Etoxazole stereoselective determination, bioaccumulation, and resulting oxidative stress in Danio rerio (zebrafish)

Etoxazole stereoselective determination, bioaccumulation, and resulting oxidative stress in Danio rerio (zebrafish)

Ecotoxicology and Environmental Safety 192 (2020) 110287 Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal ho...

1MB Sizes 0 Downloads 57 Views

Ecotoxicology and Environmental Safety 192 (2020) 110287

Contents lists available at ScienceDirect

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

Etoxazole stereoselective determination, bioaccumulation, and resulting oxidative stress in Danio rerio (zebrafish)

T

Weixia Changa, Jiyun Niea,b,∗, Yue Gengc, Danyang Zhangc, Qi Wangc, Saqib Farooqa a Institute of Pomology, Chinese Academy of Agricultural Sciences/Laboratory of Quality & Safety Risk Assessment for Fruit (Xingcheng), Ministry of Agriculture and Rural Affairs, Xingcheng, 125100, China b College of Horticulture, Qingdao Agriculture University, Qingdao, 266109, China c Key Laboratory for Environmental Factors Control of Agro-Product Quality Safety, Ministry of Agriculture and Rural Affairs, Agro-Environmental Protection Institute, Ministry of Agriculture and Rural Affairs, Tianjin, 300191, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Etoxazole Stereoselective determination SFC-MS/MS Zebrafish Bioaccumunation Oxidative stress

An environmentally-friendly and fast analytical method for the stereoselective determination of etoxazole was developed and then applied to estimate stereoselective bioaccumulation and elimination in zebrafish using SFCMS/MS. Optimal enantioseparation conditions were determined using a Chiralpak IG-3 column and CO2/MeOH mobile phase (80/20, v/v), at 3.0 mL/min within 1 min, 30°Me and 18 MPa. A modified QuEChERS method was developed for zebrafish sample pretreatment, and mean recoveries were 88.43–110.12% with relative standard deviations ranging from 0.32 to 5.34%. The enantioselectives of etoxazole enantiomers in zebrafish during uptake and depuration phases were evaluated. Significant enantioselective bioaccumulation was observed, with preferential accumulation of (−)-R-etoxazole compared to its antipode, during uptake at both low and high exposure concentrations. The toxic effects of etoxazole on zebrafish were further explored, and activities of antioxidant enzymes were determined in liver of zebrafish. Significant changes were observed in the SOD and GST activities and in the MDA levels, which indicated the occurrence of oxidative stress in liver of zebrafish. The toxic effects exhibited time- and dose-dependent properties. These results can facilitate the accurate risk evaluation of etoxazole and provide basic knowledge for further study of biotoxicity mechanisms.

1. Introduction Etoxazole,2-(2,6-difluorophenyl)-4-[4-(1,1-dimethylethyl)-2-ethoxyphenyl]-4,5-dihyd-rooxazole, is a member of the oxazoline class of organofluorine insecticides, and is active against mite eggs, nymphs, and prevent the laying of viable eggs by adult mites (Dekeyser, 2005). Due to its extremely high efficacy toward target pests such as Tetranychus spp., Panonychus spp., and Eotetranychus spp., this chemical has been used worldwide for treatment of ornamental and agricultural crops including fruits, vegetables, tea, and cotton (Li et al., 2014). However, etoxazole exhibits extreme toxicity to aquatic invertebrates including Daphnia magna, Sheepshead minnow, Mysid shrimp and oyster (Arena et al., 2017), and also is potentially harmful to non-target arthropods including Typhlodromus pyri, Chrysoperla carnea, and Orius laevigatus (Arena et al., 2017). The Pesticide Properties Database (PPDB) of University of Hertfordshire (UH) (UH) reports experiments of etoxazole bioaccumulation in fish with a bio-concentration factor of 2800 L/kg, a level that is extremely dangerous for both fish and human.



Therefore, etoxazole is considered to be bioaccumulative and toxic. A previous study also reported that etoxazole exhibits potential genotoxicity against human peripheral lymphocytes (Rencuzogullari et al., 2004). Given all of these properties in addition to its possible persistence in soil sediment, etoxazole was banned in the European Union (EU) in 2018, and the environmental risks presented by this compound have drawn increasing recent attention. Etoxazole is a racemic mixture that consists of two stereoisomers (Fig. 1). Traditional risk assessments of chemicals lack discrimination between enantiomers for chiral compounds, resulting in the potential underestimation of risks and inaccurate evaluation for environmental assessment. Therefore, it is of great importance to perform comprehensive risk evaluation of etoxazole at the enantiomeric level. Stereoselective behaviors of etoxazole enantiomers have been partially described in previous studies (Chang et al., 2019; Sun et al., 2016; Yao et al., 2016). Sun et al. (2016) reported that (+)-(S)-etoxazole preferentially dissipates in open field citrus. Yao et al. (2016) described the enantioselective metabolism of etoxazole enantiomers in human

Corresponding author. College of Horticulture, Qingdao Agriculture University, Qingdao, 266109, China. E-mail address: [email protected] (J. Nie).

https://doi.org/10.1016/j.ecoenv.2020.110287 Received 22 November 2019; Received in revised form 29 January 2020; Accepted 31 January 2020 0147-6513/ © 2020 Elsevier Inc. All rights reserved.

Ecotoxicology and Environmental Safety 192 (2020) 110287

W. Chang, et al.

Fig. 1. Chemical structure of etoxazole enantiomers.

and rat liver microsomes in vitro with β-nicotinamide adenine dinucleotide phosphate (NADPH), in which (−)-(R)-etoxazole degrades more quickly than its enantiomer in rat liver microsomes and slower in human liver microsomes. Moreover, past studies have indicated a higher bioactivity of (+)-(S)-etoxazole than that of (−)-(R)-etoxazole toward Tetranychus cinnabarinus eggs and higher toxicity of (−)-(R)etoxazole against Danio rerio (Chang et al., 2019). Aquatic organisms accumulate chemicals via several mechanisms, including direct uptake from water through the skin or gills, and the ingestion of contaminated food and suspended particles. The bioaccumulation of chemicals in organisms usually acts as a prerequisite for adverse influence on environments (Franke et al., 1994). Zebrafish (Danio rerio) is a model organism, and has been widely used in studying the potential biological effects of chemical contaminants (Lu et al., 2018; Chai et al., 2018; Batel et al., 2018; Liu et al., 2016; Wang et al., 2015). As mentioned above, etoxazole has been shown to be bioaccumulative in fish. However, no previous attempts have been made to evaluate the bioaccumulation of etoxazole enantiomers. Therefore, monitoring etoxazole enantiomers in zebrafish would enable a better risk evaluation of this chiral insecticide. Additionally, increased description of the potential bioconcentration of etoxazole enantiomers would be a useful contribution to the aquatic organism database. The acute toxicity of etoxazole toward aquatic organisms has been partially studied in our previous work (Chang et al., 2019). However, limited knowledge has been reported on the chronic toxicity of etoxazole exposure to zebrafish. Endpoints in acute toxicity experiment measured mortalities, which do not allow the mechanism exploration of chemical pollutant-induced effects. Therefore, there is a significant need for additional insight into interactions between etoxazole exposure and living organisms. The metabolism of toxicants in vivo usually results in the generation of reactive oxygen species (ROS) (Avallone et al., 2015; Pavagadhi et al, 2012) and then cells limit the damage from ROS via antioxidant defense systems. These systems involve antioxidant enzymes that act to remove the ROS, such as catalase (CAT), superoxide dismutase (SOD), and glutathioneS-transferase (GST). When the equilibrium between the elimination and production of ROS in an organism is disrupted, oxidative stress is triggered (Valavanidis et al, 2006). Thus, antioxidative enzyme activities are investigated as biomarkers of oxidative stress in zebrafish. Another compound, malondialdehyde (MDA), is often used as a marker to evaluate lipid peroxidation (Wang et al., 2015; Valko, et al, 2007). The activities of these antioxidative enzymes would vary with exposure time and concentration, so to better understand the defense and response mechanisms in zebrafish in this study, the antioxidative enzyme activities were determined at three time points for two exposure concentrations of etoxazole. The chiral separation of etoxazole enantiomers has been performed

