larval zebrafish (Danio rerio)

Chemosphere 243 (2020) 125416

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The pyrethroid esfenvalerate induces hypoactivity and decreases dopamine transporter expression in embryonic/larval zebrafish (Danio rerio) Xiao H. Wang a, b, Christopher L. Souders II b, Priscilla Xavier b, Xiao Y. Li a, Bing Yan a, **, Christopher J. Martyniuk b, * a

Institute of Environmental Research at Greater Bay Area, Key Laboratory for Water Quality and Conservation of the Pearl River Delta, Ministry of Education, Guangzhou University, Guangzhou, 510006, China Center for Environmental and Human Toxicology, Department of Physiological Sciences, College of Veterinary Medicine, UF Genetics Institute, Interdisciplinary Program in Biomedical Sciences Neuroscience, University of Florida, Gainesville, FL, 32611, USA

b

h i g h l i g h t s
Esfenvalerate is a pyrethroid insecticide that affects zebrafish behaviors and is neurotoxic.
Dopamine active transporter mRNA was decreased by environmentally-relevant levels of esfenvalerate.
Esfenvalerate at environmentally-relevant levels induced hypoactivity, effects that were dependent upon age.
A comprehensive table summarizes data on pyrethroids in zebrafish as a resource for future experiments.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 September 2019 Received in revised form 15 November 2019 Accepted 19 November 2019 Available online 22 November 2019

Esfenvalerate is a pyrethroid insecticide used widely for agricultural and residential applications. This insecticide has been detected in aquatic environments at concentrations that can induce sub-lethal effects in organisms. In this study, zebrafish embryos were used to examine the effects of environmentally-relevant concentrations of esfenvalerate on development and behavior. It was hypothesized that esfenvalerate exposure would impair locomotion due to its effects on the central nervous system. We also measured mitochondrial bioenergetics and the expression of genes (dopamine system) as putative mechanisms of locomotor impairment. Concentrations of 0.02, 0.2 and 2 mg/L esfenvalerate did not induce significant mortality nor deformity in zebrafish, but there was an acceleration in hatching time for zebrafish exposed to 2 mg/L esfenvalerate. As an indicator of neurotoxicity, the Visual Motor Response (VMR) test was conducted with 5, 6, and 7 dpf zebrafish after continuous exposure, and higher concentrations were used (4 and 8 mg/L esfenvalerate) to better discern age-and dose dependent responses in behavior. Experiments revealed that, unlike the other stages, 6 dpf larvae showed evidence for hypo-activity with esfenvalerate, suggesting that different stages of larval development may show increased sensitivity to pyrethroid exposure. This may be related to age-dependent maturation of the central nervous system. We hypothesized that reduced larval activity may be associated with impaired production of ATP and the function of mitochondria at earlier life stages, however dramatic alterations in oxidative phosphorylation were not observed. Based on evidence that dopamine regulates behavior and studies showing that other pyrethroids affect dopamine system, we measured transcripts involved in dopaminergic signaling. We found that dopamine active transporter was down-regulated with 0.2 mg/L esfenvalerate. Lastly, we comprehensively summarize the current literature (>20 studies) regarding the toxicity of pyrethroids in zebrafish, which is a valuable resource to those studying these pesticides. This study demonstrates that esfenvalerate at environmentally-relevant levels induces hypoactivity that are dependent upon the age of the zebrafish, and these behavioral changes are hypothesized to be related to impaired dopamine signaling. © 2019 Elsevier Ltd. All rights reserved.

Handling Editor: David Volz Keywords: Aquatic toxicology Zebrafish Pyrethroid Locomotor behavior Dopamine system

* Corresponding author. ** Corresponding author. E-mail address: cmartyn@ufl.edu (C.J. Martyniuk). https://doi.org/10.1016/j.chemosphere.2019.125416 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

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X.H. Wang et al. / Chemosphere 243 (2020) 125416

1. Introduction Pyrethroid insecticides have replaced organophosphates as the most frequently used class of insecticides for agricultural and urban use (Deanovic et al., 2018). The widespread use of pyrethroids subsequently leads to the detection of pyrethroids in aquatic environments, at concentrations that can cause sub-lethal effects in aquatic organisms (Siegler et al., 2015; Awoyemi et al., 2019). Fenvalerate is an extensively used pyrethroid insecticide developed in 1976, and is a chiral compound with four enantiomers, i.e. aS-2S; aR-2S; aS-2R; aR-2R. The aS-2S is the most biologically active enantiomer, and is named esfenvalerate. This chemical has a high efficacy against target species, and is also the most toxic enantiomer to non-target aquatic species (Danio rerio and Daphnia magna) (Ma et al., 2009; Rasmussen et al., 2013). According to US EPA, 2008 and EFSA, 2014, esfenvalerate was identified as one with high toxicity to fish, on the basis of acute medium lethal concentrations, with LC50 values ranging from 0.07 to 0.69 mg/L. In aquatic habitats, surface runoff and spray drift can place aquatic organisms at risk, and according to the maximal application rate, esfenvalerate has been estimated at concentrations ranging from 0.02 mg/L to 6.46 mg/L in the environment (agricultural applications, ranging from 0.02 mg/L to 1.11 mg/L, and for non-agricultural uses, ranging from 0.05 mg/L to 6.47 mg/L) (US EPA, 2008). Additionally, esfenvalerate has been detected in aquatic environments in the USA (American River, Colusa Basin Drain, Sacramento River, and San Joaquin River) ranging from 0.025 mg/L to 0.76 mg/L (Cooper et al., 2003; Bacey et al., 2005; Brady et al., 2006; Hladik and Kuivila, 2009; Fojut et al., 2017; Münze et al., 2017). Therefore, esfenvalerate remains an environmental concern in some aquatic environments. The mode of action of the pyrethroids is binding and modulation of the activity of the voltage-gated sodium channels, leading to prolonged opening of sodium channels, and continuous firing of action potential (Soderlund, 2012). This neurotoxic propensity impairs animal locomotor behaviors, commonly noted as hyperactivity and convulsions, followed by lethargy, paralysis and death (Palmquist et al., 2012; Awoyemi et al., 2019). Thus, esfenvalerate acts by impairing normal feeding behaviors of target insects, and this is related to its mode of action. In fact, esfenvalerate has been classified as one of the fastest acting insecticides that cause feeding cessation (Rodrigues et al., 2017). The high potential for accumulation of esfenvalerate due to weak activity of metabolizing enzymes in some fish species, coupled with potential negative impacts on other neuronal systems, suggests that esfenvalerate may be harmful to non-target fish species (Suvetha et al., 2015; Wang et al., 2016). For example, larval fathead minnows (Pimephales promelas) exposed to 0.455 and 1.142 mg/L esfenvalerate for 4 h showed evidence for abnormal behaviors, including impaired swimming and feeding-related activities (Palmquist et al., 2012). In another study, bluegill sunfish (Lepomis macrochirus) chronically exposed to 0.01e0.05 mg/L esfenvalerate for 90 days were observed to have a reduced number of aggressive interactions between individual fish (Palmquist et al., 2012). Beyond these behavioral effects, developmental and reproductive capacities in fish species have also been noted with esfenvalerate exposure. For example, a significant negative correlation was observed between the fecundity of Australian rainbowfish (Melanotaenia fluviatilis) and the dose of esfenvalerate (1 mg/L, 3.2 mg/L, 10 mg/L, 32 mg/L), according to an averaged breeding regime within 6-days of monitoring (Prusty et al., 2015). In the same study, the hatch rate of zebrafish embryos during the 6-day period was significantly decreased under all tested concentrations of esfenvalerate. Moreover, bluegill sunfish (Lepomis macrochirus) were observed to have retarded growth and

