Lethal toxicity and gene expression changes in embryonic zebrafish upon exposure to individual and mixture of malathion, chlorpyrifos and lambda-cyhalothrin

Chemosphere 239 (2020) 124802

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Lethal toxicity and gene expression changes in embryonic zebrafish upon exposure to individual and mixture of malathion, chlorpyrifos and lambda-cyhalothrin Weifeng Shen a, Bao Lou a, Chao Xu b, Guiling Yang a, Ruixian Yu a, Xinquan Wang a, Xinfang Li a, Qiang Wang a, Yanhua Wang a, * a

State Key Laboratory for Quality and Safety of Agro-products / Key Laboratory for Pesticide Residue Detection of Ministry of Agriculture / Laboratory (Hangzhou) for Risk Assessment of Agricultural Products of Ministry of Agriculture, Institute of Quality and Standard for Agro-products / Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, Zhejiang, China b College of Environment, Zhejiang University of Technology, Hangzhou, 310021, Zhejiang, China

h i g h l i g h t s
Lambda-cyhalothrin was the most toxic to zebrafish among three tested pesticides.
Lambda-cyhalothrin in combination with malathion or chlorpyrifos exerted synergism.
Expressions of 5 genes exerted greater changes in mixtures compared to individuals.
Evaluation of single-compound data may underrate the risk of pesticide mixtures.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 July 2019 Received in revised form 5 September 2019 Accepted 6 September 2019 Available online 7 September 2019

Pesticides are usually present as mixtures in water environments. Evaluating the toxic effects of individual pesticide may not be enough for protecting ecological environment due to interactions among substances. In this study, we aimed to examine the lethal doses and gene expression changes in zebrafish (Danio rerio) upon exposure to individual and mixture pesticides [malathion (MAL), chlorpyrifos (CHL) and lambda-cyhalothrin (LCY)]. Individual pesticide toxicity evaluation manifested that the toxicity of the three pesticides to D. rerio at various developmental stages (embryonic, larval, juvenile and adult stages) followed the order of LCY > CHL > MAL. On the contrary, the least toxicity to the animals was discovered from MAL. Most of the tested pesticides displayed lower toxicities to the embryonic stage compared with other life stages of zebrafish. Synergistic effects were monitored from two binary mixtures of LCY in combination with MAL or CHL and ternary mixture of MAL þ CHL þ LCY. The expressions of 16 genes involved in oxidative stress, immunity system, cell apoptosis and endocrine disruption at the mRNA level revealed that embryonic zebrafish were influenced by the individual or mixture pesticides. The expressions of Tnf, P53, TRa, Crh and Cyp19a exerted greater variations upon exposure to pesticide mixtures compared with their individual compounds. Collectively, the transcriptional responses of these genes might afford early warning biomarkers for identifying pollutant exposure, and the data acquired from this study provided valuable insights into the comprehensive toxicity of pesticide mixtures to zebrafish. © 2019 Elsevier Ltd. All rights reserved.

Handling Editor: David Volz Keywords: Mixture toxicity Aquatic toxicity Synergistic response Gene expressions

1. Introduction

* Corresponding author. State Key Laboratory for Quality and Safety of Agroproducts, Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, Zhejiang, China. E-mail address: [email protected] (Y. Wang). https://doi.org/10.1016/j.chemosphere.2019.124802 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

During the growth period of crops, many pesticides are frequently applied to prevent pest and damage harm worldwide (Cordova et al., 2017). It has become very extensive to employ pesticide mixtures with different modes of action to overcome resistance to an individual compound (Sewell et al., 2016).

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Therefore, multiple pesticide residues likely co-occur in the same water sample via runoff (Wu et al., 2018). In addition, although the concentrations of the individual substances are low, their mixture effects may be ecotoxicologically significant (Levine and Borgert, 2018). Accordingly, the effects of pesticide multi-residues on the ecosystem have drawn a global attraction (Belden and Brain, 2018). However, regulatory risk assessment of pesticides and the majority of ecotoxicological studies in the aquatic environment mainly focus on the toxicity of individual compounds under controlled conditions (Hassold and Backhaus, 2014; Cui et al., 2017). Therefore, it is crucial to determine the toxic effect of pesticide mixtures when performing risk assessments and evaluating the quality of water ecosystems. Aquatic organisms are usually exposed chemical mixtures in the natural environment rather than an individual compound (Ge et al., 2015; Maharajan et al., 2018). Currently, zebrafish (Danio rerio) are used for water quality assessment as an OECD and ISO standard test organism due to several characteristic, such as easy availability, small size, short test cycle, low maintenance cost and breeding under laboratory conditions (ISO, 1996; OECD, 2013). Moreover, the zebrafish has been used for environmental toxicological assessment because of its high homology with human genome. The toxic results from zebrafish can indicate the risks to human health in some extent. Assays of zebrafish at early life stage are particularly suitable for chemical evaluating since early developmental stages are very sensitive to compounds (Braunbeck et al., 2015; Klüver et al., 2015). Because of these distinct advantages, zebrafish have turned into a preferred choice for toxicity assessment of chemicals (Mu et al., 2017). To date, many mixture toxicity studies on D. rerio have only determined the extent of the interaction, whereas the reason for the mixture toxicity has been seldom reported (Chen et al., 2016; Wang et al., 2017; Velki et al., 2017). Understanding the cause of mixture toxicity for normally co-existing pesticide mixtures would help us understand the underlying mechanisms of mixture toxicity for other similar combinations and ultimately
ndez et al., 2013; accelerate the accuracy of risk assessment (Herna Rizzati et al., 2016). Two organophosphate insecticides malathion (MAL) and chlorpyrifos (CHL) as well as one pyrethroid insecticide lambda-cyhalothrin (LCY) have been used extensively in agriculture to improve crop production (He et al., 2008; Lu et al., 2016; Jeon et al., 2016; Li et al., 2018a, b). Co-occurrence of MAL, CHL and LCY (the levels of concentrations for three pesticides range from 10 to 1000 ng a.i. L1) has been usually detected in aquatic ecosystems, especially the area surrounding intensive agricultural site as a result of spray drift or surface transport following application, and combined pollution of these pesticides has recently drawn great attention (Grung et al., 2015; Schreiner et al., 2016). Despite the co-existence of these pesticides in the environment, their ecological risk assessment has been primarily focused on the toxicological effects of individual substances (Belden and Brain, 2018). In the current study, we investigated the lethal toxicity and gene expression changes coexposure to these pesticides using D. rerio as a model organism. Gene expression analysis is a more sensitive method than conventional toxicity test in environment toxicology assessment, and it can detect the changes at the transcription level, which occur earlier than those at the phenotype level. The expression changes of related genes, including oxidative stress, innate immune system, apoptosis and endocrine system, in embryonic zebrafish exposed to three pesticides individually and their mixtures were analyzed. The results supplied new insights into the toxic mechanism of pesticide mixtures towards non-target organisms.

