2.0, 2.6 and 3.2 mg/L) at 48 h and 72 hpf. The asterisks indicate significant differences from the control group (determined by Dunnett post-hoc comparison, p < 0.05*; p < 0.01**).

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Fig. 2. Effects of penthiopyrad on embryonic development of zebrafish. A: The number of spontaneous movements at 24 hpf. B: Rate of yolk Yse and Pe caused by penthiopyrad at 48 and 72 hpf. The asterisks indicate significant differences from the control group. C: Heartbeat of zebrafish in all treatments at each observation time point. D: The hatching rate at each observation time point. Asterisks denote significant difference between the treatment and control groups (determined by Dunnett's post-hoc comparison, p < 0.05*; p < 0.01**).

3.3. Sub-lethal developmental toxicity of penthiopyrad in early-life stages of zebrafish 3.3.1. LOEC values of penthiopyrad to zebrafish embryo and sac-fry stages Based on the results of statistical analysis, the LOEC values of penthiopyrad to spontaneous movement of zebrafish embryos were 2.5 mg/L (Table S5). The LOEC values of penthiopyrad to yolk sac and pericardium at 72 hpf were 0.9 and 0.6 mg/L, respectively. Penthiopyrad could induce an observed effect on the heartbeat of zebrafish in the sac-fry stage at 96 hpf at 0.9 mg/L and had no observed effect at 0.6 mg/L or lower concentrations. Penthiopyrad induced obvious changes in hatching rate at 1.8 mg/L. The LOEC value of penthiopyrad to melanin deposition was 0.9 mg/L. The LOEC value of penthiopyrad to body length of the surviving larvae was 0.6 mg/L.

3.3.2. Effect of penthiopyrad on the swimming behaviour of zebrafish in early-life stages During the 8 days of penthiopyrad exposure, the locomotor activities of larvae displayed remarkably different responses in different groups at 5, 6, 7 and 8 dpe (Fig. 4). From the results, penthiopyrad significantly inhibited the average velocity and showed a significant concentration dependent effect in the 0.3, 0.6 and 1.2 mg/L penthiopyrad groups after 6, 7 and 8 days of exposure (Fig. 4A). Similarly, the average acceleration of larvae was also inhibited by penthiopyrad after 6, 7 and 8 days of exposure (Fig. 4B), as well as distance moved (Fig. 4C). Additionally, the response for active time of larvae exposed to penthiopyrad was significantly lower relative to the control group at 5, 6, 7 and 8 dpe (Fig. 4D).

3.3.3. Effects of penthiopyrad on body length, cholesterol and triglycerides As shown in Fig. 5A, penthiopyrad significantly inhibited the body length of larvae in a concentration-dependent manner. The content of cholesterol and triglycerides increased by 1.2 mg/L penthiopyrad at 4 dpe (Fig. 5B and C), and there were no obvious changes in any of the treated groups at 8 dpe. 3.3.4. Effects of penthiopyrad on the expression of genes related to lipid metabolism and melanin At 4 dpe, exposure to increasing concentrations of penthiopyrad resulted in an increase in expression of srebf1, with 1.74-fold at 0.3 mg/L, 1.81-fold at 0.6 mg/L and 1.68-fold at 1.2 mg/L, relative to the control (Fig. 6A). Consistently, expression of acca1 was increased by 1.53-fold in the 0.6 mg/L and 1.60-fold in the 1.2 mg/L treated groups. Expression of cyp51 was upregulated by 1.29-fold in the 1.2 mg/L treated group, whereas expression of hmgcra and fas were significantly downregulated by 0.57-fold in the 0.6 mg/L and 0.71-fold in the 1.2 mg/L treated group, respectively. Gene transcription of ppara1 was not significantly affected at all test concentrations. Transcription level of hmgcra and srebf1 was significantly inhibited after 8 days of exposure to 0.6 and 1.2 mg/L penthiopyrad (Fig. 6B). Significant downregulation of ppara1, fas and acca1 was observed in the 1.2 mg/L treated group with 0.14-, 0.16- and 0.20fold, respectively. Obvious up-regulation of ppara1 was additionally observed in 0.6 mg/L penthiopyrad group with 0.14-fold change. No significant alteration in the expression of cyp51 occurred in any treatment groups. As shown in Fig. 7, downregulation of the mc1r was observed in a concentration-dependent manner after exposure to penthiopyrad for 8 days, with down regulation of 0.64-, 0.80- and 0.35-fold in the

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0.3, 0.6 and 1.2 mg/L groups relative to the control group, respectively. As for the expression of slc24a5 and pkc-b, significant alteration occurred in the 0.3 and 1.2 mg/L treated groups, with upregulation of 1.71- and 1.30-fold for 0.3 mg/L and downregulation of 0.55- and 0.43-fold for 1.2 mg/L. The mRNA expression of asip and foxd3 were significantly upregulated at 0.6 mg/L with 1.37-fold but 1.38-fold and down-regulated at 1.2 mg/L with 0.45fold and 0.41-fold. Transcription levels of myosin-Va, kita and tyrp1a were significantly inhibited at 1.2 mg/L of penthiopyrad. Transcription level of mitfa increased at 0.6 and 1.2 mg/L with 1.48fold and 1.39-fold. The mRNA expression of tyrp1b decreased at 0.3 mg/L penthiopyrad. There was no obvious change in the expression of dct in any of the treated groups. 4. Discussion

Fig. 3. The external morphological images of larvae after exposure for 48 h (A) and 72 h (B). A1/A2: Embryos in control group with normal pericardium, yolk sac, gas bladder and pigmentation at 48 and 72 h (100  ). BeH: Abnormities in larvae caused by penthiopyrad at 48 and 72 h.