delayed spawning with 1 mg/L esfenvalerate exposure over ~ two months, and the results showed that the sunfish young of the year growth were reduced by 62%, 57% and 86%, respectively, after exposure to 0.08 mg/L, 0.20 mg/L and 1 mg/L esfenvalerate (Prusty et al., 2015). Thus, there can be multiple adverse effects following esfenvalerate exposure that are related to development, reproduction, and behavior impairments. Zebrafish are a widely applied toxicological model for studying the effects of environmental chemicals in aquatic organisms, and zebrafish embryos/larvae have been used to investigate the toxicity of pyrethroid insecticides. These studies have revealed significant oxidative damage responses following exposure to these chemicals. For example, in pyrethroid-exposed embryo-larval zebrafish, the total reactive oxygen level (ROS) was increased and there was a positive correlation with dose and carboxylesterase (CES) activities (Awoyemi et al., 2019). This is significant as CES is considered to be one of the key esterases that metabolize pyrethroid insecticides. In other studies, superoxide dismutase and catalase activity, total glutathione (as a non-enzymatic antioxidant defense), lipid peroxidation, and malonaldehyde (measure of oxidative damage), have been measured to be lower (or higher in some cases) following different pyrethroid exposures compared to control groups (Burgess and Granato, 2007; DeMicco et al., 2010; Fernandes et al., 2012; Han et al., 2017; Mendis et al., 2018; Mu et al., 2014; Mueller and Neuhauss, 2012; Shi et al., 2011; Strungaru et al., 2019; Xu et al., 2008; Yang et al., 2014). These studies support the hypothesis that oxidative stress is a relevant event that underlies pyrethroid-induced toxicity. This study examined the effects of environmentally relevant concentrations of esfenvalerate on embryonic-larval zebrafish. Development, mitochondrial bioenergetics, and locomotor behaviors were measured as well as the expression of genes associated with the dopamine system. Mitochondrial bioenergetics was assessed because of evidence that oxidative stress responses and ROS production are increased with pyrethroid exposure in fish, and due to evidence that other pesticides affect oxygen consumption rates in zebrafish embryos (Wang et al., 2018; Perez-Rodrigues et al., 2019). Locomotor activity was measured using a Visual Motor Response (VMR) assay as studies show pyrethroids affect feeding behavior of fish, as well as locomotor activity (Frank et al., 2018; Awoyemi et al., 2019). Locomotor behavior is regulated in part by dopamine, and we hypothesized that changes in the dopamine system may be relevant to pyrethroid-induced changes in behavior. Noteworthy is that pyrethroids affect dopamine active transporter expression in mammals and studies suggest that pyrethroids may also affect the dopamine system in fish, such as zebrafish and rainbow trout (Crago and Schlenk, 2015; Kung et al., 2015; Bertotto et al., 2018). This study presents novel data on sublethal biological responses to esfenvalerate to inform risk assessments of esfenvalerate in aquatic organisms early in development.

2. Materials and methods 2.1. Breeding of zebrafish Adult wildtype zebrafish (AB crossed to Tu strain, Danio rerio) were housed in the Cancer Genetics Research Center (CGRC) of University of Florida. The University of Florida Institutional Animal Care and Use Committee approved the experimental protocols, and experiments adhere to all relevant National Institutes of Health guidelines on the use of animals. Adult zebrafish are maintained at temperatures ~28 ± 1  C with a photoperiod cycle of 16:8 h of light and dark for breeding. Embryos were collected immediately in the morning, transferred to the laboratory from CRGC, and placed into

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embryo rearing medium (ERM) for exposure experiments as per Perez-Rodrigues et al. (2019). 2.2. Animal exposures Esfenvalerate (66230-04-4; purity  99.5%; Sigma, USA) stock solution was prepared in dimethyl sulfoxide (DMSO), and diluted into ERM, to achieve the final exposure concentrations (0.01% DMSO). We first tested 0.02, 0.2, 2 and 20 mg/L esfenvalerate for our exposure, and the results showed zebrafish were healthy without significant mortality and deformity after 96 h exposure with 0.02, 0.2, 2 and 20 mg/L, but were all dead with exposure to 20 mg/L insecticide. We therefore selected 0.02, 0.2, 2 mg/L for our final exposure, as we aimed to capture sub-lethal effects of the insecticide, and these concentrations were also environmentally relevant in aquatic habitats (Cooper et al., 2003; Bacey et al., 2005; Brady et al., 2006; Hladik and Kuivila, 2009; Fojut et al., 2017; Münze et al., 2017). The exposure solutions were prepared fresh daily and renewed every day. Toxicity of esfenvalerate in fish embryos was first assessed with morphological development, mortality rate and hatch time over 4 days. Ninety-six embryos at ~6 h post fertilized (hpf) were transferred into a 96-well plate for exposure (N ¼ 24), and images were recorded by an EVOS FL Auto Imaging System (Life Technologies) to obtain bright field images every hour to assess hatch rate. For assessing locomotor behaviors, we included two higher exposure concentrations of 4 and 8 mg/L based on our earlier experiments. Fish embryos were exposed in glass beakers continuously for 6 days, and then were subsequently transferred into a 96-well plate. For experiments to assess mitochondrial bioenergetics, fish embryos were first exposed in beakers for 24 or 48 h, and then transferred into a 24-well Islet Capture Microplate for the assay. Lastly, for gene expression analysis, fish embryos/larvae were exposed in glass beakers for 96 h, and then flash-frozen using liquid nitrogen and stored at 80  C. 2.3. Mitochondrial bioenergetics The oxygen consumption rate (OCR) of whole fish embryos, after a 24 or 48 h exposure, was determined by the XFe24 Extracellular Flux Analyzer (Seahorse Bioscience, Agilent Technologies). A 24well Islet Capture Microplate was used to accommodate fish embryos, and one embryo was placed into each well with 0.01% DMSO, or one dose of either 0.02, 0.2, 2 mg/L of esfenvalerate (N ¼ 5). The basal respiration was first measured for 10 measurement cycles. Oligomycin, FCCP (carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone) and sodium azide were immediately added sequentially to assess respiratory sources. There were 18 cycles performed after oligomycin injection to inhibit ATP production, and 8 cycles were performed after FCCP injection to maximize embryonic oxidative respiration. Lastly, non-mitochondrial respiratory rate was determined for 24 measurement cycles after sodium azide injection to completely inhibit mitochondrial oxidative respiratory. The final concentrations per well were 9.4 mM oligomycin, 6 mM FCCP, and 20 mM sodium azide. This protocol consisted of the following time cycles: 2 min for mixing, 1 min pause, and then 2 min oxygen levels determination. Detailed procedures are provided in Wang et al. (2018). 2.4. Larval visual motor response test To assess behavioral responses in larval zebrafish, a visual motor response (VMR) test was conducted with different staged embryos to better define the scope of impact for esfenvalerate. This is important as larvae can show unique patterns of response based on their age (de Esch et al., 2012). Behavioral tests were conducted in