using a high performance liquid chromatography-ultraviolet (HPLCUV) system with a relatively long measurement time (> 12 min) (Sun et al., 2016). In recent years, LC-tandem mass spectroscopy (LC-MS/ MS) and ultrahigh performance (UP) LC-MS/MS methods have been applied for the analysis of etoxazole enantiomers with high velocity, selectivity, and sensitivity (Chang et al., 2019; Yao et al., 2015, 2016). In addition, researchers have established chiral determination methods of etoxazole enantiomers in many matrices, including fruit, soil, and liver microsome samples (Chang et al., 2019; Sun et al., 2016; Yao et al., 2015, 2016). However, there is no currently available method to measure etoxazole enantiomers in aquatic environmental samples. In this study, supercritical fluid chromatography combined with tandem mass spectrometry/mass spectrometry (SFC-MS/MS) was developed for the effective chiral analysis of etoxazole enantiomers. This method is more effective than the currently reported UPLC-MS/MS method, with shorter analysis time, higher efficiency, and lower solvent costs (Chang et al., 2019). Here, SFC-MS/MS was developed as a novel method for the detection of etoxazole enantiomers. In addition, an efficient QuEChERS (quick, easy, cheap, effective, rugged, and safe) purification method was developed for the determination of etoxazole enantiomers in zebrafish and water samples. These new methods were then applied to evaluate the bioaccumulation behavior of etoxazole enantiomers in zebrafish. This is the first report of the effects of sub-lethal doses of racetoxazole on adult zebrafish. 2. Materials and methods 2.1. Chemicals and reagents The standards of (+)-(S)-etoxazole and (−)-(R)-etoxazole were acquired from Shanghai Chiralway Biotech Co., Ltd. (Shanghai, China). The racemic (rac)-etoxazole standard was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). CO2 and N2 (purity≥99.99%) were supplied by Taiyuan Gas (Tianjin, China). HPLC-grade acetone and ethanol, methanol, isopropanol, and acetonitrile (EtOH, MeOH, IPA, and ACN, respectively) were obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). The sorbents including octadecylsilane (C18, 50 μm) and Florisil of 100–200 mesh were provided by Tianjin Bonna Agela Technologies Co., Ltd. (Tianjin, China). Analytical grade anhydrous magnesium sulfate (MgSO4) and sodium chloride (NaCl) were obtained from Bonna-Agela Technologies Inc. (Wilmington, DE, USA). Polytetrafluoroethylene (PTFE) syringe filter membranes (0.2 μm) were obtained from Tengda Scientific Inc. (Tianjin, China). A stock solution of rac-etoxazole (1000 mg/L) was prepared in ACN and was diluted to prepare working standard solutions at 0.1, 1, 10, 100, and 500 μg/kg. All these solutions were stored 2

Ecotoxicology and Environmental Safety 192 (2020) 110287

W. Chang, et al.

wrapped in aluminum foil (to protect against light exposure) at −20 °C until further analysis.

2.5. Sample preparation Blank samples were generated of zebrafish (water) without etoxazole. Each zebrafish was homogenized in a mixer mill using a 3 mm steel-ball. Aliquots of 1.0 g of the powder in 1.0 mL pure water were added to a 10 mL centrifuge tube. A series of etoxazole standard solutions were added into the tubes, and then the tube was vortexed and allowed to equilibrate at room temperature for 2 h equilibration. The sample was extracted using 1 mL of ACN, and then the tube was vigorously vortexed for 3 min. Next, 0.5 g of NaCl was added and the tube was vortexed for another 3 min, followed by centrifugation at 8000 rpm for 2 min. Then, 0.5 mL of the upper layer was transferred to a 2 mL centrifuge tube containing 50 mg of MgSO4, 150 mg of Florisil, and 150 mg of C18. The resulting supernatant was finally filtered through a PTFE filter membrane (0.22 μm) and then analyzed using SFC-MS/MS. For water samples, 0.5 mL of the upper layer was filtered through a 0.22 μm PTFE filter membrane and then directly injected for SFC-MS/ MS analysis.

2.2. Experimental fish and acclimation Adult 120-day-old zebrafish (0.30 ± 0.15 g) were obtained from a local fish supplier in Liaoning, China. All fish were acclimated in aerated tap water for one week under laboratory conditions at 27.5 ± 1.2 °C under a photoperiod of 14 h/10 h (light/dark). The water quality was tested once a week. Throughout the assay, the parameters of water were monitored as follows: pH 8.0 ± 0.5; dissolved oxygen 6.2 ± 0.5 mg/L; hardness 8.5 ± 1.3 mg/L, expressed as CaCO3 equivalents. All fish were healthy and fed a commercial food twice a day. The animal experiment conformed to the Chinese guidelines and was approved by the animal ethics committee of the institute.

2.3. Experimental treatments Two experiments were conducted (Fig. S1). Experiment 1 was performed to evaluate the stereoselective bioaccumulation and elimination behavior of etoxaozle in whole zebrafish. Experiment 2 was performed using liver of zebrafish, and was carried out to explore the effect of etoxaozle on oxidative stress in zebrafish. Based on the standard of the People's Republic of China (The National Standard of the People's Republic of China, 2014), zebrafish were exposed at doses of 0.145 mg/ L and 1.45 mg/L racemic-etoxazole since our previous results measured an LC50 value of 14.55 mg/L with a 95% confidence interval at 11.55–17.65 mg/L6. The uptake experiment was conducted in duplicate and each experiment was performed in a tank (15 L) containing one hundred zebrafish. The water in the tank during the uptake period was manually renewed once per 12 h to keep a consistent exposure concentration ( ± 20% of the nominal concentration). In a pretest study, zebrafish were tested for 4 weeks to evaluate the steady state time period for rac-etoxazole exposure. However, the concentration of etoxazole did not achieve the steady-state equilibrium during the exposure period but instead exhibited an “increase–decrease” effect. The results indicated highest concentrations of etoxazole enantiomers at 8 d during the exposure. Based on this, for experiment 1, an 8 d exposure was performed. For the depuration phase, zebrafish after 8 d of exposure were moved to other vessels containing dechlorinated and aerated tap water without etoxazole, and the water in the vessels was renewed once per 24 h. Additionally, two other tanks each containing one hundred zebrafish were used as controls for which the zebrafish were only exposed to acetone (< 0.01%). Zebrafish and water were sampled for bioaccumulation and depuration testing after 1, 3, 5, 7, 8, 9, 11, 13, and 15 d of exposure. In experiment 2, 270 adult zebrafish were kept in nine glass aquaria and exposed to nominal concentrations of rac-etoxazole at 0 (control), 0.145 mg/L, and 1.45 mg/L, for a period of 15 d. Fish were sampled (n = 10) at 4, 8, and 15 days. Briefly, fish were anaesthetized with MS-222, sacrificed, and dissected on ice. The livers were immediately separated and frozen in liquid nitrogen before storage at −85 °C until subsequent analysis. Samples were stored in triplicate from each treatment.