The early-life stages of zebrafish, mainly including embryo and larvae, are popular experimental models in aquatic toxicological studies. The embryogenesis of zebrafish is usually completed within 72 hpf, discrete organs and tissues are developed by 120 hpf (He et al., 2014). Zebrafish embryos usually hatch between 48 and 72 hpf at 28.5  C (Hwang and Chou, 2013). The sensitivity of zebrafish in early-life stages to the effects of environmental pollutants is more easily exerted, as important processes in the development of body are occurring (Guo et al., 2018). Besides, due to the differences between embryo and larvae in the development of discrete organs and tissues, the sensitivity to pollutants might be distinct. According to the previous study, the lethal sensitivity (96 h-LC50 value) sequence of the zebrafish caused by difenoconazole during three stages was larvae (1.17 mg/L) > adult fish (1.45 mg/ L) > embryo (2.34 mg/L) (Mu et al., 2013). However, in our study, the lethal sensitivity sequence of the zebrafish caused by penthiopyrad was embryo (2.77 mg/L) > larvae (2.38 mg/L). Morphological techniques play an important role in the evaluation of the effects of pollutants on the development of organisms

Fig. 4. Effect of penthiopyrad exposure on swimming activity of larvae at 5, 6, 7 and 8 dpe. A: Average velocity; B: Average acceleration; C: Distance moved; D: Active time (%). Asterisks denote significant difference between the treatments and control (determined by Dunnett's post-hoc comparison, p < 0.05*; p < 0.01**).

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Fig. 5. Effects of penthiopyrad on body length, cholesterol and triglycerides. A: Changes in body length of the hatched larvae of control and penthiopyrad-treated groups at 4 and 8 dpe. B and C: The content of TG (B) and TC (C) of the control and penthiopyrad-treated groups at 4 and 8 dpe. The asterisks indicate significant differences from the control group (determined by Dunnett's post-hoc comparison, *p < 0.05; **p < 0.01).

in early-life stages. Many environmental model organisms, such as Daphnia magna and zebrafish, whose embryos are fully transparent, can be used to observe morphological changes caused by pollutants directly or microscopically (Mu et al., 2013; Toumi et al., 2015). In the present study, the adverse effects of penthiopyrad on the development of zebrafish in early-life stages were investigated by morphological techniques. From these results, penthiopyrad also caused developmental effects of zebrafish in early-life stage, such as hatching inhibition, abnormal spontaneous movement, slow heartbeat and growth regression. In addition, penthiopyrad resulted in pronounced morphological deformities, Pe and Yse in embryo, and the disorders in synthesis and distribution of melanin and

uninflated gas bladders of larvae. The same malformation has been reported in zebrafish embryos exposed to environmental toxicants, such as carbendazim, 2,4-dinitrotoluene (Schmidt et al., 2016), perfluorinated alkylated substances (Giari et al., 2015) and hexabromocyclododecane (Hong et al., 2017). Among these malformations, heart and swim bladders in earlylife stages of zebrafish were the main organs affected by penthiopyrad. The heart is the first organ to develop and function in vertebrate organisms (Wang et al., 2006). Heart formation is a complex, dynamic and highly coordinated process of molecular, morphogenetic and functional factors with each interacting and contributing to the formation of the mature organ (Matrone et al., 2015). Correspondingly, heart malformations by pollutants can result in abnormal development or even death. Significant heart malformation and dysfunction induced by acrylamide in zebrafish embryos resulted in a deficient cardiovascular system (Huang et al., 2018). Heart malformation induced by 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) in embryonic zebrafish led to functional deficits in the developing hearts, including blood regurgitation and a striking ventricular standstill (Antkiewicz et al., 2005). The gas bladder, as a specific organ in teleosts, has important functions in the abdomen that protects other visceral organs from injury by external hydraulic pressure and provides oxygen when the fish is in an anoxic condition (Li et al., 2011; Yamashita-Ichimura et al., 2018). It is also crucial for survival in most fish species because it minimises the energy required to maintain the vertical position in the water column. Previous studies have shown that environmental pollutants could cause developmental malformation in gas bladder (de Oliveira et al., 2016; Iwasaki et al., 2017). The gas bladder has been widely applied as an indicator in many environmental risk assessments (Li et al., 2011; Yue et al., 2015; Stinckens et al., 2016). According to the apparent abnormities in larvae, including Yse, Pe, pigmentation and failure of swim bladder inflation by penthiopyrad which might be related to behaviour, lipid metabolism and pigmentation, therefore, a futher study related these endpoints were carried out in our study. Locomotor activity is used extensively as a quantitative endpoint for measuring behavioural toxicity in the zebrafish (Kluver et al., 2015; Nusser et al., 2016; Velki et al., 2017). Locomotor activity of zebrafish larvae exposed to penthiopyrad was evaluated in our study. Results showed that penthiopyrad significantly inhibited the behaviour of larvae, including average velocity, average acceleration and distance moved. Previous results have shown that swimming activity of Daphnia magna was a measurable effect and a sensitive biological endpoint induced by increasing toxicity and temperature stress (Bahrndorff et al., 2016). Behavioural swimming effects were studied in Jenynsia multidentata exposed to chlorpyrifos and cypermethrin individually and in mixtures (Bonansea et al., 2016). For aquatic organisms, swimming ability is the most important part of behavioural research. The swimming ability can be evaluated from many aspects, and the most commonly used indicator is the swimming speed. In addition, the previous studies have shown that developmental toxicity could lead to abnormal or dysfunctional behaviours (Velki et al., 2017). Correspondingly, changes in the gas bladder were found in our study, which could have a direct impact on the swimming ability of zebrafish. Lipid metabolism plays an important role in the development of zebrafish in early-life stages, providing the nutrients needed to sustain their growth and survival and involving the body's energy supply and storage, biofilm composition and other important life processes (Fraher et al., 2016). Triglycerides and cholesterol are the main representative forms of lipids. The changes in triglycerides and cholesterol might cause disorders of lipid metabolism, according to the report in male albino Wistar rats by the methanol