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late morning-early afternoon. We performed three independent VMR tests for zebrafish exposed in 0.02, 0.2 and 2 mg/L esfenvalerate (at 5 dpf, 6 dpf or 7 dpf, respectively, and then performed two independent VMR tests for zebrafish exposed in 2, 4, and 8 mg/L esfenvalerate from 6 hpf to 6 dpf (N ¼ 24). After exposure, larvae were transferred into separate wells in a 96-well plate and transferred into a DanioVision™ Observation Chamber (Noldus Information Technology, Wageningen, Netherlands). In the chamber, fish larvae were simultaneously and individually tracked using an infrared analog camera, and were situated in a 26 ± 1  C circulating water bath throughout the experiment. Following the first period of dark to acclimate to the instrument, the Noldus White Routine was used to test the VMR by exposing larvae to a series of 10 min alternating dark and light periods for a total of 50 min to determine total distance travelled (Wang et al., 2018; Perez-Rodrigues et al., 2019). Group data were binned into a mean value for all zebrafish every minute within an experiment, and data from each of the five time periods were analyzed independently. 2.5. Real-time PCR analysis Zebrafish larvae, after 96 h exposure of 0.02, 0.2 and 2 mg/L esfenvalerate, were pooled from each glass beaker for gene expression analysis (N ¼ 6). Total RNA was extracted from each sample using TRIzol® Reagent (Life Technologies, Carlsbad, CA, USA). After extraction, RNA pellets were reconstituted in the nuclease-free water, and purified using the RNeasy Mini Kit column (Qiagen, Valencia, CA, USA). The Qiagen columns also effectively remove any genomic DNA. The purified RNA samples were assessed for quality using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The RNA integrity number (RIN) for samples ranged from 8.8 to 10 (mean 9.48 ± 0.16). The concentrations of RNA from samples were measured using the Qubit 3.0 Fluorometer (ThermoFisher Scientific, USA). Lastly, 1.5 mg column purified RNA from each sample was used for cDNA synthesis by using iScript (BioRad, USA) based on the manufacturer’s protocol. Real-time PCR analysis was performed using the CFX Connect System with SsoAdvanced Universal SYBR Green Supermix (Biorad, Hercules, CA, USA). Six biological replicates in duplicate were measured for each group. The genes investigated in this study consisted of superoxide dismutase 1 (sod1), superoxide dismutase 2 (sod2), tyrosine hydroxylase 1 (th1), dopamine active transporter (dat), dopamine receptor D1 (drd1b), dopamine receptor D2a (drd2a) and dopamine receptor D2b (drd2b). Three housekeeping genes, ribosomal subunit 18 (rps18), beta-actin (b-actin) and ribosomal protein L13 (rpl13), were assessed for stability and appropriateness for normalization. Using the CFX Manager™ software, a combined M-value of 0.72 with CV-value of 0.24 was achieved. Target genes were therefore normalized to the geometric mean of the three housekeeping genes. Primer sets for target genes were collected from the literature and are listed in Table S1. Normalized gene expression was extracted using the CFX Manager™ software with the relative DDCq method based on Pfaffl (2001). 2.6. Statistical analysis Statistical analysis was performed by Graph-Pad Prism version 6.0 (GraphPad Software Inc., La Jolla, CA, USA). All quantitative data are expressed as mean values ± standard error. For zebrafish behavioral responses, differences between data from each treatment were analyzed with One-way ANOVA following by a Dunnett’s post hoc test. For hatch time, mitochondrial respiration and transcripts analysis, differences between data from each treatment were analyzed with Kruskal-Wallis test following by a Dunn’s post hoc test. A probability of p < 0.05 was considered to be statistically

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significant. All figures were prepared by using Graph-Pad Prism version 6.0 (GraphPad Software Inc., La Jolla, CA, USA). 3. Results 3.1. Esfenvalerate accelerated zebrafish hatching Development of fish embryos were monitored over 96 h, and neither significant mortality nor overt malformation was noted after exposure to esfenvalerate at environmentally-relevant levels. Fish hatched out successfully during the exposure time, but esfenvalerate-treated fish escaped from the chorion earlier than untreated fish. Fish exposed to 2 mg/L of the insecticide hatched out significantly earlier compared to control fish (K ¼ 10.70, p ¼ 0.014) (Table 1).

presented as Fig. 3B and C. In the initial dark period to allow for acclimation and to minimize disturbances, fish behaviors were different among groups (F(3, 36) ¼ 3.19, p ¼ 0.0173 and F (3, 36) ¼ 20, p < 0.0001 in two separate tests). In the following alternating dark and light periods, fish larvae moved more in the dark compared to in the light as expected (Burgess and Granato, 2007; Fernandes et al., 2012; Mueller and Neuhauss, 2012), and fish larvae showed different locomotor behaviors due to esfenvalerate treatment in both periods (F(3, 36) ¼ 7.3, p ¼ 0.0006; F(3, 36) ¼ 15, p < 0.0001; F(3, 36) ¼ 4.4, p ¼ 0.0095; F(3, 36) ¼ 6.3, p ¼ 0.0014 in each period of experiment 1, and F(3, 36) ¼ 10, p < 0.0001; F(3, 36) ¼ 7.7, p ¼ 0.0004; F(3, 36) ¼ 12, p < 0.0001; F(3, 36) ¼ 27, p < 0.0001 in each period of experiment 2). 3.4. Esfenvalerate disrupted dopaminergic signaling in zebrafish embryos

3.2. Effects of esfenvalerate on oxygen consumption rates The measurement of energy consumption, through electron transport chain (ETC) activity, provides insight into the metabolic activities of fish species under stressful conditions. We therefore determined the oxygen consumption rate from mitochondrial ETC of whole embryos. Fig. 1A depicts the profiles of OCR of fish embryos at 48 hpf throughout the assay. The basal respiration, maximal respiration and non-mitochondrial respiration of zebrafish at 48 hpf was not affected by esfenvalerate at the concentrations measured (Fig. 1 B, D, E). However, the ATP-linked respiration of embryos exposed to 0.2 mg/L at 48 hpf was decreased significantly (K ¼ 3.53, p ¼ 0.039) (Fig. 1 C). The OCR of fish embryos at 24 hpf were also determined, but OCR for mitochondrial respiratory endpoints were not affected. Additionally, the expression of genes related to oxidative stress were assessed because of evidence that oxidative stress responses and ROS production are increased with pyrethroid exposure in fish. The transcript levels of sod1 and sod2 in fish were measured as endpoints related to oxidative stress, and embryonic expressions of these two genes at measured concentrations were not altered by any exposure to esfenvalerate (Fig. 2 A, B). 3.3. Esfenvalerate impaired locomotor behaviors in zebrafish larvae Zebrafish exposed continuously to esfenvalerate for 5 dpf, 6 dpf and 7 dpf were collected to assess behavioral responses to esfenvalerate. Zebrafish at 6 dpf, in treatments (0.02, 0.2, 2 mg/L), were behaviorally different from zebrafish at the same age, in vehicle control (statistical data in each period of the test were F (3, 36) ¼ 32, p < 0.0001; F (3, 36) ¼ 6.8, p ¼ 0.0010; F (3, 36) ¼ 11, p < 0.0001; F (3, 36) ¼ 4.3, p ¼ 0.0110; F (3, 36) ¼ 9.4, p < 0.0001, respectively (Fig. 3A). The behavioral changes of zebrafish at 6 dpf were more notable when compared with zebrafish at 5 dpf and 7 dpf (Fig. S2). Our data showed that zebrafish at 6 dpf showed lower variability and revealed significant differences between treatment and control fish in locomotor activities. We therefore selected this time point to do additional independent VMR tests, and also included two higher concentrations of 4 and 8 mg/L esfenvalerate. Fish locomotor behaviors collected from two additional independent experiments are

The transcript levels of th1, dat, drd1b, drd2a and drd2b were measured to determine if dopaminergic signaling was affected due to esfenvalerate exposure during development. Zebrafish exposed to environmentally relevant concentrations of esfenvalerate did not show any significant changes in expressed genes for those related to dopamine synthesis and receptor signaling. However, zebrafish exposed to 0.2 mg/L esfenvalerate showed a reduction in dopamine active transporter (dat) with exposure (K ¼ 8.228; p ¼ 0.0415) (Fig. 4 B). 4. Discussion 4.1. Esfenvalerate did not induce zebrafish deformities and accelerated zebrafish hatching Zebrafish embryos were not noted to have any significant deformity nor mortality relative to vehicle control zebrafish under tested concentrations of esfenvalerate over the 96 h exposure duration (Table 2). In a study by Klüver et al. (2015), zebrafish embryos were exposed to esfenvalerate ranging from 0 to 5 mg/L, and no observable mortalities and deformities were observed when exposure concentrations were equal to or below 2.5 mg/L. As exposures approached ~5 mg/L, ~30% fish suffered from curved body axis, thus it appeared zebrafish were somewhat more sensitive to esfenvalerate compared to those from our study. Moreover, studies that report on the toxicities of esfenvalerate to zebrafish embryos vary greatly. In the study by Ma et al. (2009), 96-h LC50 of esfenvalerate to zebrafish embryos was 3.48 mg/L, but Awoyemi et al. (2019) exposed zebrafish embryos in 1000 mg/L esfenvalerate, and reported less than 20% mortality. This could be due to differences in experimental design, husbandry, and the genetics of fish used in different laboratories that may confer increased sensitivity or resistance to exposure. Fenvalerate is the racemate of four enantiomers, including esfenvalerate, and this racemate is believed to be less toxic than esfenvalerate to zebrafish embryos (~96 hpf), having an LC50 value of 8.29 mg/L, and to zebrafish larvae (8 dpf-12 dpf), having an LC50 value of 6.25 mg/L (Ma et al., 2009; Gu et al., 2010). Zebrafish in our study did not exhibit significant mortality nor deformity from controls up to ~8 mg/L esfenvalerate exposure.