2.6. SFC-MS/MS A Shimadzu SFC-MS/MS system (Shimadzu Corp., Kyoto, Japan) combined with a Chiralpak IG-3 column (silica coated with amylose tris-3-chloro-5-methylphenylcarbamate, 3 × 100 mm and 3 μm; Daicel Corp., Osaka, Japan) were employed to perform the etoxazole enantiomer separation. Four additional chiral columns were tested: Chiralpak IB-3 [cellulose tris-(3,5-dimethylphenylcarbamate), 3 μm], Chiralpak IE-3 [amylose tris-(3,5-dichlorophenylcarbamate), 3 μm], Chiralpak OJ-H [cellulose tris-(4-methylbenzoate), 5 μm], and Chiralpak AD-H [(amylose tris (3,5-dimethylphenylcarbamate), 5 μm). A LabSolutions workstation V5.86 was applied to control the system. The separation of etoxazole enantiomers was fulfilled in 1 min with a CO2/methanol ratio of 80/20 (v/v), at a constant flow rate of 3 mL/ min, column temperature of 35 °C, and injection volume at 1 μL for each run. MeOH was used as the post-column additive and applied at a flow rate of 0.1 mL/min and the auto back-pressure regulator (ABPR) pressure was maintained at 18 MPa. An LC-MS 8050 TQ mass spectrometer (Shimadzu Corp., Kyoto, Japan) combined with an electrospray ionization source was applied for the quantification of etoxazole stereoisomers in ESI+ mode. The ESI parameters were set as follows: capillary voltage, 3.0 kV; desolvation temperature, 500 °C; source temperature, 150 °C. A desolvation gas flow at 1000 L/h and cone gas flow at 50 L/h were employed. The MS source conditions were set as follows: 10 L/min for heating gas flow; 3 L/min for nebulizing gas flow; 10 L/min for drying gas flow; 300 °C for interface temperature; 400 °C for heating block; 250 °C for desolvation lines. Multiple reaction monitoring (MRM) was used for MS detection. The optimized MS/MS parameters for the analyte are listed in Complementary Material Table S1. Under these conditions, the retention times were 0.47 and 0.82 min for the (+)-S-etoxazole and (−)-R-etoxazole, respectively. The parameters to evaluate the separation such as the retention factor (k), selectivity factor (α), and resolution (Rs) were determined using the following equation (Chen et al., 2014):

k= 2.4. Enzymatic assays

(t − t0) t0

(1) (2)

α = k2/ k1

Liver tissues were homogenized in cooled saline (m/v 1:10) using a hand held homogenizer (Tiangen, Beijing, China), and then centrifuged at 20,000 r/min at 4 °C for 15 min. The supernatant was collected for enzyme activity analysis. Enzymatic assays were performed using a UV–vis plate reader (Bio-Rad, USA). The CAT, SOD, GST activity, and MDA content were measured using specific assay kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and performed according to the instructions from the manufacturer.

Rs = 1.177 ×

t2 − t1 w1/2 + w2/2

(3)

where retention time is expressed as t, void time as t0 (t0 determined with 1, 3, 5-tritert-butylbenzene), retention factor as expressed by k, and the peak width at half height as w/2. 3

Ecotoxicology and Environmental Safety 192 (2020) 110287

W. Chang, et al.

dimethylphenylcarbamate) (Chiralpak IE-3), and amylose tris-3-chloro5-methylphenylcarbamate (Chipark IG-3) were tested using the same chromatographic conditions for the chiral separation of etoxazole enantiomers. Successful chromatographic separation of etoxazole enantiomers was attained by use of Chiralpak AD-H and Chiralpak IG-3 columns, but the IB-3 and IE-3 columns had inadequate recognition aptitude for etoxazole enantiomers (Fig. S2. A). The advantages of SFC analysis coupled with smaller-particle columns for chiral analysis have been previously discussed (Biba et al., 2014). Compared with the 5 μm particles in Chiralpak AD-H, Chiralpak IG-3 contains 3 μm particles and demonstrated an increased productive capacity for etoxazole enantiomer separation. Specifically, the separation of chiral etoxazole was achieved on the Chiralpak IG-3 column in < 1 min with a resolution of 5.80. In contrast, chiral separation on a Chiralpak AD-H column required over 2.5 min, which was three-fold longer than the IG-3 column (0.8 min). Polar functional groups and backbone structures of CPSs are considered essential to enantiorecognition (Roccaldo et al., 2013). The structure and functional groups of the chiral compounds also affect enantiorecognition. The analyte can interact with CPSs through hydrogen-bonding with NH groups via O, N, and F atoms and dipole-dipole interactions with C]O groups (Qiu et al., 2011). In addition, phenyl groups interact with ester moieties and phenyl carbamates of the CPSs (Klerck et al., 2012). Considering the lower solvent consumption and shorter retention time, the Chiralpak IG-3 column was selected for the subsequent optimization experiments.

2.7. Method verification and statistical analysis The performance of the proposed method was ascertained by measuring the following parameters: linearity, matrix effects, sensitivity, accuracy, and precision. Linearity was assessed by analyzing the standards and matrix-standards solutions at five concentrations (0.05, 0.5, 5, 50, and 250 μg/kg for single enantiomer). The matrix-effect was computed from the equation: Matrix effect (%) = ((slope of matrix – slope of solvent)/slope of solvent) × 100%. The limit of detection (LOD) and the limit of quantification (LOQ) were obtained with the blank and calibration solution standards consisting of the zebrafish and water matrices without etoxazole. Specifically, LOD was calculated as the lowest fortified level of the analyte that was detectable under acceptable certainty. LOQ was set as the lowest chemical concentration that was confirmed with acceptable precision (RSDr≤20%) and good recoveries (70–120%) using the proposed method (SANTE, 2017). The precision and accuracy were evaluated via recovery tests performed according to SANCO, 2011 guidelines (). Spiked samples (zebrafish and water) and six replicates at various concentrations (5, 50, and 250 μg/ kg for each enantiomer) were primed independently on different three days. The precision was determined as the relative standard deviation (RSD) and estimated via intra and inter-day experiments. The bioconcentration factor (BCF) for fish is defined as the ratio of the fish (CB) and water (CW) concentrations at sufficiently long exposure to achieve equilibrium: BCF = CB/CW (Butte and Blum, 1984) A lack of equilibrium (balanced state) can be determined with a bioaccumulation model (Gobas and Xin, 1992), which calculates the uptake and clearance rates by means of a first-order kinetic model, according to the following equations:

dCB = k1⋅Cw − k2⋅CB dt

dCB = −k2⋅CB dt

(uptake) 

3.1.2. Mobile phase Generally, the use of a certain organic modifier with CO2 can enhance the elution of polar analytes by a solvent (Wolrab et al., 2013). The modifiers (MeOH, IPA, EtOH, and ACN) most commonly used in chiral separations on SFC (Klerck et al., 2014) were tested here for the optimal separation of etoxazole enantiomers on the Chiralpak IG-3 column. Among the evaluated modifiers, MeOH exhibited the highest efficiency for chiral separation, followed by EtOH and ACN, and IPA presented the worst efficiency (Fig. S2. B). Specifically, compared with MeOH, IPA, and EtOH required much more solvent consumption due to longer retention times, and use of ACN resulted in abnormal peak shapes with lower resolution. The ratio of the organic modifier was also shown to affect enantioseparation (Roccaldo et al., 2013). Different proportions of MeOH from 5 to 20% were evaluated and the results indicated shorter retention times of the two enantiomers and lower resolution with increased ratio of the modifier. MeOH exhibited strong elution strength for etoxazole separation on the Chiralpak IG-3 column. However, as the content of the modifier continued to increase (> 20%), the resolution was still acceptable, but the high system pressure exceeded the range of the column. Based on these results, 20% MeOH was chosen as the proposed ratio for the modifier.