2. Materials and methods 2.1. Chemicals and reagents MAL (CAS: 121-75-5, purity of 95%) was bought from Zhejiang Jiahua Group Co., Ltd. (Haiyan, Zhejiang, China). CHL (CAS: 292188-2, purity of 96%) was provided by Yannong Agrochemical Group (Yangzhou, Jiangsu, China). LCY (CAS: 91465-08-6, purity of 97%) was gifted by Jiangsu Changlong Chemical Industrial Group (Changzhou, Jiangsu, China). Stock solutions of three tested pesticides were confected with acetone AR and Tween-80 and then stored at 4  C. All stock solutions were further diluted to designed concentrations adopting standard water involving in 2 mmol L1 Ca2þ, 0.5 mmol L1 Mg2þ, 0.75 mmol L1 Na2þ and 0.074 mmol L1 Kþ (ISO, 1996). The RNA extraction kit, reverse transcriptase kit and the SYBR Green system were supplied by Takara (TransGen Biotech Ltd., China). 2.2. Test organism Wild-type zebrafish adults (AB strain) were bought from Chinese Academy of Sciences (Wuhan, Hubei, China). They were raised in an aquarium giving circulating and aerated tap water at ambient temperature of 26 ± 1  C under a photoperiod of 14 h/10 h (light/ dark). These zebrafish were fed three times every day with brine shrimp. Embryos were acquired from zebrafish adults with a ratio of 1:2 (female to male) in a spawning box set with grids next the bottom (Esen Corp, Beijing, China). Spawning was induced by the light stimulation in the next morning. Embryos were collected within 1 h of light exposure and cultured using reconstituted water. All fish-related tests were conducted in accordance with the Experimental Animal Management Law of China and confirmed by the Animal Ethics Committee of Zhejiang Academy of Agricultural Sciences. 2.3. Individual pesticide toxicity test Toxicity assessments of individual pesticide to zebrafish at multiple developmental stages (embryonic, larval, juvenile and adult stages) were performed in accordance with the procedure as previously described (OECD, 1992, 2013; Wang et al., 2017). Exposure solutions were renewed every 12 h to keep the appropriate concentration of pesticides and water quality. The external circumstances during exposure, including the temperature and light cycle were the same as the husbandry environment. Mortality was checked after exposure for 96 h. Details about toxicity test method of pesticides to zebrafish at different life stages were provided in the supplemental materials. 2.4. Mixture toxicity test Toxicities of pesticide mixtures were examined with embryonic zebrafish. The toxicities of individual pesticides were directly compared with their mixtures. To illuminate the interaction of pesticide mixtures, embryonic zebrafish were exposed to serial dilutions of each pesticide with a fixed constant equitoxic ratio on the basis of the determined individual LC50 values. The total concentration of each mixture was systematically varied, whereas all the above-mentioned ratios were maintain constant for estimating the relationship of concentration-response (Table S2). All measurements were conducted out in triplicate for each concentration. 2.5. Mixture toxicity evaluation The LC50 and 95% confidence interval (CI) were evaluated with a

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profit analysis using a computer-based program developed by Chi (1997). Significant level of mean separation (P < 0.05) was based on non-overlap between the 95% CI of two LC50 values. To evaluate mixture toxicity, Marking's additive index (AI) method was adopted (Marking, 1985; Su et al., 2016): S ¼ (Am/Ai) þ (Bm/Bi) þ (Cm/Ci)

2.6. Expression analysis of related genes 2.6.1. Exposure procedures About 300 selected embryos of blastula stage at about 2 hpf were placed into a breaker containing 500 mL test solutions and incubated under semi-static conditions at 26 ± 1  C with a photoperiod of 14 h:10 h light/dark for 96 h. Because of the sensitivity of embryo to pesticides, concentrations of 1/20, 1/80 and 1/320 of 96h LC50 were set for gene expression analysis, respectively. The test solution was daily refreshed to keep the chemical concentrations. The test for each concentration was conducted for three times. After exposure for 96 h, 25 hatched zebrafish larvae from each concentration were randomly collected and stored at 80  C. 2.6.2. RNA isolation Total RNA of homogenized zebrafish embryos was extracted using the TransZol Up Plus RNA Kit (TransGen Biotech Ltd., China) in accordance with the manufacturer's protocols. The quality of RNA samples was assessed on the basis of the OD260/OD280 ratios and patterns of RNA electrophoretogram on 1% agarose gel. 2.6.3. Quantitative real-time (qRT-PCR) analysis First-strand cDNA was synthesized using TransScript Firststrand cDNA Synthesis SuperMix (TransGen Biotech Ltd., China). The specific primer pairs were obtained from published sequences and synthesized by Sangon Biotech Shanghai Co., Ltd. (Table S1). Briefly, the PCR thermal cycling conditions were 95  C for 15 s, and then the amplifications were carried out with 40 cycles at a melting temperature of 95  C for 5 s, an annealing temperature of 60  C for 15 s, and an extension temperature of 72  C for 20 s. The b-actin gene was chosen as a housekeeping gene. For each target gene, test was performed on three replicates. The relative expressions of target genes were computed with the 2eDDCT method. 2.6.4. Statistical analysis SPSS 18.0 software was used for the statistical analysis of qRTPCR. All of the data were presented as means ± standard error (SE). Differences of treatments were compared by one-way ANOVA and Tukey's post-hoc test. A probability of P < 0.05 or P < 0.01 was considered statistically significant (labeled with single asterisk) or extremely significant (labeled with double asterisks), respectively. 2.7. Heatmap drawing The heatmap of gene expression changes was constructed by importing the analyzed qRT-PCR data into MEV 4.9 (Multi Experiment Viewer) software.