Table 1 The mortality rate, deformity rate and hatch time of zebrafish embryos exposed to esfenvalerate.

Mortality Deformity Hatch Time (hpf)

Vehicle Control

0.02 mg/L esfenvalerate

0.2 mg/L esfenvalerate

2 mg/L esfenvalerate

8% 8% 67.14 ± 1.94

4% 4% 66.26 ± 2.43

8% 8% 65.23 ± 1.96

16% 16% 61.35 ± 1.75*

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Fig. 1. Mitochondrial respiration of zebrafish embryos exposed in 0.01% DMSO, 0.02, 0.2 and 2 mg/L esfenvalerate from 6 hpf up to 54 hpf. (A) Diagram depicting mean oxygen consumption rate for zebrafish for each treatment over time. (B) Basal respiration, (C) Oligomycin-induced ATP linked respiration, (D) FCCP-induced maximal respiration, (E) Nonmitochondrial respiration. Data are presented as mean value ± standard error (N ¼ 5). Asterisks (*) indicate a significant difference between the treatment and the control at p < 0.05.

Fig. 2. Transcript levels for genes (A) sod1 and (B) sod2 in zebrafish embryos exposed to either vehicle control, or one dose of 0.02, 0.2, or 2 mg/L of esfenvalerate from 6 hpf up to 96 h. Data are expressed as mean ± standard error (N ¼ 6). Asterisks (*) indicate a significant difference between control fish and treated fish at p < 0.05.

Differences among studies may be due to the strain used (we used wildtype AB crossed to a Tu strain from ZIRC) or differences in experimental design. In another study, Zhang et al. (2017) treated zebrafish embryos to one dose of either 2.5 nM, 10 nM, 25 nM, 125 nM, or 500 nM (1.05 mg/L, 4.20 mg/L, 10.50 mg/L, 52.49 mg/L, or 209.95 mg/L, respectively) fenvalerate racemate, and fish mortalities (7.5%) were significantly increased relative to control mortality (0%) when exposure concentrations were equal to or higher than 10 nM (4.20 mg/L). Zebrafish embryos exposed to 500 nM (209.95 mg/L) fenvalerate began to exhibit significant deformities, including pericardial edema, yolk sac edema, curved body axis, and cytochrome synthetic anomaly, with a deformity rate of 25%. The developmental toxicity, including mortality and malformation of pyrethroids on zebrafish are summarized in Table 2, including type I pyrethroids bifenthrin, permethrin, resmethrin and tetramethrin, and type II pyrethroids cypermethrin, deltamethrin, cyphenothrin, l-cyhalothrin, fenvalerate and esfenvalerate, demonstrating that there is a wide range in response that is dependent upon chemical species, life stage, and exposure paradigm. Although zebrafish were not observed with significant

mortalities and deformities in this study at environmentally relevant doses (<2 mg/L), our results showed that esfenvalerate accelerated zebrafish hatching, and there was an acceleration in hatching time of zebrafish with 2 mg/L esfenvalerate. Others have also observed that pyrethroids at low exposure concentrations accelerated zebrafish hatching, and evidentially through increased spontaneous movement, such as bifenthrin, cypermethrin, deltamethrin (Jin et al., 2009; Zhang et al., 2017; Li et al., 2019). However, these insecticides, at higher doses, induced fish malformations, inhibited fish spontaneous movement when they were in chorion, and reduced locomotor activity after hatching. 4.2. Effects of esfenvalerate on oxygen consumption rates were not overly evident Mitochondrial respiratory data showed that ATP production was decreased only in zebrafish embryos exposed to 0.2 mg/L esfenvalerate following 48 h exposure. The energy production of mitochondrial oxidative respiratory can affect fish development, and mitochondrial dysfunction-based neurotoxicity can potentially

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Fig. 3. Behavioral analysis of zebrafish locomotors following 6-day exposure over the 50 min in a visual motor response test. (A) Group mean data of distance moved per minute intervals and per dark-light period intervals for zebrafish exposed in 0.02, 0.2 and 2 mg/L esfenvalerate. (B) Group mean data of distance moved per minute intervals and per darklight period intervals for zebrafish exposed in 2, 4 and 8 mg/L esfenvalerate in experiment 1. (C) Group mean data of distance moved per minute intervals and per dark-light period intervals for zebrafish exposed in 2, 4 and 8 mg/L esfenvalerate in experiment 2. Different letters indicate significant differences among groups within an interval at p < 0.05.

affect fish behaviors. There have been several studies reporting that mitochondrial function in vivo or vitro mammalian is regulated by pyrethroid insecticides. He et al. (2019) exposed both murine macrophage RAW 264.7 cells and murine peritoneal macrophages to 25 mM, 50 mM and 100 mM b-cypermethrin, and both cell lines were reported to exhibit decreased mitochondrial membrane potential and ATP production. The authors used molecular docking method to screen potential target site of b-cypermethrin, and the molecular docking showed that b-cypermethrin docked with mouse respiratory chain complex I. Another type II pyrethroid deltamethrin can modify rat liver mitochondrial function, and reduced mitochondrial enzyme activities, such as NADH oxidase, NADH cytochrome c reductase, succinate oxidase, and succinate cytochrome c reductase (Braguini et al., 2004). In the same study, mitochondrial state III and state IV oxygen consumption were significantly decreased by deltamethrin, and the target site of the insecticide was located between Complex II and Complex III of the electron transport chain. Additionally, deltamethrin affected the

quality of mouse oocytes by inducing abnormal mitochondrial distribution and decreasing mitochondrial membrane potential (Jia et al., 2019). Gasmi et al. (2017) invested the neurotoxicity induced by deltamethrin at 0.32 mg/kg/day in two main regions of the Wistar rat brain (hippocampus and striatum). The results showed that deltamethrin caused a significant increase of mitochondrial metabolite level (protein, lipids, and carbohydrates) and enzyme activity (glutathione S-transferase and superoxide dismutase). The study also reported a decrease of mitochondrial glutathione levels as well as and catalase and glutathione peroxidase activities; and an increase of malondialdehyde (MDA) acid levels of the two regions. Furthermore, mitochondrial functional assessment in the brains of treated rats revealed an increase in permeability followed by mitochondrial swelling. There was also a significant decrease in mitochondrial respiration (O2 consumption) in the striatum and hippocampus. However, in this paper, the mitochondrial respiratory responses of zebrafish embryos to environmentally relevant levels of esfenvalerate were not overly evident.

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Fig. 4. Transcript levels for genes related to dopamine synthesis, transport and receptors, (A) th1, (B) dat, (C) drd1b, (D) drd2a, (E) drd2b in zebrafish embryos exposed to either vehicle control, or one dose of 0.02, 0.2, or 2 mg/L of esfenvalerate from 6 hpf up to 96 h. Data are expressed as mean ± standard error (N ¼ 6). Asterisks (*) indicate a significant difference between control fish and treated fish at p < 0.05.