(4)

(clearance)

(5)

where k1 represents the uptake constant (ml/g/day), k2 is the elimination rate constant (ml/g/day), and t is the exposure time (d). Assuming t = 0, the analyte concentration in the water solution is constant and equations (4) and (5) can be written as:

Cf = (k1/ k2)⋅CW (1 − exp(−k2⋅t ) Cf = Cf,0⋅exp(−k2⋅t )

(uptake)

(clearance)

(6) (7)

where Cf,0 is the concentration of the chemical in fish at the start of clearance. Therefore, the BCF was defined as the ratio of k1 to k2, and k1 and k2 were determined from equation (6) and (7) via nonlinear regression. The BCF was calculated from

Cf / Cw = BCF = k1/ k2

(8)

The OriginPro V.9.0 software (OriginLab Corp., Northampton, MA, USA) was used to process the statistical data. Statistical differences were determined by one-way ANOVA and Tukey's multiple range test.

3.1.3. Auto back pressure regulator device An ABPR device is used in the SFC system to achieve a constant mobile phase density condition. Further, the density of the supercritical fluid directly affected the solvent robustness and influenced both enantioseparation and rentention time. As the ABPR pressure increased from 10 to 18 MPa, the retention time declined from 1.74 to 0.83 min and the resolution decreased from 7.89 to 5.81 (Fig. S2. C). However, the SFC-MS/MS system could not be used at higher ABPR pressures. Furthermore, increased ABPR pressure can reduce column lifespan. Thus, an ABPR pressure of 18 MPa was selected for further examination.

3. Results and discussion 3.1. Optimization of enantiomeric separation conditions 3.1.1. Selection of CSPs Chiral separation phases (CSPs) play a significant role in chiral recognition. According to previous work, coated or immobilized polysaccharide-derived CSPs, such as cellulose and amylose, are most frequently used due to their long-range helical subordinate structure and individual chiral carbohydrate monomers, which exhibit excellent discrimination ability for chiral compounds (Nováková and Douša, 2017; Zhao et al., 2018). Here, four promising CSPs, namely, cellulose tris(3,5-dimethylphenylcarbamate) (Chiralpak IB-3), amylose tris-(3,5-dimethylphenylcarbamate) (Chiralpak AD-H), amylose tris- (3,5-

3.1.4. Column temperature The column temperature can significantly affect retention and selectivity in chiral separation by SFC-MS/MS. Here, the temperature of the column was tested from 20 to 40 °C. The results for separation efficiency and retention time showed that temperature of the Chiralpak IG-3 column exhibited slight effects on enantiomer separation. 4

Ecotoxicology and Environmental Safety 192 (2020) 110287

W. Chang, et al.

Etoxazole enantiomers were well-separated at each evaluated temperature, so according to the temperature recommended by the manufacturer, the temperature was set at 30 °C. 3.1.5. Mobile phase flow rate The mobile phase flow rate is another significant factor that can affect elution efficiency. With high-level diffusion rate and low viscosity of supercritical fluids, a high-level flow rate can be applied in an SFC system, which improves throughput capacity and reduces analysis time. In the present study, different flow rates were tested using a binary mobile phase with CO2/MeOH (80/20, v/v). The retention time decreased with increasing flow rates (Fig. S2. D). As described above, MeOH exhibited strong elution strength for etoxazole enantiomer separation on the Chiralpak IG-3 column. The MeOH content increased with increasing flow rates, improving the elution strength. However, a high flow rate often results in high system pressure. Therefore, to optimize better resolution, relatively short analysis time, and acceptable system pressure, a flow rate of 3 mL/min was selected for subsequent experiments.

Fig. 3. Comparison of recoveries of etoxazole enantiomers (at spiking level of 50 μg/kg) with different sorbents in zebrafish (n = 6).

3.2. Elution order of etoxazole enantiomers

reported previously (Chang et al., 2019; Yao et al., 2015).

The relationship between the optical rotations (ORs) and absolute configurations for etoxazole enantiomers has been previously reported (Chang et al., 2019; Yao et al., 2015). Pure single isomers (with identified optical rotations) and rac-etoxazole were individually analyzed with SFC-MS/MS, which was performed under the conditions described above. The result showed that (+)-S-etoxazole eluted first, followed by (−)-R-etoxazole (Fig. 2), which was consistent with the separation

3.3. Optimization of the purification procedure A dispersive solid-phase extraction (D-SPE) procedure is generally utilized in a QuEChERS method with various dispersive sorbents (e.g., C18; primary secondary amine, PSA; graphitized carbon black, GCB; and

Fig. 2. SFC-MS/MS chromatograms for the separation of etoxazole enantiomers with a Chiralpak IG-3 column: A, chromatogram of racemic etoxazole; B, chromatogram of (+)-S-etoxaozle; C, chromatogram of (−)-R-etoxazole. 5

Ecotoxicology and Environmental Safety 192 (2020) 110287

W. Chang, et al.

enantiomers were all > 88% in the testing matrices (Table 2). Additionally, the intraday RSD values (n = 6) for the established method were in the range of 0.32–5.42% while the interday RSD values (n = 12) were in the range of 1.36–4.61%. These results revealed that the developed method could be applied to attain precise data for the analysis of etoxazole enantiomers in water and zebrafish samples.

Florisil). C18 can remove fat and lipophilic substances from the ACN extract, PSA removes fatty acids and polar interference, and GCB is mainly used to remove pigment. Florisil, a kind of normal phase sorbent, is primarily applied for the removal of lipid residues. With the high lipid residue in zebrafish extracts, lipid removal was essential as part of the cleanup step. Therefore, Florisil and C18 were tested in this process. Four combinations of sorbents (each supplemented with an extra 50 mg of MgSO4) were evaluated for the clean-up procedure. They were: A, 25 mg of Florisil +25 mg of C18; B, 50 mg of Florisil +50 mg of C18; C, 100 mg of Florisil +100 mg of C18; and D, 150 mg of Florisil +150 mg of C18. To determine the efficiency for interference removal, the zebrafish samples were extracted with ACN and then treated with these different sorbent mixtures. The average recovery of etoxazole enantiomers increased with the adsorbent amount (Fig. 3). Therefore, combination D, containing 150 mg of Florisil, 150 mg of C18 and 50 mg of MgSO4, was used for the purification process. The control water samples contained were few impurities, so these samples were directly extracted with ACN without purification.