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Fig. 6. Gene expression levels of hmgcra, ppara1, fas, acca1, cyp51 and srebf1 in embryos after exposure to penthiopyrad for 4 and 8 d. The asterisks indicate significant differences from the control group (determined by Dunnett's post-hoc comparison, *p < 0.05; **p < 0.01).

Fig. 7. Gene expression levels of the slc24a5, asip1, pkc-b, mc1r, myosin-Va, dct, foxd3, kita, mitfa, tyrp1a and tyrp1b in embryos exposed to penthiopyrad for 8 dpe. The asterisks indicate significant differences from the control group (determined by Dunnett's post-hoc comparison, *p < 0.05; **p < 0.01).

bark extract of Terminalia chebula (Reddy et al., 2015). b-Oxidation is the main catabolic process of fatty acid that splits long carbon chains of the fatty acid into acetyl CoA, which can eventually enter the TCA and produce a lot of energy (Poirier et al., 2006). Peroxisome proliferator-activated receptor alpha (PPARa) plays key roles in the catabolism of fatty acids and enhances fatty acid combustion in the liver by upregulating the genes encoding enzymes involved in b-oxidation and also participates in lipid synthesis working with sterol regulatory element-binding proteins (SREBPs) (Kliewer et al., 1997; Reddy and Hashimoto, 2001; Sun et al., 2013). SREBPs are lipid synthetic transcription factors in the synthesis of endogenous cholesterol, fatty acids, triglycerides and phospholipid synthesis (Eberle et al., 2004). Gene srebp1 is one of the upstream critical lipogenic transcripts. In our study, lipogenic transcripts might be promoted at 4 dpe but inhibited at 8 dpe by penthiopyrad, as the expression of srebp1 was upregulated by all the treated groups at 4 dpe but downregulated by 0.6 and 1.2 mg/L at 8 dpe. Fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) are two critical lipogenic enzymes. FAS is a key enzyme that catalyses the synthesis of fatty acid that is essential to energy production and storage, cellular structure and as intermediates in the biosynthesis of hormones and other biologically important molecules (Zeng et al., 2011). ACC is the rate-limiting enzyme to catalyse the carboxylation of acetyl-CoA to malonyl-CoA in the lipid metabolism, which is the rate-limiting step in fatty acid synthesis. In this study, changes

in the expression of fas and acca1 indicated that penthiopyrad could stimulate fatty acid synthesis at 4 dpe, according to the increase in cholesterol and triglycerides content. Similarly, in our previous study, boscalid decreased the expression of fas and increase in cholesterol and triglycerides content at 4 dpe, which has similar mode of action with penthiopyrad (Qian et al., 2018). HMGCoA reductase (HMGCRa) is the rate-limiting enzyme for cholesterol synthesis and works via a negative feedback mechanism (Mangravite et al., 2010). Lanosterol 14-demethylase (CYP51) is a critical enzyme of the late portion of cholesterol biosynthesis (Fink et al., 2005), which was significantly affected by penthiopyrad at 1.2 mg/L at 4 dpe in this study. It may be hypothesised that exposure to penthiopyrad affected several genes that regulate lipid metabolism and stimulated the biosynthesis of cholesterol and triglycerides in accordance with the remarkable increase in content of cholesterol and triglycerides at 4 dpe. With the prolonged exposure time, the content and biosynthesis of cholesterol and triglycerides decreased with the significant downregulation of hmgcra, srebf1, acca1 and fas at 8 dpe. In addition, an increase in boxidation could be caused by 0.6 mg/L penthiopyrad with the upregulation of ppara1, but a decrease in b-oxidation caused by 1.2 mg/L. Similar results were obtained in a study which indicated a significant decrease in b-oxidation of zebrafish embryos by triclosan (TCS) exposure (Ho et al., 2016). Besides, changes in b-oxidation caused by penthiopyrad ultimately affected energy metabolism in