4.3. Esfenvalerate induced hypoactivity in zebrafish larvae At tested exposure doses of 0.02, 0.2 and 2 mg/L esfenvalerate, zebrafish at 6 dpf showed evidence for locomotor impairment. Zebrafish at 5 and 7 dpf showed few changes in locomotor behavior, and larvae at specific ages may show different sensitivity in locomotor behaviors to esfenvalerate. We pursued 6 dpf as a time point to conduct additional behavioral tests with zebrafish larvae, and included two higher exposure doses of 4 mg/L and 8 mg/L esfenvalerate. Taken together, esfenvalerate induced hypoactivity in larval zebrafish, although responses showed variability based upon clutch. This variability may relate to genetics or epigenetic factors
n et al., 2018). Several studies provide compelling evidence (Roma that pyrethroids modify zebrafish locomotor behaviors (Kung et al.,

2015; Frank et al., 2018; Liu et al., 2018). Klüver et al. (2015) used behavioral effects of 4 dpf zebrafish to improve prediction of acute toxicity for neurotoxic compounds including esfenvalerate. The authors found that the EC50 values of the locomotor response for neurotoxic compounds in the fish embryo toxicity test were closer to the acute fish toxicity-LC50. Frank et al. (2018) treated zebrafish with low doses of 1, 10, 50 ng/L bifenthrin, and the results showed that 5 dpf zebrafish did not differ in the locomotor response to predator cue or predator compared to untreated fish. However, after 2 weeks of recovery, zebrafish exposed to 1 and 10 ng/L pyrethroid showed hyperactivity in the response to predator cue or predator. Additionally, zebrafish larvae exposed to 0.33 and 0.50 mg/ L deltamethrin from 3 hpf to 72 hpf showed decreased locomotor behaviors, although fish recovered 10 days later from the exposure

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Table 2 Summary of developmental toxicity conducted in zebrafish at various life stages and under different exposure paradigms in zebrafish. Asterisk indicates that the corresponding endpoint in treated fish is significantly different from control fish. Pyrethroids

Exposure duration

Exposure Mortality concentration

Bifenthrin

30 dpf-34 dpf (adult fish) 3 hpf-99 hpf 3 hpf-99 hpf

0.15 mg/L 1.5 mg/L 0.15 mg/L 1.5 mg/L 50 mg/L, 100 mg/L, 200 mg/L

Bifenthrin Bifenthrin

10 mg/L, 50 mg/L, 100 mg/L, 200 mg/L, 300 mg/L

1s-bifenthrin 1r-bifenthrin

3 hpf-99 hpf

Permethrin

3 hpf-144 100 mg/L, hpf 200 mg/L, 300 mg/L, 400 mg/L, 600 mg/L, 800 mg/L

10 mg/L, 20 mg/L, 30 mg/L, 50 mg/L, 100 mg/L, 150 mg/L

Cypermethrin

Cypermethrin

b-cypermethrin

Deltamethrin

Deltamethrin

4 hpf-100 25 mg/L hpf 50 mg/L 100 mg/L 200 mg/L 400 mg/L 3 hpf-99 0.01 mg/L, hpf 0.02 mg/L, 0.03 mg/L, 0.04 mg/L, 0.05 mg/L, 0.06 mg/L, 0.07 mg/L

0.25 mg/L 0.33 mg/L 0.50 mg/L 0.25 mg/L, 6e7 month old 0.5 mg/L, (adult 1 mg/L, zebrafish) 2 mg/L

3 hpf-72 hpf

Hatching

Reference

Mortality low under tested Malformations were not observed concentrations under tested concentrations

NA

Bertotto et al., 2018

Mortality low under tested Malformations were not observed concentrations under tested concentrations 200 mg/L Pericardial edema Mortality* 96-h EC50 ¼ 256 mg/L 200 mg/L Pericardial edema* Curved body axis 96-h EC50 ¼ 109 mg/L 50 mg/L Curved body axis >10.1%* 100 mg/L Curved body axis > ~20%* 200 mg/L Curved body axis >35.5%* 1r-bifenthrin: 200 mg/L, 300 mg/L 1rCurved body axis: EC50 ¼ 145 mg/L bifenthrin Mortality* Pericardial edema: EC50 ¼ 226 mg/L 300 mg/L 1s-bifenthrin 1s-bifenthrin: 300 mg/L: no significant curved body Mortality was pretty low axis and pericardial edema were observed. LC50 ¼ 467.5 mg/L Crooked body: EC50 ¼ 409.5 mg/L; Spasms: EC50 ¼ 443.0 mg/L; Pericardial edema EC50 ¼ 526.1 mg/L; Non-inflation of swim bladder EC50 ¼ 400.5 mg/L LC50 ¼ 73.0 mg/L Crooked body: EC50 ¼ 68 mg/L; Spasms: EC50 ¼ 64.5 mg/L; Pericardial edema EC50 ¼ 84.2 mg/L; Non-inflation of swim bladder EC50 ¼ 69.9 mg/L NA Pericardial edema Yolk sac edema (concentration NA) 100 mg/L > 80% fish Body-axis curvature*

NA

Control: Mortality ¼ 0.00 ± 0.00%, 0.01 mg/L: Mortality ¼ 3.89 ± 0.98%, 0.02 mg/L: Mortality ¼ 4.44 ± 3.46%, 0.03 mg/L: Mortality ¼ 55.00 ± 6.03%, 0.04 mg/L: Mortality ¼ 78.33 ± 5.99%, 0.05 mg/L: Mortality ¼ 80.00 ± 6.03%, 0.06 mg/L: Mortality ¼ 88.90 ± 7.88% 0.07 mg/L: Mortality ¼ 100.00 ± 0.00% 96-h LC50 ¼ 0.029 ± 0.002 mg/L Mortality low under tested concentrations

Deformity

0.3 mg/L Tail malformation 0.4 mg/L Tail malformation 0.5 mg/L Tail malformation 0.3 mg/L Pericardial edema 0.4 mg/L Pericardial edema 0.5 mg/L Pericardial edema

¼ 16.67 ± 7.64% ¼ 27.78 ± 12.89% ¼ 37.78 ± 5.93%

Jin et al. (2009)

50% fish hatched at time: Control: 52 hpf 1s-bifenthrin: 55 hpf* 1r-bifenthrin:51 hpf

Jin et al., 2009

Yang et al., 2014

NA

Shi et al., 2011

Hatch rate at 53 hpf: Control: 27.59 ± 3.79% 0.01 mg/L-0.03 mg/L Increase to 60 ± 10% 0.03 mg/L-0.07 mg/L Decrease to 30 ± 5%

Zhang et al. (2017)

NA

Kung et al. (2015)

NA

Strungaru et al., 2019

¼ 20 ± 10% ¼ 32.78 ± 6.23% ¼ 45 ± 8.49%

Malformations were not observed under tested concentrations

NA 3.125 mg/L 72-h mortality ¼ 25% 2 mg/L 15-d: Mortality was pretty low

Hatch rate at 50 hpf: Control: 54.3% 50 mg/L: 72.9%* 100 mg/L: 86.2%* 200 mg/L: 93.3%*

X.H. Wang et al. / Chemosphere 243 (2020) 125416

9

Table 2 (continued ) Pyrethroids

Exposure duration

Deltamethrin

3 hpf-72 hpf

Deltamethrin

l-cyhalothrin

l-cyhalothrin

Exposure Mortality concentration

0.01 mg/L, 0.1 mg/L, 1 mg/L, 10 mg/L 100 mg/L 10 hpf-72 0.1 mg/L, hpf 1 mg/L, 10 mg/L, 25 mg/L, 50 mg/L