3.5. SFC-MS/MS analysis In this work, we established a green, fast, and sensitive method for the etoxaozle measurement in zebrafish and water samples by SFC/MS/ MS. As an analytical technique, SFC has been increasingly applied due to its advantages for environment and health (HoguetVanessaChartonJulieHecquetPaul-Emileet al.). Compared with LC/GC approaches, use of a supercritical fluid in SFC, such as CO2 (with high diffusivity and low viscosity), allows higher flow rates resulting in shorter analysis time, higher efficiency, and lower solvent costs (Tao et al., 2014). Thus, SFC-MS/MS has been applied for fast and highly sensitive detection (Paola et al., 2017; Pan et al., 2016). In addition, owing to the high proportion of volatile CO2, SFC can be more easily applied than LC. In the present study, SFC-MS/MS analysis (< 1 min) decreased the measurement time by half and showed higher sensitivity for the detection of etoxazole enantiomers than the recently reported UPLC-MS/MS method requiring nearly 3 min (Chang et al., 2019). Importantly, aquatic samples containing low concentrations of etoxazole were accurately detected and quantified with this method. Other recent reports have described the use of SFC-MS/MS for highly sensitive detection (Fujito et al., 1508; Grand-Guillaume Perrenoud et al., 2014), indicating this is an effective alternative to existing solvent and timeconsuming methods. However, the relatively higher cost of the SFC equipment compared with the cost of the common HPLC/GC device (Ren et al., 2000) make reduce the attractiveness of use of the proposed method, especially in some small research departments. Aside from this potential cost concern, SFC-MS/MS is a potential method for practical etoxazole analysis.

3.4. Method validation 3.4.1. Specificity, linearity, and matrix effects The developed method was evaluated for specificity, linearity, and matrix effects. Analysis of control zebrafish and water samples was performed to test the specificity of the selected ion chromatograms for etoxazole stereoisomers and no interference was found. Linear regression analysis ranged from 5.0 μg/kg to 250 μg/kg for a single enantiomer and good linearity was obtained with R (Li et al., 2014)≥ 0.9953 for each enantiomer (Table 1). The matrix effect can include enhancement or suppression of the target chemical ion (Chambers et al., 2007). Thus, water and zebrafish samples compared to matrixmatched standards in pure ACN. Obvious signal suppression was noticed for the two stereoisomers in the tested matrices, ranging from 17.45 to 48.93% (Table 1). The relatively high suppression effects observed here might have resulted from the insufficient elimination of endogenous chemicals (Li et al., 1300)(e.g., fatty acids, lipids, saccharides, pigment, or phenol). Therefore, sample analysis was performed using external matrix-matched standards.

3.6. Bioaccumulation and elimination of etoxazole enantiomers in zebrafish The bioaccumulation and depuration behavior of etoxazole enantiomers were examined in zebrafish. Representative SFC-MS/MS chromatograms of (+)-S- and (−)-R-etoxazole extracted from zebrafish samples during the accumulation and depuration period were obtained and are shown in the Supporting Information (Fig. S3). During the 8 d uptake period, the average concentrations of (−)-R-etoxazole in zebrafish were significantly higher (p < 0.05, ANOVA) than those of (+)-S-etoxazole at the same time points under both low and high exposure concentrations (Fig. 5). The results indicated that the etoxazole enantiomers showed enantioselective bioaccumulation, with the preferential accumulation of (−)-R-etoxazole. BCF values were highly variable for the two enantiomers and also between different treatments (Table 3). Specifically, BCFs of (+)-S-etoxazole and (−)-R-etoxazole were both higher in high-dose treatment (138.53 and 211.73, respectively) than those in low-dose treatment (121.52 and 184.84,

3.4.2. Determination of limit of detection and the limit of quantification The LODs for each etoxazole enantiomer were obtained within the range of 0.025–0.055 μg/kg, and were estimated at the lowest spiked levels (n = 6). Similarly, the LOQs were all below 0.15 μg/kg, with an acceptable relative standard deviation (RSD, < 5.42%; Table .1). 3.4.3. Accuracy and precision Recovery assays were conducted to assess the performance of the analysis method by testing spiked samples (n = 6) for each individual stereoisomer at several different levels (5, 50, and 250 μg/kg) on three different days. Samples were prepared on three sequential days and analyzed to determine method reproducibility. To test repeatability, spiked samples were examined on the same day to evaluate the standard deviation of recovery values. The mean recoveries of the two

Table 1 Linear regression of calibration curves and matrix effects for etoxazole enantiomers. Compound

Matrix

Regression equation

R2

(+)-S-etoxazole

Solvent Zebrafish Water Solvent Zebrafish Water

y y y y y y

0.9985 0.9998 0.9999 0.9953 0.9991 0.9999

(−)-R-etoxazole

a

= = = = = =

410878x+30625.2 301543x+5050.38 176224x+99115.8 405273x+254433 334555x+4335.48 196957x+40155.6

Slope ratio = matrix/ACN. 6

a Slope ratio

Matrix effect (%)

1.36 1.09

−26.61 −47.13

1.21 1.02

−17.45 −48.93

LOD (μg/kg)

LOQ (μg/kg)

0.025 0.040 0.035 0.025 0.050 0.055

0.05 0.12 0.10 0.05 0.15 0.15

Ecotoxicology and Environmental Safety 192 (2020) 110287

W. Chang, et al.

Table 2 Percent Recovery and RSDs for analysis of enantiomers in varied matrices for three spiked levels. Compound

Matrix

Intra-day

Inter-day

Spiked level (μg/kg)

(+)-S-etoxazole

Zebrafish

Water

(−)-R-etoxazole

Zebrafish

Water

a b

5 50 250 5 50 250 5 50 250 5 50 250

Day 1

Day 2 a

Day 3 a

Recovery (%)

RSD (%)

Recovery (%)

RSD (%)

Recovery (%)

RSDa (%)

RSDb (%)

96.21 90.13 97.64 110.12 102.42 107.01 93.03 89.76 90.13 95.43 99.30 90.52

5.34 3.03 4.08 4.22 4.01 1.23 3.24 4.47 0.65 3.05 4.21 2.04

92.01 99.05 97.86 100.24 109.02 105.53 90.21 89.45 88.43 96.23 99.31 90.11

2.15 0.44 2.31 1.02 0.48 1.72 4.33 4.07 5.21 2.04 1.98 5.42

98.47 95.74 91.63 109.34 104.14 102.49 90.41 90.55 91.08 92.35 94.09 95.04

0.73 2.63 2.00 1.99 2.92 2.50 1.44 1.43 0.32 2.34 4.99 4.01

3.22 1.63 3.55 4.03 2.87 2.34 3.45 1.91 4.61 1.35 4.06 3.17

Intra-day RSD (n = 6). Inter-day RSD (n = 12).

Yao et al. reported that the metabolism of etoxazole enantiomers exhibits enantioselection in the liver microsomes of human and rat, with different enantioselectivity in elimination performance (Yao et al., 2016). Specifically, the (+)-(S)-etoxazole degraded more quickly than its antipode in human liver microsomes, but the opposite degradation enantioselectivity was observed in rat liver microsomes. These stereoselective processes might reflect differences in structural characteristics, content, and enzyme compositions in the different organisms (Arora et al., 2015; Narimatsu et al., 2000). Overall, further research will be needed to clarify the mechanism of stereoselective kinetics of etoxazole in various organisms.