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investigated, LCY had the greatest toxicity to various life stages of zebrafish with 96-h LC50 values ranging from 0.0038 to 0.18 mg a.i. L1, followed by CHL with 96-h LC50 values ranging from 0.41 to 1.53 mg a.i. L1 [except for embryonic stage with 96-h LC50 value of 6.15 mg a.i. L1]. On the contrary, MAL displayed the lowest toxicity to the animals with 96-h LC50 values ranging from 8.04 to 12.45 mg a.i. L1 [except for adult stage with 96-h LC50 value of 2.90 mg a.i. L1]. Based on the 96-h LC50 values of different life stages, the lethal toxicity of three evaluated pesticides could be ranked as follows: adult fish > juvenile, larval fish, embryos for MAL; larval fish > adult, juvenile fish > embryos for CHL; juvenile fish > adult fish > embryos, larval fish for LCY. Overall, zebrafish adults were more sensitive to most of the examined pesticides, while embryos were the least sensitive stages. 3.2. Mixture toxicity test To explicit the mixture toxicity of three pesticides against embryos of D. rerio, the LC50 values0 of different binary and ternary mixtures after administration for 96 h were determined (Table 2). The two binary mixtures of MAL þ LCY and CHL þ LCY, and the ternary mixture of MAL þ CHL þ LCY showed synergistic responses with AI values of 5.91, 9.39 and 4.89, respectively. On the contrary, the calculated AI values of MAL þ CHL was 0.36, indicating antagonistic response (Table 2). 3.3. Gene expression analysis 3.3.1. Oxidative stress-related genes The expression of Cat was obviously down-regulated in the binary mixture of CHL þ LCY compared with the control. Moreover, its expression was also apparently reduced in the groups of LCY, MAL þ LCY and MAL þ CHL þ LCY at high concentrations (Fig. 1a). The expression of CuSOD was pronouncedly induced in the LCYtreated group at low and medium concentrations compared with the control, while it was inhibited in the LCY-treated group at the high concentration. In addition, its expression was markedly increased in the binary group of MAL þ CHL in a dose-dependent manner (Fig. 1b). The expression of MnSOD exhibited no significant changes in all individual and mixture groups compared with the control, except for the individual exposure of LCY at 0.0055 mg a.i. L1 (Fig. 1c). 3.3.2. Immunity-related genes The expression of Cxcl was notably down-regulated upon exposure to CHL compared with the control, while it was enhanced in the groups of MAL þ LCY, CHL þ LCY and MAL þ CHL þ LCY at medium and high concentrations (Fig. 2a). The expression of IL was dramatically changed in all treated groups compared with the control, except for the MAL individual exposure (Fig. 2b). The expression of Tnf was not obviously altered in the individual groups of CHL and LCY compared with the control, while it was distinctly decreased in the group of CHL þ LCY. Additionally, its expression was significantly reduced in the MAL þ LCY-treated group in a dose-dependent manner, but elevated in the groups of MAL þ CHL at medium and high concentrations and MAL þ CHL þ LCY at the high concentration (Fig. 2c).

3. Results 3.1. Individual pesticide toxicity test Results from individual pesticide toxicity test displayed that three tested pesticides had obvious toxic selectivities for different life stages of zebrafish (Table 1). Among these pesticide

3.3.3. Cell apoptosis-related genes The expression of Cas8 was significantly inhibited in the LCYtreated groups compared with the control, while no obvious changes were detected in all combined groups (Fig. 3a). The expression of Cas9 was not apparently changed in three individual groups compared with the control, while it was pronouncedly

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Table 1 Acute toxicity of three selected pesticides to D. rerio at various life stages. Pesticide

Malathion Chlorpyrifos Lambda-cyhalothrin

LC50 (95% confidence interval) mg a.i. L1 Embryo

Larvae

Juvenile

Adult

12.45 (6.98e17.48) 6.15 (4.29e8.37) 0.11 (0.075e0.17)

8.54 (3.91e11.94) 0.41 (0.31e0.54) 0.18 (0.091e0.27)

8.04 (5.91e11.12) 1.53 (1.06e2.18) 0.0038 (0.0024e0.0053)

2.90 (1.51e4.09) 1.01 (0.77e1.45) 0.028 (0.021e0.038)

Table 2 Mixture toxic effects of three tested pesticides on the embryonic zebrafish. LC50 (95% confidence interval) mg a.i. L1

AI value

Type of combined action

0.29 (0.11e0.53)

0.0078 (0.0049e0.011) 0.0051 (0.0024e0.0081)

0.36 5.91 9.39

Antagonism Synergism Synergism

0.35 (0.19e0.54)

0.0065 (0.0041e0.0093)

4.89

Synergism

Malathion

Chlorpyrifos

Binary mixtures 8.46 (5.29e11.69) 0.92 (0.64e1.27)

4.21 (2.17e6.38)

Ternary mixture 0.67 (0.39e0.98)

Lambda-cyhalothrin

Fig. 1. Expression of oxidative stress-related genes in zebrafish expose to three pesticides and their mixtures. M: Malathion; C: Chlorpyrifos; L: Lambda-cyhalothrin *P < 0.05; **P < 0.01.

increased in the combined groups of MAL þ LCY, CHL þ LCY and MAL þ CHL þ LCY at medium and high concentrations (Fig. 3b). The expression of P53 was markedly enhanced in the LCY-treated group at 0.0014 mg a.i. L1, but decreased by about 6-fold in the LCY-

treated group at 0.0055 mg a.i. L1 compared with the control. Furthermore, its expression was notably down-regulated in most of the combined groups (Fig. 3c). The expression of Bax was dramatically reduced in the LCY- or CHL þ LCY-treated groups compared

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Fig. 2. Expression of immunity-related genes in zebrafish expose to three pesticides and their mixtures. M: Malathion; C: Chlorpyrifos; L: Lambda-cyhalothrin *P < 0.05; **P < 0.01.

with the control, while no significant changes were found in the individual groups of MAL and CHL (Fig. 3d). 3.3.4. Endocrine system genes The expression of TRa had no noticeable alterations in the individual groups of MAL, CHL and LCY compared with the control. However, its expression was obviously increased in the binary group of MAL þ CHL, and reduced in the binary group of CHL þ LCY (Fig. 4a). The expression of TRb was apparently weakened in the individual groups of MAL and LCY compared with the control, except for their exposures at the medium concentration. In addition, its expression was pronouncedly diminished in the ternary group of MAL þ CHL þ LCY and the binary group of MAL þ LCY at medium and high concentrations (Fig. 4b). The expression of ERa was markedly elevated in the individual groups of CHL and LCY at the medium concentration compared with the control, but diminished in the LCY-treated group at low and high concentrations (Fig. 4c). The expression of Tsh was notably decreased in the individual groups of CHL and LCY as well as the binary groups of MAL þ LCY and CHL þ LCY. Furthermore, its expression was decreased by about 9e10-fold in the MAL þ LCY-treated group at medium and high concentrations compared with the control (Fig. 5a). The expression of Crh was dramatically up-regulated in the binary group of MAL þ CHL compared with the control. Besides, the expression of Crh was obviously increased in the ternary group

of MAL þ CHL þ LCY (Fig. 5b). The expression of Cyp19a was distinctly elevated in the CHL-treated group compared with the control, but reduced in the CHL þ LCY-treated group in a dosedependent manner (Fig. 5c). 3.3.5. Overall mRNA expression changes A heatmap analysis was made with all transcripts and all pesticide groups to compare overall changes of gene expressions. The individual pesticide group of CHL built a separate group, while the three MAL-treated groups and three LCY-treated groups were separated from each other in heatmap. The binary groups of MAL þ CHL built a separate group. The locations of other combined groups crossed each other and showed no obvious regularity in heatmap (Fig. 6). 4. Discussion Results from various life stages of D. rerio exhibited that the greatest toxicity was detected from LCY, followed by CHL except for the embryonic stage. In contrast, MAL elicited the lowest toxicity to the zebrafish except for the adult stage. MAL showed a 96-h LC50 value of 8.54 mg a.i. L1 to the larvae of D. rerio, which was in agreement with previous findings (Guo et al., 2017). The 96-h LC50 value of MAL to adult zebrafish estimated in this study (2.90 mg a.i. L1) was within the previously reported range between 1.05 mg a.i.