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our study, which might result in developmental malformations and abnormal behavioural effects of zebrafish in early-life stages. The pigment cells of vertebrates serve several functions and generate a stunning variety of patterns, which are also implicated in human pathologies including melanoma (Budi et al., 2011). Pigment patterns function in distinguishing between individuals, groups and species, which play crucial roles in the appropriate adaptation to the environment, such as camouflage and social signalling (Ceinos et al., 2015). Skin pigmentation results from the production and distribution of melanin in the epidermis and is the major physiological defence against solar irradiation (Kim et al., 2015). Pigmentation in fish is a complex process that involves a series of cellular, genetic and physiological factors that together determine the external appearance of a fish at a given developmental stage (Colihueque, 2009). Melanin is the most important pigment and functions in photoabsorption, photoprotection and body pigmentation. According to a previous study, synthesis of melanin is regulated by tyrosine (tyr) via an enzymatic cascade that is controlled by tyr, tyrp1 and dct/tyrp2 (Braasch et al., 2009). In addition, the microphthalmia transcription factor (MITF) is a key transcription factor that promotes the expression of tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2) in melanocytes and promotes melanoma (Wan et al., 2011). Tyr and mitf are considered important molecular targets in the screening of inhibitors of melanin synthesis (Braasch et al., 2009; Shin et al., 2016; Kwon and Kim, 2017). In this study, there were no changes in transcription of dct, but a significant decrease in tyr caused by penthiopyrad (tyr1a at 1.2 mg/L and tyr1b at 0.3 mg/L), which might inhibit the synthesis of melanin. However, the expression of mitfa was increased in the presence of penthiopyrad at 0.6 and 1.2 mg/L, which stimulated melanin synthesis. The expression of asip1 functions in regulating the activity of agouti-signalling protein (ASP), which inhibits the differentiation and proliferation of melanoblasts and creates pigmentation patterns and is predominantly expressed in the ventral skin with residual levels in the dorsal skin (Cerda-Reverter et al., 2005). The expression of asip1 was up-regulated by 0.6 mg/L but downregulated by 1.2 mg/L penthiopyrad in this study, which indicated that differentiation and proliferation of melanoblasts regulated by ASP were inhibited at 0.6 mg/L, while they were stimulated at 1.2 mg/L. The solute carrier family 24 member 5 (SLC25A5), as a potassium-dependent sodium/calcium exchanger, affects pigmentation in zebrafish (Lamason et al., 2005). The expression of kita affects the migration and survival of embryonic neural crestderived melanocytes, which are expressed by their precursors (Rawls and Johnson, 2001). Myosin-Va interacting with melanophilin involves regulation of melanosome transport (Chang et al., 2012). Protein kinase C (PKC) is involved in inducing the inhibition of melanogenesis (Ando et al., 1990). Obviously, changes induced by penthiopyrad in the expression of slc24a5, kita and myosin-Va affected the regulation of melanin synthesis and inhibited the distribution of melanin in embryos. Forkhead transcription factor 3 (Foxd3) is a robust marker of pre-migratory neural crest throughout vertebrates and involved in controlling melanophore specification in the zebrafish neural crest by regulation of microphthalmia-associated transcription factor (MITF) (Curran et al., 2009). Foxd3 is expressed in putative glial cells of the peripheral nervous system. During post-embryonic development, a proliferative population of receptor tyrosine-protein kinase Erbb3dependent foxd3-and sox10-expressing cells associated with the peripheral nervous system differentiate into adult melanophores (Dooley et al., 2013). Penthiopyrad-induced foxd3 inhibition at 1.2 mg/L at 8 dpe might reflect changes in the balance of melanocyte stem cell proliferation/differentiation. From these results, penthiopyrad induced an increase in melanin synthesis and