Mortalities were all significantly increased under tested concentrations*

2 hpf-98 hpf 72 hpf168 hpf ~96-h exposure (adult fish) ~96-h exposure

96-h LC50 ¼ 0.066 (0.048 e0.093) mg/L 96-h LC50 ¼ 0.38 (0.21 e0.53) mg/L 96-h LC50 ¼ 0.0031 (0.0017 e0.0042) mg/L

10 mg/L, 50 mg/L, 100 mg/L, 200 mg/L, 300 mg/L

50 mg/L ()-enantiomer Mortality* 100 mg/L ()-enantiomer Mortality* 200 mg/L racemate and ()-enantiomer Mortality* 300 mg/L racemate and ()-enantiomer Mortality* 96-h LC50 (racemate) ¼ 1.30 mg/L; 96-h LC50 (()-enantiomer) ¼ 0.18 mg/L

Deformity

Hatching

Reference

10 and 100 mg/L: Body length* Eye area* Head-body angle*

Hatch rate at 72 hpf: Hatch rate of zebrafish in all treatments were all increased*

Liu et al. (2018)

25 mg/L, 50 mg/L, Hatch rate at 48 10 mg/L, 25 mg/L, 50 mg/L Body length* hpf* 10 mg/L, 25 mg/L, 50 mg/L Head area* 1 mg/L, 10 mg/L, 25 mg/L, 50 mg/L Eye area* 10 mg/L, 25 mg/L, 50 mg/L Pericardial area* Yolk areas were not different in treated fish； 1 mg/L, 10 mg/L, 25 mg/L, 50 mg/L Length of dorsal longitudinal anastomotic vessel* 10 mg/L, 25 mg/L, 50 mg/L Length of internode blood vascular* 1 mg/L, 10 mg/L, 25 mg/L, 50 mg/L SV-BA distance* 10 mg/L Pericardial edema >60% 25 mg/L Pericardial edema ¼ ~90% 50 mg/L Pericardial edema ¼ ~90% 50 mg/L Stroke volume* 10 mg/L, 25 mg/L, 50 mg/L Heart rate* 25 mg/L, 50 mg/L Cardiac output* 0.1 mg/L, 1 mg/L, 10 mg/L, 25 mg/L, 50 mg/ L Thigmotaxis %* NA NA

50 mg/L racemate and ()-enantiomer NA Pericardial edema* 100 mg/L racemate and ()-enantiomer Pericardial edema* 200 mg/L racemate, (þ)-enantiomer and ()-enantiomer Pericardial edema* 300 mg/L racemate, (þ)-enantiomer and ()-enantiomer Pericardial edema* 96-h pericardial edema EC50 (racemate) ¼ 0.05 mg/L, ((þ)-enantiomer) ¼ 0.34 mg/L, (()-enantiomer) ¼ 0.09 mg/L 10 mg/L ()-enantiomer Body crook* 50 mg/L racemate and ()-enantiomer Body crook* 100 mg/L racemate, (þ)-enantiomer and ()-enantiomer Body crook* 200 mg/L racemate, (þ)-enantiomer and ()-enantiomer Body crook* 300 mg/L racemate, (þ)-enantiomer and ()-enantiomer Body crook*

Li et al. (2019)

Wang et al., 2018

Xu et al., 2008

(continued on next page)

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X.H. Wang et al. / Chemosphere 243 (2020) 125416

Table 2 (continued ) Pyrethroids

Fenvalerate

Exposure duration

8 dpf-12 dpf

Exposure Mortality concentration

0e100 mg/L

Fenvalerate (FV: ~96 hpf racemate; FV1: aR-2R; FV2: aS-2R; FV3: aR2S; FV4: aS-2S)

1 mg/L, 10 mg/L, 50 mg/L, 100 mg/L, 500 mg/L

Esfenvalerate

2 hpf-98 hpf

0.01 mg/L10 mg/L

l-cyhalothrin

2 hpf-96 hpf

2.5 nM 10 nM 25 nM 125 nM 500 nM

Fenvalerate

Permethrin

Bifenthrin Permethrin Deltamethrin

l-cyhalothrin

5 hpf-5 dpf

0.01 mg/L, 0.1 mg/L, 10 mg/L, 1000 mg/L

Deformity

Hatching

96-h body crook EC50 (racemate) ¼ 0.03 mg/L, ((þ)-enantiomer) ¼ 0.09 mg/L, (()-enantiomer) ¼ 0.03 mg/L 10 mg/L racemate and ()-enantiomer Heart beat* 50 mg/L racemate and ()-enantiomer Heart beat* 300 mg/L ()-enantiomer Heart beat* No yolk sac edema was observed under tested concentrations LC50 ¼ 6.25 (5.04e7.75) mg/ 25 mg/L L Body axis curvature ¼ 90%, 50 mg/L Body axis curvature > 90% FV LC50 ¼ 8.29 mg/L 100 mg/L FV, FV1, FV4 NA FV1 LC50 ¼ 10.5 mg/L Crooked body* FV2 LC50 ¼ 193 mg/L 500 mg/L FV, FV1, FV4 FV3 LC50 ¼ 105 mg/L Crooked body* FV4 LC50 ¼ 3.48 mg/L 50 mg/L FV4 Pericardial edema* 100 mg/L FV, FV1, FV4 Pericardial edema* 500 mg/L FV, FV1, FV4 Pericardial edema* FV, FV1, FV2, FV3, FV4 Yolk sac edema: No effects at 500 mg/L 0.3 mg/L Yolk sac edema ¼ 15 ± 5.00% 0.4 mg/L Yolk sac edema ¼ 26.67 ± 7.64% 0.5 mg/L Yolk sac edema ¼ 43.33 ± 5.87% NA Mortality low under tested ~5 mg/L Curved body axis ¼ ~30% concentrations 2.5 nM Mortality ¼ 9.82%* 10 nM Mortality ¼ ~10% 25 nM Mortality ¼ ~20%* 125 nM Mortality ¼ 26.58%* 500 nM Mortality ¼ 31.93%* 10 nM Mortality ¼ 7.5%* 25 nM Mortality ¼ 10%* 125 nM Mortality ¼ 20%* 500 nM Mortality ¼ 25%* Mortality were pretty low under tested concentrations

1000 mg/L Mortality < 20% 1000 mg/L Mortality < 20% 1000 mg/L Mortality > 20% 1000 mg/L Mortality > 20%

Pericardial edema Yolk sac edema Curved body Cytochrome synthetic anomaly 125 nM Malformation rate ¼ ~35%* 500 nM Malformation rate ¼ 65.91%*

125 nM Hatch rate ¼ 80.41%*; 500 nM Hatch rate ¼ ~70%*

Pericardial edema Yolk sac edema Curved body Cytochrome synthetic anomaly 500 nM Malformation rate ¼ 25%*

125 nM Hatch rate ¼ 88.75%* 500 nM Hatch rate ¼ ~80%

Pericardial edema Yolk sac edema Curved body Cytochrome synthetic anomaly 500 nM Malformation rate ¼ 9.94%* NA

Hatch rates were not affected under tested concentrations

NA 1000 mg/L Pericardial edema; Body axis curvature 1000 mg/L Pericardial edema; Body axis curvature

NA

Reference

Gu et al. (2010)

Ma et al. (2009)

Klüver et al. (2015) Zhang et al. (2017)

Awoyemi et al. (2019)