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Fig. 3. Expression of cell apoptosis-related genes in zebrafish expose to three pesticides and their mixtures. M: Malathion; C: Chlorpyrifos; L: Lambda-cyhalothrin *P < 0.05; **P < 0.01.

L1 and 4.32 mg a.i. L1 (Kumar and Ansari, 1984; Lu et al., 2016). Previous tests have shown that the 96-h LC50 value for CHL to the adult D. rerio is 0.71 mg a.i. L1, suggesting similar toxic effects compared with our study (Jeon et al., 2016). Additionally, the 96-h LC50 values of LCY to two types of fresh water fish are 0.00021 and 0.00024 mg a.i. L1 (He et al., 2008), implying that LCY has the extremely high toxicity to fish, which is consistent with our findings. Therefore, the application of LCY in the agricultural fields should be made judiciously to protect non-target aquatic organisms from the hazards of the insecticide. Some studies have indicated that embryonic zebrafish are more susceptible than adults to pesticides (Lammer et al., 2009; Wu et al., 2018). On the contrary, embryos of D. rerio demonstrated higher tolerance to MAL, CHL and LCY than adults in this study. Our results were in accordance with that reported by Mu et al. (2013). The discrepancy in sensitivity between embryos and adults of zebrafish could be explained by that embryos may not have the fully developed metabolic pathways to degrade pesticides compared with adult zebrafish (Embry et al., 2010). Additionally, certain higherlevel structures targeted by MAL, CHL and LCY are not fully developed in embryonic zebrafish (Domingues et al., 2010). Lastly, the embryonic membrane can effectively insulate the external

environment (Mu et al., 2013). Consequently, it is essential to test the elements which determine the sensitivity of pesticides to the different stages of zebrafish (Yang et al., 2016). However, because of increasing ethical attention worldwide, many reporters have exhibited that zebrafish embryo toxicity evaluation can be an accepted alternative for adult toxicity test in the risk assessment of chemicals (Icoglu and Ciltas, 2018). Moreover, various practical advantages, such as relatively easy maintenance and propagation, the transparent embryos and most organ systems are fully developed within 96 hpf, make them become perfect vertebrate models (Belanger et al., 2013). Therefore, we evaluated mixture toxicities of MAL, CHL and LCY to zebrafish embryos in the present study. The co-application of pyrethroid and organophosphate insecticides is a common phenomenon in agricultural productions through both deliberate co-exposures, for example tank mixes and sequential application (Sanches et al., 2017; Shukla et al., 2017). Additionally, modern farming practices have progressively increased the number of active ingredients that are adopted to crops during the growing season (Grung et al., 2015; Belden and Brain, 2018). During the last decade, synergistically enhanced toxicities of pyrethroid insecticide in combination with organophosphate insecticide to the target organisms have been reported

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Fig. 4. Expression of endocrine disruption-related genes in zebrafish expose to three pesticides and their mixtures. M: Malathion; C: Chlorpyrifos; L: Lambda-cyhalothrin *P < 0.05; **P < 0.01.

(Bielza et al., 2007; Wang et al., 2017). However, our results demonstrated that these binary mixtures of LCY in combination with MAL or CHL exhibited synergistic responses on the non-target organisms of zebrafish, indicating that we might underestimate the
ndez et al., 2017). The ortoxicities of these combinations (Herna ganism's ability to detoxify pyrethroids can be diminished by cytochrome P450 (CYP450)eactivated organophosphates because of esterase inhibition (Bielza et al., 2007). Consequently, synergistic toxicities are frequent found. Because organisms are normally exposed to pesticide mixtures rather than a single compound in the natural ecosystem, the results from individual pesticides can lead to the inaccurate evaluation on the toxicity of pesticides present in the field (Dalhoff et al., 2016). The detected synergistic interactions underline the essential to revise water quality criteria (Wang et al., 2017). Hence, the combined toxic effects of organophosphate and pyrethroid insecticides should be incorporated in the risk assessment of pesticides (Cedergreen et al., 2017). When exposed to pesticides, organisms will be in the state of oxidative stress, leading to the production of reactive oxygen species (ROS) (Shukla et al., 2017). Meanwhile, antioxidant enzymes will be activated to scavenge ROS for maintaining the oxidant/ antioxidant balance (Wang et al., 2019). In this study, the expressions of Cat, CuSOD and MnSOD had no distinct variation upon exposure to MAL and CHL compared with the control, implying that

zebrafish were not under too much oxidative stress in these groups (Ge et al., 2015). However, zebrafish could suffer oxidative stress upon LCY exposure according to the significantly changed expressions of antioxidants. It was worth noting that the expressions of Cat, CuSOD and MnSOD were all apparently diminished in the LCY exposure group at 0.0055 mg a.i. L1 compared with the control, implying that high concentration of LCY could disrupt the expressions of antioxidants in zebrafish and result in reduced antioxidant capacity (Hong et al., 2018). At the same time, the expression of CuSOD in the MAL þ CHL-treated group was increased, while the expression of Cat in the CHL þ LCY-treated group was suppressed. These results indicated that these two mixtures affected the development of zebrafish by regulating the synthesis of different antioxidants (Jia et al., 2018). The expressions of Cat and CuSOD were pronouncedly enhanced upon the LCY exposure at the low concentration compared with the control, whereas it was weakened upon the LCY exposure at the high concentration. These results might illustrate that the antioxidative ability of zebrafish was enhanced to compensate the elevated oxidative harm caused by the low concentration of LCY. However, such ability was destroyed by the high concentration of LCY (Liu et al., 2013). Innate immune system of fish plays a crucial role in elementary defense during the early life stage (Lam et al., 2004; Mason et al., 2014). In the injury site, many inflammatory cytokines (such as IL

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Fig. 5. Expression of endocrine disruption-related genes in zebrafish expose to three pesticides and their mixtures. M: Malathion; C: Chlorpyrifos; L: Lambda-cyhalothrin *P < 0.05; **P < 0.01.

and Tnf) can mediate the recruitment process of phagocytes, and chemokines (such as Cxcl) can activate the leukocytes in inflammatory response (Baggiolini et al., 1994; Jugan et al., 2010). Previous studies have revealed that the expressions of immune-related genes may be regulated by environmental compounds (Eder et al., 2008; Li et al., 2012; Rymuszka, 2013). The results of this study showed that the expressions of IL and Tnf had obvious changes in most of the individual and combined groups compared with the control, implying that the defense function of immune system in zebrafish was attempted when exposed to these pesticides. Additionally, the expressions of IL and Tnf were markedly decreased in the MAL þ LCY-treated group compared with the control, implying that strong inflammatory response was triggered by this binary exposure (Yang et al., 2018). It was worth noting that the expression of Cxcl was induced by over 5-fold in the MAL þ CHL þ LCY group at the high concentration. These findings indicated that zebrafish needed more leukocytes to eliminate the inflammation triggered by this ternary mixture of pesticides (Wang et al., 2019). The above-mentioned results might warn that the combined application of MAL, CHL and LCY should be cautious because of potential risk to immune system of humans in the waxberry production. Apoptosis exerts a crucial effect on the development of cell and tissue, which is mediated by a series of related genes, including