inhibition of melanin distribution in larvae, in accordance with the results observed under the microscope. Changes in melanin synthesis and distribution may be another possible mechanism for penthiopyrad to cause abnormal development in early-life stages of zebrafish. Previous study has shown that zebrafish are social animals and their social behaviour heavily depends on visual colour (pigment) patterns (He et al., 2014). It can be hypothesised that disorders in melanin synthesis and distribution by penthiopyrad might be the cause of behavioural response. 5. Conclusions In summary, this is the first study revealing penthiopyradinduced toxic effects in the development of zebrafish in early-life stages. Results indicated that penthiopyrad had an acute toxicity to early-life stages of zebrafish and induced a series of malformations, especially Yse, Pe, pigmentation and failure of swim bladder inflation. In addition, an obvious inhibition in locomotor behaviours of larvae and a dysregulation of gene expression of lipid metabolism and melanin deposition were found in zebrafish in early-life stages by penthiopyrad under sub-lethal exposure. Results in our study could help achieve a clear understanding of the environmental risk of penthiopyrad in aquatic systems. Acknowledgements This work was supported by the National Key Research and Development Program of China (2016YFD0200504). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2018.09.117. References ~ asco, N., Koyama, J., Uno, S., 2009. Pesticide residues in coastal waters affected by An rice paddy effluents temporarily stored in a wastewater reservoir in southern Japan. Arch. Environ. Contam. Toxicol. 58, 352e360. ~ asco, N., Uno, S., Koyama, J., Matsuoka, T., Kuwahara, N., 2008. Assessment of An pesticide residues in freshwater areas affected by rice paddy effluents in Southern Japan. Environ. Monit. Assess. 160, 371e383. Ali, S., Champagne, D., Spaink, H., Richardson, M., 2011. Zebrafish embryos and larvae: a new generation of disease models and drug screens. Birth Defects Res. Part C Embryo Today - Rev. 93, 115e133. Ando, H., Oka, M., Ichihashi, M., Mishima, Y., 1990. Protein kinase C and linoleic acid-induced inhibition of melanogenesis. Pigm. Cell Res. 3, 200e206. Antkiewicz, D., Burns, C., Carney, S., Peterson, R., Heideman, W., 2005. Heart malformation is an early response to TCDD in embryonic zebrafish. Toxicol. Sci. Offic. J. Soc. Toxicol. 84, 368e377. Atchison, G., Henry, M., Sandheinrich, M., 1987. Effects of metals on fish behavior: a review. Environ. Biol. Fish. 18, 11e25. Bahrndorff, S., Michaelsen, T., Jensen, A., Marcussen, L., Nielsen, M., Roslev, P., 2016. Automated swimming activity monitor for examining temporal patterns of toxicant effects on individual Daphnia magna. J. Appl. Toxicol. 36, 896e902. Bonansea, R., Wunderlin, D., Ame, M., 2016. Behavioral swimming effects and acetylcholinesterase activity changes in Jenynsia multidentata exposed to chlorpyrifos and cypermethrin individually and in mixtures. Ecotoxicol. Environ. Saf. 129, 311e319. Braasch, I., Liedtke, D., Volff, J., Schartl, M., 2009. Pigmentary function and evolution of tyrp1 gene duplicates in fish. Pigment Cell. Melanoma Res. 22, 839e850. Budi, E., Patterson, L., Parichy, D., 2011. Post-embryonic nerve-associated precursors to adult pigment cells: genetic requirements and dynamics of morphogenesis and differentiation. PLoS Genet. 7. Cambier, S., Rogeberg, M., Georgantzopoulou, A., Serchi, T., Karlsson, C., Verhaegen, S., Iversen, T., Guignard, C., Kruszewski, M., Hoffmann, L., Audinot, J., Ropstad, E., Gutleb, A., 2018. Fate and effects of silver nanoparticles on early lifestage development of zebrafish (Danio rerio) in comparison to silver nitrate. Sci. Total Environ. 610e611, 972e982. -Reverter, J., Josep, R., 2015. Pigment patterns in Ceinos, R., Raúl, G., Kelsh, R., Cerda adult fish result from superimposition of two largely independent pigmentation mechanisms. Pigment Cell. Melanoma Res. 28, 196e209. Cerda-Reverter, J., Haitina, T., Schioth, H., Peter, R., 2005. Gene structure of the goldfish agouti-signaling protein: a putative role in the dorsal-ventral pigment