X.H. Wang et al. / Chemosphere 243 (2020) 125416

11

Table 2 (continued ) Pyrethroids

Exposure duration

Exposure Mortality concentration

Fenvalerate Esfenvalerate Permethrin

3 hpf-144 0e800 mg/L hpf

1000 mg/L Mortality < 20% 1000 mg/L Mortality < 20% LC50 ¼ 300 ± 0.08 mg/L

Resmethrin

0e3000 mg/L 3000 mg/L Mortality < 20%

Bifenthrin

0e3000 mg/L LC50 ¼ 190 ± 0.45 mg/L

Deltamethrin

0e250 mg/L

LC50 ¼ 40 ± 0.08 mg/L

Cypermethrin

0e500 mg/L

LC50 ¼ 65 ± 0.16 mg/L

l-cyhalothrin

0e500 mg/L

LC50 ¼ 110 ± 0.24 mg/L

0.01 e1.2 mmol/L 0.02 e3.2 mmol/L 0.02 mg/L; 0.2 mg/L; 2 mg/L; 4 mg/L; 8 mg/L

72-h LC50 ¼ 0.03 mmol/L

D-tetramethrin

2 hpf-74 hpf

Cyphenothrin Esfenvalerate

6 dpf-6 dpf

Deformity

Hatching

Reference

NA NA 100 mg/L Pericardial edema >20% 200 mg/L Curvature body axis ¼ ~30% 200 mg/L, 300 mg/L Low jaw length* 300 mg/L Pericardial edema <20% Curvature body axis ¼ 0% 50 mg/L Pericardial edema >20% Curvature body axis ¼ ~20% 10 mg/L Pericardial edema ¼ ~50% Curvature body axis <20% 50 mg/L Curvature body axis ¼ ~70% Low jaw length* 50 mg/L Pericardial edema ¼ ~50% 100 mg/L Curvature body axis ¼ ~80% 50 mg/L Pericardial edema ¼ ~55% Curvature body axis ¼ ~80%

DeMicco et al., 2010

Mendis et al., 2018

72-h LC50 ¼ 0.09 mmol/L Mortality low under tested Malformations were not observed concentrations. under tested concentrations.

(Kung et al., 2015). Zebrafish larvae treated at 4 dpf with the same insecticide for 12 h (0.01, 0.1, 1, 10, 50 or 100 mg/L) showed increased waking activity and decreased total rest in the 48 consecutive hours following the exposure (at doses higher than 0.1 mg/L) (Liu et al., 2018), suggesting negative effects of pyrethroids on circadian rhythm. Behavioral effects can be detected with low doses exposure, and the effects are not always linearly correlated with exposure doses. In the study of Jin et al. (2009), zebrafish at 4 dpf and 5 dpf showed different responses in average speed to 50, 100 and 200 mg/L bifenthrin. The average speeds of zebrafish at 4 dpf in each of the three treatments were all significantly increased; however, while the average speeds of zebrafish at 5 dpf exposed to 50 mg/L pyrethroid were increased, the speeds of fish at 100 and 200 mg/L pyrethroid exposure were decreased. Taken together, zebrafish can show varied responses to pyrethroid insecticides in terms of behavior, and this is dependent upon the chemical and the age of the fish. Behavioral responses of pyrethroids in zebrafish from the literature are summarized in Table 2.

4.4. Esfenvalerate decreased dopamine active transporter expression in zebrafish larvae Based on evidence that dopamine regulates behavior and studies showing that other pyrethroids affect dopamine system, we measured transcripts involved in dopaminergic signaling. We detected reduced levels of dopamine active transporter mRNA in zebrafish larvae exposed to 0.2 mg/L esfenvalerate, but no change in other dopamine-associated transcripts (synthesis and receptor

Hatch rate of zebrafish at 3 dpf were This study increased compared with control fish, when exposure doses 2 mg/L*

signaling). Esfenvalerate is the (S)-enantiomer of fenvalerate and in rodents, there is good evidence that dopamine release is modulated by this pyrethroid. Husain et al. (1991) treated female rats with fenvalerate for 21 days at doses of 5, 10, or 20 mg/kg and reported inhibition of dopamine production and related metabolites (HVA and DOPAC) in different brain regions. Conversely, in another study, tissue slices of male rabbit striatum were treated with different type II pyrethroids (deltamethrin, cypermethrin or fenvalerate) and the authors reported that release of dopamine from rabbit striatal slices was stimulated by the SS isomer of fenvalerate (esfenvalerate) (Eells and Dubocovich, 1988). The mechanism appeared to be via the activation of sodium channels, as the use of tetrodotoxin (a sodium channel blocker), abolished any effect of esfenvalerate on dopamine release from the striatum. Lastly in rodents, there can exist multi-generational and gestational effects on dopamine due to fenvalerate exposure. Malaviya et al. (1993) exposed mothers to 10 mg fenvalerate/kg bw and 15 mg cypermethrin/kg bw and assessed neonatal rats. Exposed pups showed a significant increase in the levels of dopamine receptors, perhaps suggestive of sensitization to dopamine. The authors pointed out that changes in the dopamine system in early development can be detrimental for normal brain formation and function. Long term consequences, if any, for such neonatal exposures to pyrethroids are not fully elucidated at this time. While there are no data available regarding the effects of esfenvalerate on the zebrafish dopamine system, there is evidence from other studies demonstrating that pyrethroids can modify the dopaminergic system at both the neurotransmitter and gene

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X.H. Wang et al. / Chemosphere 243 (2020) 125416

Table 3 Summary of behavioral endpoints conducted in zebrafish at various life stages and under different exposure paradigms in zebrafish. Asterisk indicates that the corresponding endpoint in treated fish is significantly different from control fish. Pyrethroids

Exposure duration

Exposure concentration

Locomotor behaviors

Reference

Bifenthrin

2 hpf-5 dpf (exposure) 5 dpf-19 dpf (recovery)

1 ng/L, 10 ng/L, 50 ng/L

Frank et al. (2018)

Bifenthrin

3 hpf-24 hpf

50 mg/L, 100 mg/L, 200 mg/L

Response to predator cue: 5 dpf zebrafish: no change 19 dpf zebrafish: locomotor behaviors were significantly increased after exposure of 1 and 10 ng/L insecticide. Response to predator: 5 dpf zebrafish: no change 19 dpf zebrafish: locomotor behaviors were significantly increased after exposure of 1 ng/L insecticide. Spontaneous movement: Control fish ¼ 3.84 movements/60 s, 50 mg/L: 4.84 movements/60 s*, 100 mg/L: 6.02 movements/60 s*, 200 mg/L: 8.83 movements/60 s* Averaged speed: Control fish ¼ 0.29 ± 0.07 mm/s, 50 mg/L ¼ 0.82 ± 0.16 mm/s*, 100 mg/L ¼ 1.6 ± 0.17 mm/s*, 200 mg/L ¼ 1.75 ± 0.11 mm/s* Averaged speed: Control fish ¼ 2.155 ± 0.138 mm/s, 50 mg/L ¼ 2.384 ± 0.144 mm/s*, 100 mg/L ¼ 1.894 ± 0.136 mm/s*, 200 mg/L ¼ 1.549 ± 0.160 mm/s* Spontaneous movement: Zebrafish exposed in 1r-bifenthrin at most of the time points were increased significantly. Zebrafish exposed in 1s-bifenthrin at most of the time points were decreased significantly. Total distance moved: 1s-bifenthrin: 5, 10, 20 mg/L insecticide did not change zebrafish total distance moved. 1r-bifenthrin: 5, 10, 20 mg/L insecticide made zebrafish total distance moved significantly decreased. Total distance moved: Zebrafish exposed in 0.33 mg/L and 0.50 mg/L insecticide showed significantly decreased locomotor behaviors.