Cas8, Cas9, Bax and P53 (Lockshin and Williams, 1965). The analysis of caspase gene expression is believed to be an effective method to estimate the progress of apoptosis (Wang et al., 2018). Previous studies have proved that Cas8 is involved in the extrinsic pathway, and Cas9 plays a fundamental role in the intrinsic pathway (Jin and El-Deiry, 2005). In the present study, the expression of Cas8 was dramatically inhibited in the LCY individual group compared with the control. However, the expression of Cas9 was obviously increased in combined groups of MAL þ LCY, CHL þ LCY and MAL þ CHL þ LCY at medium and high concentrations. These results showed that cell apoptosis of zebrafish embryo was triggered in different treatment groups through the different pathways (Yang et al., 2018). The low expression of P53 is considered to benefit tumor development (Marcel et al., 2015). The expression of P53 was distinctly down-regulated in most of combined groups compared with the control, indicating that the zebrafish were under the risk of tumor development induced by these combinations (Wang et al., 2018). Over-expression of Bax can facilitate the apoptosis of mitochondria and lead to cell death (Maes et al., 2017). Therefore, the decreased Bax expression in this study was worthy to be further investigated. The results of toxicity test showed that LCY exhibited the greatest toxicity to zebrafish embryo than MAL and CHL. Meanwhile, the expressions of antioxidation and apoptosis related genes

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Fig. 6. Heat map analysis for comparing overall changes of mRNA expression. M: Malathion; C: Chlorpyrifos; L: Lambda-cyhalothrin.

exhibited more changes in the LCY group compared with MAL and CHL groups. Taken together, we surmised that LCY showed greater toxicity to zebrafish embryo through destroying antioxidant system and inducing apoptosis (Li et al., 2018a). The combined group of MAL þ CHL could improve the antioxidant ability of zebrafish by inducing the expression of CuSOD, leading to antagonistic responses in joint toxicity test. Meanwhile, the mixtures of MAL þ LCY, CHL þ LCY, MAL þ CHL þ LCY exhibited synergistic responses, which could be attributed to the expression changes of related genes, such as decrease of Cat expression, or increase of Cxcl and Cas9 expressions (Wang et al., 2019). Thyriod hormones (THs) play very important roles in the progress of growth and metabolism in fish (Crane et al., 2004; Orozco et al., 2002). THs exhibit the biological activity through binding thyroid hormone receptor (TR) isoforms (TRa and TRb), and then regulate the expressions of downstream genes (Power et al., 2001; Yang et al., 2007). The abnormal expressions of TRs may lead to the failure of THs to bind and activate the post-receptor response cascades (Liu et al., 2011). Many evidences have shown that the expressions of TRa and TRb in zebrafish are significantly elevated upon exposure to hexaconazole (Yu et al., 2013). The opposite results of TR isoform expressions in zebrafish are found when exposed to acetochlor (Shi et al., 2009). In our study, the expression of TRa was apparently diminished by over 10-fold in the binary group of MAL þ LCY compared with the individual groups of MAL and LCY, indicating that the T3 level was strongly suppressed when MAL and LCY were used in combination (Chen et al., 2012). Nevertheless, the expression of TRa was induced in the binary group of MAL þ CHL, suggesting that different pesticide combinations could regulate the T3 level in zebrafish with different effects. Additionally, the expression of TRb was pronouncedly inhibited in the MAL þ CHL þ LCY-treated group and MAL þ LCY-treated group at medium and high concentrations. These findings revealed that the T3 level was decreased by down-regulating the expressions of different TR isoforms in different exposure groups (Walter et al., 2019). TSH and CRH proteins can regulate the HPT axis in fish through altering in the concentrations of circulating THs. Therefore, Tsh and

Crh genes can be considered as biomarkers at the molecular level for evaluation of thyroid function (De Groef et al., 2006; Yu et al., 2011). In this study, the expression of Tsh was apparently weakened in the CHL-, LCY-, MAL þ LCY- and CHL þ LCY-treated groups compared with the control, indicating that the T4 level was increased in accordance with the negative feedback mechanism (Lema et al., 2009; Chen et al., 2012). However, the expression of Crh was markedly up-regulated in MAL þ CHL- and MAL þ CHL þ LCY-treated groups compared with the control, suggesting that the T4 level was down-regulated in these groups. These changes warned that the risk to thyroid system should not be neglected when exposed to individual or combinations of these pesticides. Estrogen is an important sex hormone, which can regulate the development and reproduction in zebrafish, particularly in female zebrafish (Yilmaz et al., 2018). Other studies have revealed that the abnormal expression of Cyp19a affects the estrogen synthesis, leading to the impaired development in fish (Hinfray et al., 2006; Marlatt et al., 2008). Our results exhibited that the expression of Cyp19a was notably changed in many groups of MAL, CHL, MAL þ LCY, CHL þ LCY and MAL þ CHL þ LCY. These findings indicated that different pesticide exposures caused differently altered estrogen quantity, which impeded the growth of zebrafish. ERa protein is estrogen receptor, which can bind to estrogen to exert its biological effects (Bertotto et al., 2019). The results of our study showed that the expression of ERa was increased or decreased without regularity on different pesticide exposures. These findings might imply that the balance of estrogen level in zebrafish was disturbed by pesticide exposure, leading to hindrance of growth (Cao et al., 2019). Heatmap is an effective method for comparing different gene expression patterns upon pesticide challenge (Toyota et al., 2014). The results of our study displayed that location of the different treatment groups crossed each other in the map except for CHL- and MAL þ CHL-treated group, suggesting that there were more differences in gene expression among the different pesticide exposures.