L. Qian et al. / Chemosphere 214 (2019) 184e194 pattern of fish. Endocrinology 146, 1597e1610. Chang, H., Choi, H., Joo, K., Kim, D., Lee, T., 2012. Manassantin B inhibits melanosome transport in melanocytes by disrupting the melanophilin-myosin Va interaction. Pigment Cell. Melanoma Res. 25, 765e772. Colihueque, N., 2009. Genetics of salmonid skin pigmentation: clues and prospects for improving the external appearance of farmed salmonids. Rev. Fish Biol. Fish. 20, 71e86. Curran, K., Raible, D., Lister, J., 2009. Foxd3 controls melanophore specification in the zebrafish neural crest by regulation of Mitf. Dev. Biol. 332, 408e417. de Oliveira, G., de Lapuente, J., Teixido, E., Porredon, C., Borras, M., de Oliveira, D., 2016. Textile dyes induce toxicity on zebrafish early life stages. Environ. Toxicol. Chem. 35, 429e434. Deng, J., Yu, L., Liu, C., Yu, K., Shi, X., Yeung, L., Lam, P., Wu, R., Zhou, B., 2009. Hexabromocyclododecane-induced developmental toxicity and apoptosis in zebrafish embryos. Aquat. Toxicol. 93, 29e36. Dooley, C., Mongera, A., Walderich, B., Nüssleinvolhard, C., 2013. On the embryonic origin of adult melanophores: the role of ErbB and Kit signalling in establishing melanophore stem cells in zebrafish. Development 140, 1003e1013. Eberle, D., Hegarty, B., Bossard, P., et al., 2004. SREBP transcription factors: master regulators of lipid homeostasis. Biochimie 86, 839e848. EFSA, 2012. Reasoned opinion on the setting of new MRLs for penthiopyrad in various crops. EFSA Journal 10. EPA, 2011. United States Environmental Protection Agency Washington, D.C. 20460: Penthiopyrad (MTF-753): Section 3 New Chemical Environmental Fate and Ecological Risk Assessment. Fink, M., Acimovic, J., Rezen, T., Tansek, N., Rozman, D., 2005. Cholesterogenic lanosterol 14alpha-demethylase (CYP51) is an immediate early response gene. Endocrinology 146, 5321e5331. Fraher, D., Sanigorski, A., Mellett, N., Meikle, P., Sinclair, A., Gibert, Y., 2016. Zebrafish embryonic lipidomic analysis reveals that the yolk cell is metabolically active in processing lipid. Cell Rep. 14, 1317e1329. Gerlai, R., Lee, V., Blaser, R., 2006. Effects of acute and chronic ethanol exposure on the behavior of adult zebrafish (Danio rerio). Pharmacol. Biochem. Behav. 85, 752e761. Giari, L., Guerranti, C., Perra, G., Lanzoni, M., Fano, E., Castaldelli, G., 2015. Occurrence of perfluorooctanesulfonate and perfluorooctanoic acid and histopathology in eels from north Italian waters. Chemosphere 118, 117e123. Guo, X., Zhang, S., Lu, S., Zheng, B., Xie, P., Chen, J., Li, G., Liu, C., Wu, Q., Cheng, H., Sang, N., 2018. Perfluorododecanoic acid exposure induced developmental neurotoxicity in zebrafish embryos. Environ. Pollut. 241, 1018e1026. He, J., Gao, J., Huang, C., Li, C., 2014. Zebrafish models for assessing developmental and reproductive toxicity. Neurotoxicol. Teratol. 42, 35e42. Ho, J., Hsiao, C., Kawakami, K., Tse, W., 2016. Triclosan (TCS) exposure impairs lipid metabolism in zebrafish embryos. Aquat. Toxicol. 173, 29e35. Hong, H., Lv, D., Liu, W., Huang, L., Chen, L., Shen, R., Shi, D., 2017. Toxicity and bioaccumulation of three hexabromocyclododecane diastereoisomers in the marine copepod Tigriopus japonicas. Aquat. Toxicol. 188, 1e9. Huang, M., Jiao, J., Wang, J., Xia, Z., Zhang, Y., 2018. Characterization of acrylamideinduced oxidative stress and cardiovascular toxicity in zebrafish embryos. J. Hazard Mater. 347, 451e460. Hwang, P., Chou, M., 2013. Zebrafish as an animal model to study ion homeostasis. Pflueg. Arch. Eur. J. Physiol. 465, 1233e1247. ISO, 1996. Water Quality e Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio rerio Hamilton and Buchanan (Teleostei, Cyprinidae)]: Part 3: Flow-through Method ISO. Iwasaki, T., Teruya, K., Mizuta, S., Hamasaki, K., 2017. Swim bladder inflation as a possible cause of saddleback-like syndrome malformation in hatchery-reared red spotted grouper Epinephelus akaara. Fish. Sci. 83, 447e454. Jin, Y., Zhang, X., Shu, L., Chen, L., Sun, L., Qian, H., Liu, W., Fu, Z., 2010. Oxidative stress response and gene expression with atrazine exposure in adult female zebrafish (Danio rerio). Chemosphere 78, 846e852. Kim, K., Yang, H., Kang, S., Ahn, G., Roh, S., Lee, W., Kim, D., Jeon, Y., 2015. Whitening effect of octaphlorethol a isolated from Ishige foliacea in an in vivo zebrafish model. J. Microbiol. Biotechnol. 25, 448e451. Kimmel, C., Ballard, W., Kimmel, S., Ullmann, B., Schilling, T., 1995. Stages of embryonic development of the zebrafish. Dev. Dynam. 203, 253e310. Kliewer, S., Sundseth, S., Jones, S., et al., 1997. Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferatoractivated receptors a and g. Proc. Natl. Acad. Sci. U.S.A. 94, 4318e4323. Kluver, N., Konig, M., Ortmann, J., Massei, R., Paschke, A., Kuhne, R., Scholz, S., 2015. Fish embryo toxicity test: identification of compounds with weak toxicity and analysis of behavioral effects to improve prediction of acute toxicity for neurotoxic compounds. Environ. Sci. Technol. 49, 7002e7011. Kwon, E., Kim, M., 2017. Agmatine modulates melanogenesis via MITF signaling pathway. Environ. Toxicol. Pharmacol. 49, 124e130. Lamason, R., Mohideen, M., Mest, J., et al., 2005. SLC24A5, a putative cation exchanger, affects pigmentation in zebrafish and humans. Science 310, 1782e1786. Li, J., Liang, Y., Zhang, X., Lu, J., Zhang, J., Ruan, T., Zhou, Q., Jiang, G., 2011. Impaired gas bladder inflation in zebrafish exposed to a novel heterocyclic brominated flame retardant tris (2,3-dibromopropyl) isocyanurate. Environ. Sci. Technol. 45, 9750e9757. Little, E., Finger, S., 1990. Swimming behavior as an indicator of sublethal toxicity in fish. Environ. Toxicol. Chem. 9, 13e19. Liu, Y., Khan, M., 2016. Penthiopyrad applied in close proximity to Rhizoctonia solani