Table 3 Kinetic parameters and BCF values of etoxazole enantiomers. Exposure conc.

Enantiomer

k1 (mL/g/ day)

k2 (mL/g/ day)

BCF

0.145 mg/L racetoxazole 1.450 mg/L racetoxazole

(+)-S-etoxazole (−)-R-etoxazole (+)-S-etoxazole (−)-R-etoxazole

25.52 35.12 23.55 31.76

0.21 0.19 0.17 0.15

121.52 184.84 138.53 211.73

respectively). In addition, BCFs of (−)-R-etoxazole were higher than those of (+)-S-etoxazole in both treatments. This phenomenon might result from the activities of certain enzymes in zebrafish that result in the privileged accretion of (−)-R-etoxazole resulting in enantioselective bioaccumulation. Previous studies have also reported the enantioselective bioaccumulation of chiral chemicals in zebrafish. Wang et al. documented enantioselective bioaccumulation of hexaconazole, with favored accretion of the (−)-enantiomer in zebrafish (Wang et al., 2015). Similarly, Liu et al. documented the enantioselective bioaccumulation of tebuconazole enantiomers in zebrafish, with faster accumulation of (−)-R-tebuconazole than (+)-S-tebuconazole (Liu et al., 2016). Interestingly, in that study, significant enantioselective bioaccumulation of tebuconazole was observed at a lower dose compared with the observed bioaccumulation at the higher dose. In contrast, in this study, no significant difference was found in the stereoselectivity of bioaccumulation between low and high dose exposure of etoxazole. The enantioselectivity bioaccumulation of chiral compounds in an organism can be associated with certain factors, including enzyme activities, the physicochemical property of the chiral compounds, and specific metabolic processes, so further work is required to further elucidate the mechanisms of this selection for etoxazole chirality in zebrafish. During the clearance period, etoxazole was eliminated from zebrafish during depuration, and the rate constants of the two enantiomers were determined. The depuration of the two enantiomers exhibited a sharp decline the first day of clearance. The depuration rate constant of (−)-R-etoxazole in zebrafish was lower than that of (+)-S-etoxazole at both low and high dose treatments. Additionally, the concentration of (−)-R-etoxazole remained higher than that of (+)-S-etoxazole throughout the clearance period. At the end of depuration, the concentration of (−)-R-etoxazole to (+)-S-etoxazole was ~4-fold greater (Fig. 4). The differences between etoxazole enantiomer concentrations at the same time points were also analyzed using SPSS 19.0 and the results showed that the elimination of (−)-R-etoxazole and (+)-Setoxazole from zebrafish was enantioselective (p < 0.05, ANOVA).

3.7. Activities of antioxidant enzymes and the MDA content Oxidative stress is regarded as a potential mechanism for pesticideinduced toxicity (Renugadevi and Prabu, 2010). Antioxidant enzymes serve to eliminate ROS and protect organisms from oxidative damage. SOD can transform O2 into H2O2, which is further converted to oxygen and water with the combined action of CAT (Pi et al., 2010). When the antioxidant system is overwhelmed or not functioning, oxidative stress follows. As shown in Fig. 5A, the enzyme activities of SOD did not show significant change after low-dose exposure for 4, 8, and 15 days compared to the control (p > 0.05, ANOVA). However, SOD activity decreased with increasing waterborne concentrations and exposure duration at high-dose treatment, with significant change at 8 and 15 days compared to the two other groups. The observed significant reduction of SOD activities implied generation of oxidative stress from the exposure to etoxazole at high-dose treatment, and reduced ability of SOD to transform ROS into H2O2. The relatively higher SOD activity in the low-dose exposure group is probably related to a lower level of excess ROS in zebrafish due to lower amount of etoxazole. However, CAT activities showed no significant change at all tested time points among different groups (Fig. 5B). This result was consistent with the result of a previous study, which examined the toxic effect of IBU on zebrafish (Yue et al. et al, 2018). In that study, ibuprofen had no influence on CAT activity within a 14-day period of exposure. Here, GST activity, was slightly enhanced at the 4th day for all dosage treatments compared to controls, but the difference was not significant (p > 0.05, ANOVA). At the second and third time points, the enzyme activity of GST was significantly increased compared to that observed in controls (Fig. 5C). Metcalf et al. (Metcalf, et al, 2000) reported that GST enzyme promotes detoxification after exposure to microcystins in zebrafish. In this study, an elevation in GST activity was detected at the 8th and 15th day after exposure, indicating an adaptive signal by the liver in response to etoxaozle. This adaption is likely an intrinsic defense 7

Ecotoxicology and Environmental Safety 192 (2020) 110287

W. Chang, et al.

Fig. 4. Enantioselective accumulation and depuration behavior of etoxazole enantiomers in zebrafish. (A) Low concentration and (B) High concentration. Data are expressed as mean values ± SD (n = 3).

balneation conditions to provide additional information on the mechanisms of biotoxicity of etoxazole in aquatic organisms.

mechanism in zebrafish. No previous work has attempted to explore the change in GST activity upon etoxazole exposure, so the results obtained in this work could provide the basis for future study of the potential detoxification mechanism of etoxazole. The findings here are consistent with those reported by Pavagadhi et al. (Pavagadhi et al, 2012). In that work, GST activity for MC-LR (0.1–1.0 μg/kg)-exposed tissue groups of zebrafish was increased in liver and brain the 7th and the 15th day after the start of exposure. For MC-RR exposed tissue groups, the GST activity continued to increase for all dosage groups in all tissues during the 15 days of treatment. A similar pattern was also reported by Song et al. (Song et al, 2018.), who studied the stereoselective effects of ibuprofen (5 μg/kg) on zebrafish. In their study, the activity of GST enzyme in zebrafish brain was significantly increased compared to controls for an exposure period of 28 days. MDA is considered as a biomarker for the damage of cellular oxidation (Valko, et al, 2007). In this study, MDA content increased with the duration of exposure in the high-dose etoxazole treated group for 4, 8, and 15 days, and significant difference was observed at 8th and 15th day between the high-dose treatment and the two other groups (p < 0.05, ANOVA). However, no change of MDA level was observed for low-dose exposure of etoxazole at tested time points (Fig. 5D). We hypothesized that the increased MDA content observed in the high dosage treatment may be related to the overwhelming enzymatic activities, i.e. SOD. Oxidative stress occurred in the zebrafish, and both the concentration of etoxazole and exposure duration influenced the antioxidant enzyme activity in liver of zebrafish. The oxidative stress observed during the balneation study is consistent with that of azoxystrobin toward zebrafish liver (Han et al., 2016), in which azoxystrobin notably induced oxidative stress and genotoxicity, indicating that extoxazole and azoxystrobin might have a similar mechanism of toxicity toward zebrafish. The results obtained in the present work are consistent with the findings reported by Zhuang et al. (ShulinZhuangZhishengZhangWenjingZhanget al.) on zebrafish embryos. In their work, pyraclofos caused concentration- and timedependent malformations in zebrafish including yolk sac edema, pericardial edema, and hatching during embryonic development. Sevgiler et al. (Oruc and Nevin., 2004) studied the effects of etoxazole after exposure of Oreochromis niloticus to five different sub-lethal etoxazole concentrations. However, in that study, no significant change was observed in the activities of antioxidant enzymes compared to controls. The different results may represent species-specific biochemical behavior towards etoxazole. Therefore, further research is needed to determine the fate and transfer of etoxazole by focusing on bioaccumulation. Additionally, more species should be investigated under