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5. Conclusions LCY had the greatest toxicity among the three determined pesticides, followed by CHL. On the contrary, MAL exhibited the least toxicity to D. rerio. The embryonic stage of D. rerio was tolerant to most of the elevated pesticides among various developmental stages. Synergistic effects were found from the two binary mixtures of LCY in combination with MAL or CHL and the ternary mixture of MAL þ CHL þ LCY. The increased toxicity of the pesticide mixtures suggested that the employ of toxicity data from individual pesticide tests might underrate the ecological risk of pesticides that actually present under the field conditions. Additionally, sensitive variation in gene transcriptions of Tnf, P53, TRa, Crh and Cyp19a might provide early warning. Furthermore, it is indispensable to survey the validity of these genes as feasible molecular biomarkers in the subsequent studies, and these results provided useful information in evaluating and ranking pesticides according to their potential impact. Collectively, our findings offered valuable insights into the balanced crop protection and best management practice guidelines to mitigate potential risks from these pesticides. Acknowledgments The authors acknowledge the technical assistance of Hongchen Wang and Jian Li (Zhejiang Academy of Agricultural Sciences). The research was supported by the National Key Research and Development Program of China (Grant No. 2018YFC1603004), the Special Fund for Agro-scientific Research in the Public Interest (Grant No. 201503107) and the Project Grant of Key Laboratory of Detection for Pesticide Residues, Ministry of Agriculture of China (2016PRG02). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.124802. References Baggiolini, M., Dewald, B., Moser, B., 1994. Interleukin-8 and related chemotactic cytokines- CXC and CC chemokines. Adv. Immunol. 55, 97e179. Belanger, S.E., Rawlings, J.M., Carr, G.J., 2013. Use of fish embryo toxicity tests for the prediction of acute fish toxicity to chemicals. Environ. Toxicol. Chem. 32 (8), 1768e1783. Belden, J.B., Brain, R.A., 2018. Incorporating the joint toxicity of co-applied pesticides into the ecological risk assessment process. Integr. Environ. Assess. Manag. 14, 79e91. Bertotto, L.B., Dasgupta, S., Vliet, S., Dudley, S., Gan, J., Volz, D.C., Schlenk, D., 2019. Evaluation of the estrogen receptor alpha as a possible target of bifenthrin effects in the estrogenic and dopaminergic signaling pathways in zebrafish embryos. Sci. Total Environ. 651 Pt 2, 2424e2431.
n, J., Contreras, J., 2007. Synergism studies Bielza, P., Espinosa, P.J., Quinto, V., Abella with binary mixtures of pyrethroid, carbamate and organophosphate insecticides on Frankliniella occidentalis (Pergande). Pest Manag. Sci. 63 (1), 84e89. Braunbeck, T., Kais, B., Lammer, E., Otte, J., Schneider, K., Stengel, D., Strecker, R., 2015. The fish embryo test (FET): origin, applications, and future. Environ. Sci. Pollut. Res. 22, 16247e16261. Cao, J., Wang, G., Wang, T., Chen, J., Wenjing, G., Wu, P., He, X., Xie, L., 2019. Copper caused reproductive endocrine disruption in zebrafish (Danio rerio). Aquat. Toxicol. 211, 124e136. Cedergreen, N., Dalhoff, K., Li, D., Gottardi, M., Kretschmann, A.C., 2017. Can toxicokinetic and toxicodynamic modeling be used to understand and predict synergistic interactions between chemicals? Environ. Sci. Technol. 51 (24), 14379e14389. Chen, Q., Yu, L.Q., Yang, L.H., Zhou, B.S., 2012. Bioconcentration and metabolism of decabromodiphenyl ether (BDE-209) result in thyroid endocrine disruption in zebrafish larvae. Aquat. Toxicol. 110, 141e148. Chen, X., Li, H., Zhang, J., Ding, Y., You, J., 2016. Does cadmium affect the toxicokinetics of permethrin in Chironomus dilutus at sublethal level? Evidence of enzymatic activity and gene expression. Environ. Pollut. 218, 1005e1013. Chi, H., 1997. Computer Program for the Probit Analysis. National Chung Hsing University, Taichung, Taiwan. Cordova, L.G., Amiri, A., Peres, N.A., 2017. Effectiveness of fungicide treatments

following the Strawberry Advisory System for control of Botrytis fruit rot in Florida. Crop Protect. 100, 163e167. Crane, H.M., Pickford, D.B., Hutchinson, T.H., Brown, J.A., 2004. Developmental changes of thyroid hormones in the fathead minnow, Pimephales promelas. Gen. Comp. Endocrinol. 139, 55e60. Cui, F., Chai, T., Liu, X., Wang, C., 2017. Toxicity of three strobilurins (kresoximmethyl, pyraclostrobin, and trifloxystrobin) on Daphnia magna. Environ. Toxicol. Chem. 36, 182e189. Dalhoff, K., Gottardi, M., Kretschmann, A., Cedergreen, N., 2016. What causes the difference in synergistic potentials of propiconazole and prochloraz toward pyrethroids in Daphnia magna? Aquat. Toxicol. 172, 95e102. De Groef, B., Van der Geyten, S., Darras, V.M., Kühn, E.R., 2006. Role of corticotropinreleasing hormone as a thyrotropin-releasing factor in nonmammalian vertebrates. Gen. Comp. Endocrinol. 146, 62e68. Domingues, I., Oliveira, R., Lourenço, J., Grisolia, C.K., Mendo, S., Soares, A.M.V.M., 2010. Biomarkers as a tool to assess effects of chromium (VI): comparison of responses in zebrafish early life stages and adults. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 152, 338e345. €hler, H.R., Werner, I., 2008. Expression of Eder, K.J., Clifford, M.A., Hedrick, R.P., Ko immune-regulatory genes in juvenile Chinook salmon following exposure to pesticides and infectious hematopoietic necrosis virus (IHNV). Fish Shellfish Immunol. 25, 508e516. Embry, M.R., Belanger, S.E., Braunbeck, T.A., Galay-Burgos, M., Halder, M.,
onard, M.A., Lillicrap, A., Norberg-King, T., Whale, G., 2010. The Hinton, D.E., Le fish embryo toxicity test as an animal alternative method in hazard and risk assessment and scientific research. Aquat. Toxicol. 97, 79e87. Ge, W., Yan, S., Wang, J., Zhu, L., Chen, A., Wang, J., 2015. Oxidative stress and DNA damage induced by imidacloprid in zebrafish (Danio rerio). J. Agric. Food Chem. 63 (6), 1856e1862. Grung, M., Lin, Y., Zhang, H., Steen, A.O., Huang, J., Zhang, G., Larssen, T., 2015. Pesticide levels and environmental risk in aquatic environments in China–A review. Environ. Int. 81, 87e97. Guo, D., Wang, Y., Qian, Y., Chen, C., Jiao, B., Cai, L., Wang, Q., 2017. Joint acute and endocrine disruptive toxicities of malathion, cypermethrin and prochloraz to embryo-larval zebrafish, Danio rerio. Chemosphere 166, 63e71. Hassold, E., Backhaus, T., 2014. The predictability of mixture toxicity of demethylase inhibiting fungicides to Daphnia magna depends on life-cycle parameters. Aquat. Toxicol. 152, 205e214. He, L.M., Trolano, J., Wang, A., Goh, K., 2008. Environmental chemistry, ecotoxicology, and fate of lambda-cyhalothrin. Rev. Environ. Contam. Toxicol. 72e91.
ndez, A.F., Gil, F., Lacasan ~ a, M., 2017. Toxicological interactions of pesticide Herna mixtures: an update. Arch. Toxicol. 91, 3211e3223.
ndez, A.F., Parro
n, T., Tsatsakis, A.M., Requena, M., Alarco
n, R., Lo
pezHerna Guarnido, O., 2013. Toxic effects of pesticide mixtures at a molecular level: their relevance to human health. Toxicology 307, 136e145. Hinfray, N., Palluel, O., Turies, C., Cousin, C., Porcher, J.M., Brion, F., 2006. Brain and gonadal aromatase as potential targets of endocrine disrupting chemicals in a model species, the zebrafish (Danio rerio). Environ. Toxicol. 21, 332e337. Hong, X., Zhao, X., Tian, X., Li, J., Zha, J., 2018. Changes of hematological and biochemical parameters revealed genotoxicity and immunotoxicity of neonicotinoids on Chinese rare minnows (Gobiocypris rarus). Environ. Pollut. 233, 862e871. Icoglu, A.F., Ciltas, A., 2018. Developmental toxicity of penconazole in Zebrfish (Danio rerio) embryos. Chemosphere 200, 8e15. ISO, 1996. Water Quality-Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio Rerio Hamilton-Buchanan (Teleostei, Cyprinidae)]-Part 3: Flow-Through Method. ISO 7346-3. Jeon, H.J., Lee, Y.H., Kim, M.J., Choi, S.D., Park, B.J., Lee, S.E., 2016. Integrated biomarkers induced by chlorpyrifos in two different life stages of zebrafish (Danio rerio) for environmental risk assessment. Environ. Toxicol. Pharmacol. 43, 166e174. Jia, Z.Q., Liu, D., Sheng, C.W., Casida, J.E., Wang, C., Song, P.P., Chen, Y.M., Han, Z.J., Zhao, C.Q., 2018. Acute toxicity, bioconcentration, elimination and antioxidant effects of fluralaner in zebrafish, Danio rerio. Environ. Pollut. 232, 183e190. Jin, Z., El-Deiry, W.S., 2005. Review overview of cell death signaling pathways. Cancer Biol. Ther. 4, 139e163. Jugan, M.L., Levi, Y., Blondeau, J.P., 2010. Endocrine disruptors and thyroid hormone physiology. Biochem. Pharmacol. 79, 939e947. €nig, M., Ortmann, J., Massei, R., Paschke, A., Kühne, R., Scholz, S., 2015. Klüver, N., Ko 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. Kumar, K., Ansari, B.A., 1984. Malathion toxicity: skeletal deformities in zebrafish (Brachydanio rerio, Cyprinidae). Pestic. Sci. 15, 107e111. Lam, S.H., Chua, H.L., Gong, Z., Lam, T.J., Sin, Y.M., 2004. Development and maturation of the immune system in zebrafish Danio rerio: a gene expression profiling, in situ hybridization and immunological study. Dev. Comp. Immunol. 28, 9e28. Lammer, E., Carr, G.J., Wendler, K., Rawlings, J.M., Belanger, S.E., Braunbeck, T., 2009. Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comp. Biochem. Phys. C: Toxicol. Pharmacol. 149, 196e209. Lema, S.C., Dickey, J.T., Schultz, I.R., Swanson, P., 2009. Thyroid hormone regulation of mRNAs encoding thyrotropin beta-subunit, glycoprotein alpha-subunit, and thyroid hormone receptors alpha and beta in brain, pituitary gland, liver, and