193

provided effective disease control in sugar beet. Crop Protect. 85, 33e37. Mangravite, L., Medina, M., Cui, J., Pressman, S., Smith, J., Rieder, M., Guo, X., Nickerson, D., Rotter, J., Krauss, R., 2010. Combined influence of LDLR and HMGCR sequence variation on lipid-lowering response to simvastatin. Arterioscler. Thromb. Vasc. Biol. 30, 1485e1492. Matrone, G., Wilson, K., Mullins, J., Tucker, C., Denvir, M., 2015. Temporal cohesion of the structural, functional and molecular characteristics of the developing zebrafish heart. Differ. Res. Biol. Divers. 89, 117e127. Meyer, M., Hausbeck, M., 2013. Using soil-applied fungicides to manage phytophthora crown and root rot on summer squash. Plant Dis. 97, 107e112. Mu, X., Chai, T., Wang, K., Zhang, J., Zhu, L., Li, X., Wang, C., 2015. Occurrence and origin of sensitivity toward difenoconazole in zebrafish (Danio reio) during different life stages. Aquat. Toxicol. 160, 57e68. Mu, X., Pang, S., Sun, X., Gao, J., Chen, J., Chen, X., Li, X., Wang, C., 2013. Evaluation of acute and developmental effects of difenoconazole via multiple stage zebrafish assays. Environ. Pollut. 175, 147e157. Nusser, L., Skulovich, O., Hartmann, S., Seiler, T., Cofalla, C., Schuettrumpf, H., Hollert, H., Salomons, E., Ostfeld, A., 2016. A sensitive biomarker for the detection of aquatic contamination based on behavioral assays using zebrafish larvae. Ecotoxicol. Environ. Saf. 133, 271e280. OECD, 1998. Fish, short-term toxicity test on embryo and sac-fry stage. Oecd Guidelines for the Testing of Chemicals. OECD, 2013. OECD guidelines for the testing of chemicals. In: Section 2: Effects on Biotic Systems Test No. 236: Fish Embryo Acute Toxicity (FET) Test. Organisation for Economic Co-operation and Development. Piechowicz, B., Wos, I., Podbielska, M., Grodzicki, P., 2018. The transfer of active ingredients of insecticides and fungicides from an orchard to beehives. J. Environ. Sci. Health Part B Pestic. Food Contam. Agric. Wastes 53, 18e24. Poirier, Y., Antonenkov, V., Glumoff, T., Hiltunen, J., 2006. Peroxisomal boxidationda metabolic pathway with multiple functions. Biochim. Biophys. Acta 1763, 1413e1426. Qian, L., Cui, F., Yang, Y., Liu, Y., Qi, S., Wang, C., 2018. Mechanisms of developmental toxicity in zebrafish embryos ( Danio rerio ) induced by boscalid. Sci. Total Environ. 634, 478e487. Qiu, S., Bai, Y., 2015. Progress on research and development of succinate dehydrogenase inhibitor fungicides (Ⅱ). Mod. Agrochem. 14, 1e20. Rawls, J., Johnson, S., 2001. Requirements for the kit receptor tyrosine kinase during regeneration of zebrafish fin melanocytes. Development 128, 1943e1949. Reddy, J., Hashimoto, T., 2001. Peroxisomal b-oxidation and peroxisome proliferator-activated receptor a: an adaptive metabolic system. Annu. Rev. Nutr. 21, 193e230. Reddy, M., Dhas Devavaram, J., Dhas, J., Adeghate, E., Starling Emerald, B., 2015. Anti-hyperlipidemic effect of methanol bark extract of Terminalia chebula in male albino Wistar rats. Pharmaceut. Biol. 53, 1133e1140. Robertson, G., McGee, C., Dumbarton, T., Croll, R., Smith, F., 2007. Development of the swimbladder and its innervation in the zebrafish, Danio rerio. J. Morphol. 268, 967e985. Schmidt, S., Busch, W., Altenburger, R., Kuster, E., 2016. Mixture toxicity of water contaminants-effect analysis using the zebrafish embryo assay (Danio rerio). Chemosphere 152, 503e512. Scott, G.R., Sloman, K.A., 2004. The effects of environmental pollutants on complex fish behaviour: integrating behavioural and physiological indicators of toxicity. Aquat. Toxicol. 68, 369e392. Shin, Y., Jang, E., Park, H., et al., 2016. Suppression of melanin synthesis by Americanin A in melan-a cells via regulation of microphthalmia-associated transcription factor. Exp. Dermatol. 25, 646e647. Stinckens, E., Vergauwen, L., Schroeder, A., Maho, W., Blackwell, B., Witters, H., Blust, R., Ankley, G., Covaci, A., Villeneuve, D., Knapen, D., 2016. Impaired anterior swim bladder inflation following exposure to the thyroid peroxidase inhibitor 2-mercaptobenzothiazole part II: Zebrafish. Aquat. Toxicol. 173, 204e217. Sun, L., Li, J., Zuo, Z., Chen, M., Wang, C., 2013. Chronic exposure to paclobutrazol causes hepatic steatosis in male rockfish Sebastiscus marmoratus and the mechanism involved. Aquat. Toxicol. 126, 148e153. Tanabe, A., Kawata, K., 2009. Daily variation of pesticides in surface water of a small river flowing through paddy field area. Bull. Environ. Contam. Toxicol. 82, 705e710. Toumi, H., Boumaiza, M., Millet, M., Radetski, C., Camara, B., Felten, V., Ferard, J., 2015. Investigation of differences in sensitivity between 3 strains of Daphnia magna (crustacean Cladocera) exposed to malathion (organophosphorous pesticide). J. Environ. Sci. Health Part B Pestic. Food Contam. Agric. Wastes 50, 34e44. Tsuda, T., Nakamura, T., Inoue, A., Tanaka, K., 2009. Pesticides in water and sediment from littoral area of Lake Biwa. Bull. Environ. Contam. Toxicol. 82, 683e689. Velki, M., Di Paolo, C., Nelles, J., Seiler, T., Hollert, H., 2017. Diuron and diazinon alter the behavior of zebrafish embryos and larvae in the absence of acute toxicity. Chemosphere 180, 65e76. Vu, H., Keough, M., Long, S., Pettigrove, V., 2016. Effects of the boscalid fungicide Filan on the marine amphipod Allorchestes compressa at environmentally relevant concentrations. Environ. Toxicol. Chem. 35, 1130e1137. Wan, P., Hu, Y., He, L., 2011. Regulation of melanocyte pivotal transcription factor MITF by some other transcription factors. Mol. Cell. Biochem. 354, 241e246. Wang, W., Huang, C., Lu, Y., Hsin, J., Prabhakar, V., Cheng, C., Hwang, S., 2006. Hearttargeted overexpression of Nip3a in zebrafish embryos causes abnormal heart development and cardiac dysfunction. Biochem. Biophys. Res. Commun. 347,