4. Conclusion In the present work, a novel analytical method, using SFC-MS/MS techniques combined with a modified QuEChERS pretreatment, was successfully established and applied for the determination of etoxazole enantiomers in zebrafish and water samples. The proposed approach is greener, with improved sensitivity and velocity of the analytical method, compared with the currently available enantioseparation method of etoxazole enantiomers. Application of this method allowed the estimation of the enantioselective behavior toward etoxaozle in zebrafish. The concentrations of etoxazole enantiomers during the uptake and clearance period in zebrafish were evaluated. Pronounced enantioselective bioaccumulation was observed in these zebrafish, with the preferential accumulation of (−)-R-etoxazole compared to (+)-Setoxazole in the uptake phase, and slower degradation of (−)-R-etoxazole during the clearance period. Moreover, oxidative stress was observed in the liver of adult zebrafish due to the toxicity of etoxazole, and this toxic effect exhibited time- and dose-dependent behavior. The results obtained in this study can facilitate accurate risk assessments of etoxazole and provide technical support for future studies to investigate the fate and metabolism of etoxazole enantiomers and guide efforts to decrease the risk posed by etoxazole. Additionally, this work also provides the basis for comprehensive evaluation of etoxazole biotoxicity mechanism in different aquatic organisms. Acknowledgment This work was supported by the earmarked fund for China Agriculture Research System (CARS-27), the National Key R&D Program of China (2016YFD0201207), the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-ASTIP), and the National Program for Quality and Safety Risk Assessment of Agricultural Products of China (GJFP2018003). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2020.110287. 8

Ecotoxicology and Environmental Safety 192 (2020) 110287

W. Chang, et al.

Fig. 5. (A)SOD, (B) CAT, (C)GST activities and (D)MDA content in the liver of zebrafish exposed to etoxazole at three time points. Values are presented as a mean ± SD (n = 3). Different letters (a, b) for each experimental group with a single exposure time indicate significant difference (p < 0.05, ANOVA) among the three treatments (control, 0.145 mg/L, and 1.45 mg/L).

Notes

Arora, S., Taneja, I., Challagundla, M., Raju, K.S., Singh, S.P., Wahajuddin, M., 2015. In vivo prediction of CYP-mediated metabolic interaction potential of formononetin and biochanin A using in vitro human and rat CYP450 inhibition data. Toxicol. Lett. 239 S0378427415300357. Avallone, B., Agnisola, C., Cerciello, R., Panzuto, R., Motta, C.M., 2015. Structural and functional changes in the zebrafish (Danio rerio) skeletal muscle after cadmium exposure. Cell Biol. Toxicol. 31, 273. Batel, A., Borchert, F., Reinwald, H., Erdinger, L., Braunbeck, T., 2018. Microplastic accumulation patterns and transfer of benzo[a]pyrene to adult zebrafish (Danio rerio) gills and zebrafish embryos. Environ. Pollut. 235, 918. Biba, M., Regalado, E.L., Wu, N., Welch, C.J., 2014. Effect of particle size on the speed and resolution of chiral separations using supercritical fluid chromatography. J. Chromatogr. A 1363, 250–256. Butte, W., Blum, J.K., 1984. Calculation of bioconcentration factors from kinetic data by non-linear iterative least-squares regression analysis using a programmable minicalculator. Chemosphere 13, 151–160. Chai, T., Cui, F., Song, Y., Ye, L., Li, T., Qiu, J., et al., 2018. Enantioselective toxicity in adult zebrafish (Danio rerio) induced by chiral PCB91 through multiple pathways. Environ. Sci. Technol. 52, 5448–5458. Chambers, E., Wagrowski-Diehl, D.M., Lu, Z., Mazzeo, J.R., 2007. Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses. J Chromatogr B Analyt Technol Biomed Life Sci 852, 22–34. Chang, W., Nie, J., Yan, Z., Wang, Y., Farooq, S., 2019. Systemic stereoselectivity study of etoxazole: stereoselective bioactivity, acute toxicity, and environmental behavior in

The authors declare no competing financial interest. Author contribution Weixia Chang: Methodology, Experiment for sample, Formal analysis. Software, Data curation, Writing - original draft. Jiyun Nie: Validation, Funding acquisition, Supervision, Data curation. Writing - review & editing. Yue Geng: Methodology, Validation, Visualization, Supervision. Danyan Zhang: Resources, Formal analysis. Writing - review & editing. Qi Wang: Resources, Sample preparation, Software. Saqib Farooq: Writing - review & editing. References Arena, M., Auteri, D., Barmaz, S., Bellisai, G., Brancato, A., Brocca, D., et al., 2017. Peer review of the pesticide risk assessment of the active substance etoxazole. Efsa Journal 15, 4988.

9

Ecotoxicology and Environmental Safety 192 (2020) 110287

W. Chang, et al.

R., 2012. Biochemical response of diverse organs in adult Danio rerio (zebrafish) exposed to sub-lethal concentrations of microcystin-LR and microcystin-RR: a balneation study. 109 0-10. Pi, J., Zhang, Q., Fu, J., Woods, C.G., Hou, Y., Corkey, B.E., et al., 2010. ROS signaling, oxidative stress and Nrf2 in pancreatic beta-cell function. 244, 77–83. Method validation and quality control procedures for pesticide residues analysis in food and feed. Document N° SANCO/12495/. Qiu, J., Dai, S., Zheng, C., Yang, S., Chai, T., Bie, M., 2011. Enantiomeric separation of triazole fungicides with 3-μm and 5-μml particle chiral columns by reverse-phase high-performance liquid chromatography. Chirality 23, 479–486. Ren, Q., Su, B., Huang, M., Wu, P., 2000. Advances in chiral separation by supercritical fluid chromatography. Chin. J. Anal. Chem. 28. Rencuzogullari, E., Ila, H.B., Kayraldiz, A., Arslan, M., Diler, S.B., Topaktas, M., 2004. The genotoxic effect of the new acaricide etoxazole. Russ. J. Genet. 40, 1300–1304. Renugadevi, J., Prabu, S.M., 2010. Cadmium-induced hepatotoxicity in rats and the protective effect of naringenin. 62, 171–181. Roccaldo, S., Federica, I., Antonella, L., Maura, M., Stefania, S., Benedetto, N., 2013. The effect of mobile phase composition in the enantioseparation of pharmaceutically relevant compounds with polysaccharide-based stationary phases. Biomedical Chromatography Bmc 28, 159–167. Sante, D.G.-, 2017. Directorate General for Health and Food Safety. Guidance Document on Analytical Quality Control and Validation Procedures for Pesticide Residues Analysis in Food and Feed. SANTE 11813. Song, Yue, Chai, Tingting, Yin, Zhiqiang, et al., 2018. Stereoselective Effects of Ibuprofen in Adult Zebrafish (Danio rerio) Using UPLC-TOF/MS-based Metabolomics. Sun, D.L., Pang, J.X., Fang, Q., Zhou, Z.Q., Jiao, B.N., 2016. Stereoselective toxicity of etoxazole to MCF-7 cells and its dissipation behavior in citrus and soil. Environ. Sci. Pollut. Res. 23, 24731–24738. Tao, Y., Dong, F., Xu, J., Liu, X., Cheng, Y., Liu, N., et al., 2014. Green and sensitive supercritical fluid chromatographic-tandem mass spectrometric method for the separation and determination of flutriafol enantiomers in vegetables, fruits, and soil. 62, 11457–11464. The National Standard of the People's Republic of China, 2014. Test Guidelines on Environmental Safety Assessment for Chemical Pesticides GB/T 31270. pp. 1. Valavanidis, A., Vlahogianni, T., Dassenakis, M., Scoullos, M., 2006. Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants. Ecotocicol Environ Saf 64 0-189. Valko, M.;Leibfritz, D.;Moncol, J.;Cronin, M. T. D.;Mazur, M.;Telser, J. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol..39,0-84. Wang, Y., Xu, L., Li, D., Teng, M., Zhang, R., Zhou, Z., et al., 2015. Enantioselective bioaccumulation of hexaconazole and its toxic effects in adult zebrafish ( Danio rerio ). Chemosphere 138, 798–805. Wolrab, D., Kohout, M., Boras, M., Lindner, W., 2013. Strong cation exchange-type chiral stationary phase for enantioseparation of chiral amines in subcritical fluid chromatography. J. Chromatogr. A 1289, 94–104. Yao, Z.L., Li, Z.G., Zhuang, S.L., Li, X.G., Xu, M.F., Lin, M., et al., 2015. Enantioselective determination of acaricide etoxazole in orange pulp, peel, and whole orange by chiral liquid chromatography with tandem mass spectrometry. J. Separ. Sci. 38, 599–604. Yao, Z.L., Qian, M.R., Zhang, H., Nie, J., Ye, J.Q., Li, Z.G., 2016. Etoxazole is metabolized enantioselectively in liver microsomes of rat and human in vitro. Environ. Sci. Technol. 50, 9682–9688. Yue, S., Tingting, C., Zhiqiang, Y., Xining, Z., Wei, Z., Yongzhong, Q., et al., 2018. Stereoselective effects of ibuprofen in adult zebrafish ( Danio rerio ) using UPLCTOF/MS-based metabolomics. Environ. Pollut. 241, 730–739. Zhao, L., Xie, J., Guo, F., Liu, K., 2018. Enantioseparation of napropamide by supercritical fluid chromatography: effects of the chromatographic conditions and separation mechanism. Chirality 30, 661–669.