W. Shen et al. / Chemosphere 239 (2020) 124802 gonads of an adult teleost, Pimephales promelas. J. Endocrinol. 202, 43e54. Levine, S.L., Borgert, C.J., 2018. Review and recommendations on criteria to evaluate the relevance of pesticide interaction data for ecological risk assessments. Chemosphere 209, 124e136. Li, G., Yan, W., Qiao, Q., Chen, J., Cai, F., He, Y., Zhang, X., 2012. Global effects of subchronic treatment of microcystin-LR on rat splenetic protein levels. J. Proteomics 77, 383e393. Li, C., Cao, M., Ma, L., Ye, X., Song, Y., Pan, W., Xu, Z., Ma, X., Lan, Y., Chen, P., Liu, W., Liu, J., Zhou, J., 2018a. Pyrethroid pesticide exposure and risk of primary ovarian insufficiency in Chinese women. Environ. Sci. Technol. 52 (5), 3240e3248. Li, H., Yu, S., Cao, F., Wang, C., Zheng, M., Li, X., Qiu, L., 2018b. Developmental toxicity and potential mechanisms of pyraoxystrobin to zebrafish (Danio rerio). Ecotoxicol. Environ. Saf. 151, 1e9. Liu, L., Jiang, C., Wu, Z.Q., Gong, Y.X., Wang, G.X., 2013. Toxic effects of three strobilurins (trifloxystrobin, azoxystrobin and kresoxim-methyl) on mRNA expression and antioxidant enzymes in grass carp (Ctenopharyngodon idella) juveniles. Ecotoxicol. Environ. Saf. 98, 297e302. Liu, S.Y., Chang, J.H., Zhao, Y., Zhu, G.N., 2011. Changes of thyroid hormone levels and related gene expression in zebrafish on early life stage exposure to triadimefon. Environ. Toxicol. Pharmacol. 32, 472e477. Lockshin, R.A., Williams, C.M., 1965. Programmed cell death-I. Cytology of degeneration in the intersegmental muscles of the Pernyi silkmoth. J. Insect Physiol. 11 (2), 123e133. Lu, N.N., Li, R., Song, W.C., Sun, S.H., Jia, R.B., 2016. Study on behavioral change of zebrafish exposed to malathion and chlorothalonil. Asian J. Ecotoxicol. 11 (1), 369e374. Maes, M.E., Schlamp, C.L., Nickells, R.W., 2017. BAX to basics: how the BCL2 gene family controls the death of retinal ganglion cells. Prog. Retin. Eye Res. 57, 1e25. Maharajan, K., Muthulakshmi, S., Nataraj, B., Ramesh, M., Kadirvelu, K., 2018. Toxicity assessment of pyriproxyfen in vertebrate model zebrafish embryos (Danio rerio): a multi biomarker study. Aquat. Toxicol. 196, 132e145. Marcel, V., Catez, F., Diaz, J.J., 2015. p53, a translational regulator: contribution to its tumour-suppressor activity. Oncogene 34 (44), 5513e5523. Marking, L.L., 1985. Toxicity of chemical mixtures. In: Rand, G., Petroceli, S. (Eds.), Fundamentals of Aquatic Toxicology. Hemisphere Publishing Corporation Washington DC pp164e176. Marlatt, V.L., Martyniuk, C.J., Zhang, D., Xiong, H., Watt, J., Xia, X., Moon, T., Trudeau, V.L., 2008. Auto-regulation of estrogen receptor subtypes and gene expression profiling of 17 beta-estradiol action in the neuroendocrine axis of male goldfish. Mol. Cell. Endocrinol. 283, 38e48. Mason, R., Tennekes, H., S_anchez-Bayo, F., Jepsen, P.U., 2014. Immune suppression by neonicotinoid insecticides at the root of global wildlife declines. J. Environ. Immunol. Toxicol. 1 (1), 3e12. 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. Mu, X., Shen, G., Huang, Y., Luo, J., Zhu, L., Qi, S., Li, Y., Wang, C., Li, X., 2017. The enantioselective toxicity and oxidative stress of beta-cypermethrin on zebrafish. Environ. Pollut. 229, 312e320. OECD, 1992. OECD Guidelines for the Testing of Chemicals, Fish, Acute Toxicity Test. OECD, Paris, France. No, p. 203. OECD, 2013. OECD Guidelines for the Testing of Chemicals, Fish Embryo Acute Toxicity (FET) Test. OECD, Paris, France. No, p. 236. Orozco, A., Villalobos, P., Valverde, R.C., 2002. Environmental salinity selectively modifies the outer-ring deiodinating activity of liver, kidney and gill in the rainbow trout. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 131, 387e395. Power, D.M., Llewellyn, L., Faustino, M., Nowell, M.A., Bjornsson, B.T., Einarsdottir, I.E., Canario, A.V.M., Sweeney, G.E., 2001. Thyroid hormones in growth and development of fish. Comp. Biochem. Physiol., C 130, 447e459. Rizzati, V., Briand, O., Guillou, H., Gamet-Payrastre, L., 2016. Effects of pesticide mixtures in human and animal models: an update of the recent literature. Chem. Biol. Interact. 254, 231e246. Rymuszka, A., 2013. Microcystin-LR induces cytotoxicity and affects carp immune cells by impairment of their phagocytosis and the organization of the