194

L. Qian et al. / Chemosphere 214 (2019) 184e194

979e987. Warner, R., 1967. Bio-assays for microchemical environmental contaminants, with special reference to water supplies. Bull. World Health Organ. 36, 181e207. Wu, S., Lei, L., Liu, M., Song, Y., Lu, S., Li, D., Shi, H., Raley-Susman, K., He, D., 2018. Single and mixture toxicity of strobilurin and SDHI fungicides to Xenopus tropicalis embryos. Ecotoxicol. Environ. Saf. 153, 8e15. Yamashita-Ichimura, M., Toyama, E., Sasoh, M., Shiwaku, H., Yamashita, K., Yamashita, Y., Yamaura, K., 2018. Bladder pressure monitoring and CO2 gasrelated adverse events during per-oral endoscopic myotomy. J. Clin. Monit. Comput. 1e6. Yang, Y., Liu, W., Mu, X., Qi, S., Fu, B., Wang, C., 2016a. Biological response of zebrafish embryos after short-term exposure to thifluzamide. Sci. Rep. 6. Yang, Y., Qi, S., Chen, J., Liu, Y., Teng, M., Wang, C., 2016b. Toxic effects of bromothalonil and flutolanil on multiple developmental stages in zebrafish. Bull.

Environ. Contam. Toxicol. 97, 91e97. -Reverter, J., Yin, Z., Guillot, R., Muriach, B., Rocha, A., Rotllant, J., Kelsh, R., Cerda 2016. Thyroid hormones regulate zebrafish melanogenesis in a gender-specific manner. PLoS One 11. Yue, M., Peterson, R., Heideman, W., 2015. Dioxin inhibition of swim bladder development in zebrafish: is it secondary to heart failure? Aquat. Toxicol. 162, 10e17. Zeng, X., Li, W., Fan, H., et al., 2011. Discovery of novel fatty acid synthase (FAS) inhibitors based on the structure of ketoaceyl synthase (KS) domain. Bioorg. Med. Chem. Lett. 21, 4742e4744. Zhu, L., Mu, X., Wang, K., Chai, T., Yang, Y., Qiu, L., Wang, C., 2015. Cyhalofop-butyl has the potential to induce developmental toxicity, oxidative stress and apoptosis in early life stage of zebrafish (Danio rerio). Environ. Pollut. 203, 40e49.