3hpf-84hpf-96hpf

3hpf-84hpf-120hpf

1s-bifenthrin 1r-bifenthrin

Deltamethrin

Deltamethrin

Deltamethrin

Deltamethrin

3 hpf-17 hpf-25 hpf

100 mg/L

3 hpf-99 hpf

5 mg/L, 10 mg/L, 20 mg/L

Exposure duration: 3 hpf-72 hpf Time point tested: 2 weeks post fertilization Exposure duration: 4 dpf-4.5 dpf Consecutive monitor: 4 dpf 6 dpf

0.25 mg/L, 0.33 mg/L, 0.50 mg/L

0.01 mg/L, 0.1 mg/L, 1 mg/L, 10 mg/L, 50 mg/L, 100 mg/L 10 hpf-72 hpf 0.1 mg/L, 1 mg/L, 10 mg/L, 25 mg/L, 50 mg/L 6e7 month old (adult 0.25 mg/L, zebrafish) 15-day exposure 0.5 mg/L, 1 mg/L, 2 mg/L

Fenvalerate

5 hpf-120 hpf

100 mg/L

Esfenvalerate

2 hpf-98 hpf

0.01 mg/L-10 mg/L

Bifenthrin

5 hpf-5 dpf

0.01 mg/L, 0.1 mg/L, 10 mg/L, 1000 mg/L, Environmental relevant levels of each insecticide

Permethrin

Deltamethrin

>0.1 mg/L: increased the waking activity and decreased the total rest in 48 consecutive hours

Jin et al. (2009)

Jin et al., 2009

Kung et al. (2015)

Liu et al. (2018)

Thigmotaxis (%): 0.1, 1, 10, 25, 50 mg/L insecticide significantly increase or decrease Li et al. zebrafish thigmotaxis. (2019)

Anxiety: Control: non-aggressive 0.25 mg/: aggressive 0.5 mg/L: aggressive 1 mg/L: aggressive and instable behavior 2 mg/L: aggressive and instable behavior 1 mg/L: rotation frequency (alteration between aggressive and non-aggressive behavior (clockwise & counterclockwise)) was significantly increased. Swimming distance and time were significantly decreased.

Strungaru et al., 2019

Han et al., 2017 Distance moved: Klüver et al. EC50 ¼ 0.15 mg/L (2015) 10 mg/L: total distance moved and swimming velocity were significantly decreased. Awoyemi 0.1 mg/L: total distance moved was significantly decreased. et al. (2019) 0.01 mg/L: swimming velocity was significantly decreased. Environmental level of 0.269 mg/L: impaired zebrafish swimming behaviors 1000 mg/L: total distance moved and swimming velocity were not changed significantly Environmental level of 0.122 mg/L: distance moved and swimming velocity were not changed significantly 10 mg/L: total distance moved and swimming velocity were not changed significantly Environmental level of 0.252 mg/L: distance moved and swimming velocity were not changed significantly

X.H. Wang et al. / Chemosphere 243 (2020) 125416

13

Table 3 (continued ) Pyrethroids

l-cyhalothrin

Fenvalerate

Esfenvalerate

Exposure duration

Exposure concentration

Locomotor behaviors

Reference

10 mg/L: total distance moved and swimming velocity were not changed significantly Environmental level of 0.053 mg/L: distance moved and swimming velocity were not changed significantly 1000 mg/L: total distance moved and swimming velocity were not changed significantly Environmental level of 0.037 mg/L: distance moved and swimming velocity were not changed significantly 1000 mg/L: total distance moved was significantly decreased 1000 mg/L: swimming velocity was not changed significantly Environmental level of 0.247 mg/L: distance moved and swimming velocity were not changed significantly

expression level. For example, Bertotto et al. (2018) exposed zebrafish embryos at 3 h post fertilization to a solvent control, or one dose of 0.15 mg/L bifenthrin or 1.5 mg/L bifenthrin (nominal). Treatments with the pyrethroid lead to a significant 30-fold decrease in homogenate concentrations of HVA in zebrafish embryos exposed to 0.34 mg/L of bifenthrin (measured). This was accompanied by a decrease in the expression of drd1 and th1 in embryos treated with bifenthrin. Although we did not measure dopamine levels in this study, it is plausible that a decrease in dat expression with esfenvalerate may lead to increased levels of dopamine, corresponding to mammalian studies demonstrating increased dopamine release with esfenvalerate (Eells and Dubocovich, 1988). Other studies in zebrafish show that pyrethroid exposure can modulate genes involved in dopamine signaling. Kung et al. (2015) exposed embryos and larvae to the pesticide deltamethrin at concentrations ranging from 0 to 0.50 mg/ L deltamethrin. Zebrafish exposed up to 72 hpf to 0.33 and 0.50 mg/L deltamethrin showed a ~33% decrease in drd1 steady state mRNA levels while 0.25 mg/l deltamethrin increased th1 almost 2-fold. We did not detect changes in drd1 nor th1 in this study with esfenvalerate but point out that the effect of pyrethroids on the expression levels of dopaminergic transcripts may be stage dependent, as the study by Kung and colleagues reported a downregulation of drd1 and drd2a after two weeks in the larvae. At neither stage was dat1 affected. The down-regulation of receptors in both embryos and larvae following exposure to deltamethrin may be a mechanism to mitigate excessive dopamine release following exposure to the pyrethroid pesticide. The effects of pyrethroids are not limited to zebrafish, and juvenile rainbow trout exposed to bifenthrin up to 1.5 ppb did not show any significant impact on th expression in the brain, but did show changes in drd2a at 96 h and 2 weeks exposure time points (Crago and Schlenk, 2015). The authors suggest that drd2a may be modulated via changes in Ca2þ or Naþ following bifenthrin exposure. Taken together, it appears that pyrethroid exposure can significantly affect expression of the dopamine system in fish, effects that appear to be pyrethroid and age dependent. 5. Conclusion In summary, zebrafish exposed in 0.02, 0.2 and 2 mg/L esfenvalerate did not exhibit significant mortality nor deformity, but they were noted to have sub-lethal responses at these environmentally relevant levels. Esfenvalerate at 2 mg/L accelerated zebrafish hatching, and this phenomenon was evidentially due to increased spontaneous movement for pyrethroids at low exposure concentrations. Zebrafish at 6 dpf showed evidence for locomotor impairment, but at 5 dpf and 7 dpf, there were no significant changes in locomotor behavior. These time periods represent an active time for central nervous system maturation and this could

explain age dependent sensitivities to chemical exposures. Zebrafish at specific ages may also show different sensitivity in locomotor behaviors to esfenvalerate, due to the recovery of zebrafish locomotor activity, similar to that observed with other pyrethroids (Frank et al., 2018). And dat expression was down-regulated by esfenvalerate, which may result in a rise in extracellular dopamine. This in turn could affect behavioral responses to the pesticide. Lastly, we comprehensively summarize data on mortality, deformity, hatch rates and behavioral endpoints for different pyrethroids in the zebrafish model as a resource for future experiments and risk assessment (Tables 2 and 3). This study demonstrates that esfenvalerate at environmentally relevant levels induces hypoactivity in larval zebrafish that are dependent upon the age of the fish, and that these behavioral changes may be related to impaired dopamine signaling. Author contributions Xiao H. Wang: Conceived and designed the analysis, Collected the data, Contributed data or analysis tools, Performed the analysis, Wrote the paper. Christopher L. Souders II: Collected the data, Contributed data or analysis tools, Performed the analysis. Priscilla Xavier: Collected the data, Contributed data or analysis tools, Performed the analysis. Xiao Y. Li: Performed the analysis, Wrote the paper. Bing Yan: Wrote the paper, Other contribution. Christopher J. Martyniuk: Conceived and designed the analysis, Collected the data, Contributed data or analysis tools, Performed the analysis, Wrote the paper. Declaration of competing interest The authors have no conflict of interest. Acknowledgments The authors have no conflict of interest to declare. We thank Edward Flynn for zebrafish husbandry and technical support. This research was funded by the University of Florida and the College of Veterinary Medicine (CJM) and supported by the National Key R&D Program of China (2016YFA0203103) and the National Natural Science Foundation of China (91543204 and 91643204). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125416. References Awoyemi, O.M., Kumar, N., Schmitt, C., Subbish, S., Crago, J., 2019. Behavioral,

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