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cytoskeleton. J. Appl. Toxicol. 33, 1294e1302. Sanches, A.L.M., Vieira, B.H., Reghini, M.V., Moreira, R.A., Freitas, E.C., Espíndola, E.L.G., Daam, M.A., 2017. Single and mixture toxicity of abamectin and difenoconazole to adult zebrafish (Danio rerio). Chemosphere 188, 582e587. €cs, E., Bhowmik, A.K., Vijver, M.G., Scha €fer, R.B., 2016. Pesticide Schreiner, V.C., Szo mixtures in streams of several European countries and the USA. Sci. Total Environ. 573, 680e689. Sewell, T.R., Moloney, S., Ashworth, M., Ritchie, F., Mashanova, A., Huang, Y.J., Stotz, H.U., Fitt, B.D.L., 2016. Effects of a penthiopyrad and picoxystrobin fungicide mixture on phoma stem canker (Leptosphaeria spp.) on UK winter oilseed rape. Eur. J. Plant Pathol. 145, 675e685. Shi, X.J., Liu, C.S., Wu, G.Q., Zhou, B.S., 2009. Waterborne exposure to PFOS causes disruption of the hypothalamus-pituitary-thyroid axis in zebrafish larvae. Chemosphere 77, 1010e1018. Shukla, S., Jhamtani, R.C., Dahiya, M.S., Agarwal, R., 2017. Oxidative injury caused by individual and combined exposure of neonicotinoid, organophosphate and herbicide in zebrafish. Toxicol. Rep. 4, 240e244. Su, L.S., Yang, G.L., Wu, S.G., Pi, T.X., Wang, Q., 2016. The single and joint toxicity of tiazophos and cyhalothrin to earthworm. Asian J. Ecotoxicol. 11, 294e301. Toyota, K., Kato, Y., Miyakawa, H., Yatsu, R., Mizutani, T., Ogino, Y., Miyagawa, S., Watanabe, H., Nishide, H., Uchiyama, I., Tatarazako, N., Iguchi, T., 2014. Molecular impact of juvenile hormone agonists on neonatal Daphnia magna. J. Appl. Toxicol. 34 (5), 537e544. Velki, M., Meyer-Alert, H., Seiler, T.B., Hollert, H., 2017. Enzymatic activity and gene expression changes in zebrafish embryos and larvae exposed to pesticides diazinon and diuron. Aquat. Toxicol. 193, 187e200. Walter, K.M., Miller, G.W., Chen, X., Yaghoobi, B., Puschner, B., Lein, P.J., 2019. Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (Danio rerio). Gen. Comp. Endocrinol. 272, 20e32. Wang, Y.H., Lv, L., Yu, Y.J., Yang, G.L., Xu, Z.L., Wang, Q., Cai, L.M., 2017. Single and joint toxic effects of five selected pesticides on the early life stages of zebrafish (Denio rerio). Chemosphere 170, 61e67. Wang, X., Wei, L., Wang, Y., He, B., Kong, B., Zhu, J., Jin, Y., Fu, Z., 2019. Evaluation of development, locomotor behavior, oxidative stress, immune responses and apoptosis in developing zebrafish (Danio rerio) exposed to TBECH (tetrabromoethylcyclohexane). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 217, 106e113. Wang, Y., Dai, D., Yu, Y., Yang, G., Shen, W., Wang, Q., Weng, H., Zhao, X., 2018. Evaluation of joint effects of cyprodinil and kresoxim-methyl on zebrafish, Danio rerio. J. Hazard Mater. 352, 80e91. Wu, S., Li, X., Liu, X., Yang, G., An, X., Wang, Q., Wang, Y., 2018. Joint toxic effects of triazophos and imidacloprid on zebrafish (Danio rerio). Environ. Pollut. 235, 470e481. Yang, X., Xie, J., Wu, T., Yue, G., Chen, J., Zhao, R., 2007. Hepatic and muscle expression of thyroid hormone receptors in association with body and muscle growth in large yellow croaker, Pseudosciaena crocea (Richardson). Gen. Comp. Endocrinol. 151, 163e171. Yang, Y., Dong, F., Liu, X., Xu, J., Wu, X., Liu, W., Zheng, Y., 2018. Crosstalk of oxidative damage, apoptosis, and autophagy under endoplasmic reticulum (ER) stress involved in thifluzamide-induced liver damage in zebrafish (Danio rerio). Environ. Pollut. 243 Pt B, 1904e1911. Yang, Y., Qi, S., Wang, D., Wang, K., Zhu, L., Chai, T., Wang, C., 2016. Toxic effects of thifluzamide on zebrafish (Danio rerio). J. Hazard Mater. 307, 127e136. Yilmaz, O., Patinote, A., Nguyen, T., Bobe, J., 2018. Multiple vitellogenins in zebrafish (Danio rerio): quantitative inventory of genes, transcripts and proteins, and relation to egg quality. Fish Physiol. Biochem. 44 (6), 1509e1525. Yu, L., Chen, M.L., Liu, Y.H., Gui, W.J., Zhu, G.N., 2013. Thyriod endocrine disruption in zebrafish larvae following exposure to hexaconazole and tebuconazole. Aquat. Toxicol. 138e139, 35e42. Yu, L.Q., Lam, J.C.W., Guo, Y.Y., Wu, R.S.S., Lam, P.K.S., Zhou, B.S., 2011. Parental transfer of polybrominated diphenyl ethers (PBDEs) and thyroid endocrine disruption in zebrafish. Environ. Sci. Technol. 45, 10652e10659.