fruits and soils. J. Agric. Food Chem. 67, 6708–6715. Chen, Z.L., Dong, F.S., Xu, J., Liu, X.G., Cheng, Y.P., Liu, N., et al., 2014. Stereoselective separation and pharmacokinetic dissipation of the chiral neonicotinoid sulfoxaflor in soil by ultraperformance convergence chromatography/tandem mass spectrometry. Anal. Bioanal. Chem. 406, 6677–6690. Dekeyser, M.A., 2005. Acaricide mode of action. Pest Manag. Sci. 61, 103–110. Franke, C., Studinger, G., Berger, G., Böhling, S., Bruckmann, U., Cohors-Fresenborg, D., et al., 1994. The assessment of bioaccumulation. Chemosphere 29 0-1514. Fujito, Y.;Hayakawa, Y.;Izumi, Y.;Bamba, T. Importance of optimizing chromatographic conditions and mass spectrometric parameters for supercritical fluid chromatography/mass spectrometry. J. Chromatogr. A.1508,138-147. Gobas, F.A.P.C., Xin, Z., 1992. Measuring bioconcentration factors and rate constants of chemicals in aquatic organisms under conditions of variable water concentrations and short exposure time. Chemosphere 25, 1961–1971. Grand-Guillaume Perrenoud, A., Veuthey, J.L., Guillarme, D., 2014. Coupling state-ofthe-art supercritical fluid chromatography and mass spectrometry: from hyphenation interface optimization to high-sensitivity analysis of pharmaceutical compounds. J. Chromatogr. A 1339, 174–184. Klerck, K.D., Parewyck, G., Mangelings, D., Heyden, Y.V., 2012. Enantioselectivity of polysaccharide-based chiral stationary phases in supercritical fluid chromatography using methanol-containing carbon dioxide mobile phases. J. Chromatogr. A 1269, 336–345. Han, Y., Liu, T., Wang, J., Wang, J., Zhang, C., Zhu, L., 2016. Genotoxicity and oxidative stress induced by the fungicide azoxystrobin in zebrafish (Danio rerio) livers. Pestic. Biochem. Physiol. 133, 13–19. Klerck, K.D., Heyden, Y.V., Mangelings, D., 2014. Generic chiral method development in supercritical fluid chromatography and ultra-performance supercritical fluid chromatography. J. Chromatogr. A 1363, 311–322. Li, M.;Liu, X.;Dong, F.;Xu, J.;Kong, Z.;Li, Y.;et al. Simultaneous determination of cyflumetofen and its main metabolite residues in samples of plant and animal origin using multi-walled carbon nanotubes in dispersive solid-phase extraction and ultrahigh performance liquid chromatography–tandem mass spectro. J. Chromatogr. A.1300, 95-103. Li, Y.Q., Yang, N., Wei, X.C., Ling, Y., Yang, X.L., Wang, Q.M., 2014. Evaluation of etoxazole against insects and acari in vegetables in China. J. Insect Sci. 14. Liu, N., Dong, F., Xu, J., Liu, X., Zheng, Y., 2016. Chiral bioaccumulation behavior of tebuconazole in the zebrafish ( Danio rerio ). Ecotoxicol. Environ. Saf. 126, 78–84. Lu, K., Qiao, R., An, H., Zhang, Y., 2018. Influence of microplastics on the accumulation and chronic toxic effects of cadmium in zebrafish ( Danio rerio ). Chemosphere 202 S0045653518305629. Metcalf, J.S., Beattie, K.A., Pflugmacher, S., Codd, G.A., 2000. Immuno-crossreactivity and toxicity assessment of conjugation products of the cyanobacterial toxin, microcystin-LR. FEMS Microbiol. Lett. 189, 155–158. Narimatsu, S., Kobayashi, N., Masubuchi, Y., Horie, T., Kakegawa, T., Kobayashi, H., et al., 2000. Species difference in enantioselectivity for the oxidation of propranolol by cytochrome P450 2D enzymes. Chem. Biol. Interact. 127, 73–90. Nováková, L., Douša, M., 2017. General screening and optimization strategy for fast chiral separations in modern supercritical fluid chromatography. Anal. Chim. Acta 950, 199–210. SEVGILER;Yusuf,Oruc, E.O., UNER,Nevin, 2004. Evaluation of etoxazole toxicity in the liver of Oreochromis niloticus. Pestic. Biochem. Physiol. 78, 1–8. Pan, X., Dong, F., Xu, J., Liu, X., Chen, Z., Zheng, Y., 2016. Stereoselective analysis of novel chiral fungicide pyrisoxazole in cucumber, tomato and soil under different application methods with supercritical fluid chromatography/tandem mass spectrometry. J. Hazard Mater. 311, 115–124. Paola, E.L.D., Montevecchi, G., Masino, F., Garbini, D., Barbanera, M., Antonelli, A., 2017. Determination of acrylamide in dried fruits and edible seeds using QuEChERS extraction and LC separation with MS detection. Food Chem. 217, 191–195. Pavagadhi, S., Gong, Z., Hande, M.P., Dionysiou, D.D., Cruz, A.A.d. l., Balasubramanian,

10