Environmental Pollution xxx (2017) 1e8
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Acute toxicity, bioconcentration, elimination and antioxidant effects of fluralaner in zebrafish, Danio rerio* Zhong-Qiang Jia a, 1, Di Liu a, 1, Cheng-Wang Sheng a, John E. Casida b, Chen Wang c, Ping-Ping Song d, Yu-Ming Chen a, Zhao-Jun Han a, Chun-Qing Zhao a, * a
Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy, and Management, University of California, Berkeley, CA, 94720-3112, United States c Tea Research Institute, Chinese Academy of Agriculture Sciences, Hangzhou, 310008, China d Jiangsu Centre for Research and Development of Medicinal Plants, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, 210095, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 June 2017 Received in revised form 9 September 2017 Accepted 11 September 2017 Available online xxx
Fluralaner is a novel isoxazoline insecticide which shows high insecticidal activity against parasitic, sanitary and agricultural pests, but there is little information about the effect of fluralaner on non-target organisms. This study reports the acute toxicity, bioconcentration, elimination and antioxidant response of fluralaner in zebrafish. All LC50 values of fluralaner to zebrafish were higher than 10 mg L1 at 24, 48, 72 and 96 h. To study the bioconcentration and elimination, the zebrafish were exposed to sub-lethal concentrations of fluralaner (2.00 and 0.20 mg L1) for 15 d and then held 6 d in clean water. The results showed medium BCF of fluralaner with values of 12.06 (48 h) and 21.34 (144 h) after exposure to 2.00 and 0.20 mg L1 fluralaner, respectively. In the elimination process, a concentration of only 0.113 mg kg1 was found in zebrafish on the 6th day after removal to clean water. After exposure in 2.00 mg L1 fluralaner, the enzyme activities of SOD, CAT, and GST, GSH-PX, CarE and content of MDA were measured. Only CAT and CarE activities were significantly regulated and the others stayed at a stable level compared to the control group. Meanwhile, transcriptional expression of CYP1C2, CYP1D1, CYP11A were significantly down-regulated at 12 h exposed to 2.00 mg L1 of fluralaner. Except CYP1D1, others CYPs were up-regulated at different time during exposure periods. Fluralaner and its formulated product (BRAVECTO®) are of low toxicity to zebrafish and are rapidly concentrated in zebrafish and eliminated after exposure in clean water. Antioxidant defense and metabolic systems were involved in the fluralaner-induced toxicity. Among them, the activities of CAT and CarE, and most mRNA expression level of CYPs showed fast response to the sub-lethal concentration of fluralaner, which could be used as a biomarker relevant to the toxicity. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Fluralaner Zebrafish Acute toxicity Bioconcentration Antioxidant response Cytochrome P450
1. Introduction The isoxazoline insecticide fluralaner (Fig. 1) has potent antiparasitic, insecticidal and acaricidal activity and has been marketed as BRAVECTO® in North America, Europe and East Asia (Casida, 2015; Ozoe et al., 2010; Zhao et al., 2015). Fluralaner can be safely
*
This paper has been recommended for acceptance by Charles Wong. * Corresponding author. E-mail address:
[email protected] (C.-Q. Zhao). 1 Both authors equally contributed to this work.
administered orally to dogs (Walther et al., 2014a, 2014c) and rabbits (Sheinberg et al., 2017). Bioassay, radioligand binding assay and electrophysiological experiments demonstrated that fluralaner has no cross-resistance with other commercial insecticides, such as fiproles (e.g., fipronil) and cyclodienes (e.g., endosulfan), etc. with unique binding site and action on the GABA receptor (Asahi et al., 2015; Casida, 2015; Gassel et al., 2014; Ozoe et al., 2010; Zhao and Casida, 2014), which indicates that fluralaner is a new generation of GABAergic insecticides with potential use in agricultural production. In addition, three isoxazoline insecticides (fluralaner, sarolaner, and afoxolaner) are marketed as BRAVECTO®, Simparica,
http://dx.doi.org/10.1016/j.envpol.2017.09.032 0269-7491/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Jia, Z.-Q., et al., Acute toxicity, bioconcentration, elimination and antioxidant effects of fluralaner in zebrafish, Danio rerio, Environmental Pollution (2017), http://dx.doi.org/10.1016/j.envpol.2017.09.032
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Abbreviation a.i. active ingredient BCF bioconcentration factor BSA bovine serum albumin CarE carboxylesterase CAT catalase cDNA complementary DNA DAD diode array detector; DEM, diethyl maleate DO dissolved oxygen DTW dechlorinated tap water GSH-PX glutathione peroxidase GST glutathione- S-transferase HC Orange No.1 2-nitro-40 -hydroxydiphenylamine HPLC high performance liquid chromatography LC50 medium lethal concentration
LOQ lower limit of detection MDA malonaldehyde PAHs polycyclic aromatic hydrocarbons PBO piperonyl butoxide PSA primary/secondary amine qPCR quantitative real-time polymerase chain reaction QuEChERS Quick, Easy, Cheap, Effective, Rugged and Safe ROS reactive oxygen species RSD relative standard deviations RT room temperature SECP Standard for Environmental Safety Evaluation of Chemical Pesticides SOD superoxide dismutase SR synergy effects TPP triphenyl phosphate
2. Materials and methods 2.1. Ethical statement The authors guarantee that all the experimental zebrafish used in the present study were maintained and manipulated in the laboratory in accordance with the code of ethics of the OECD (Code: 203e17.07.92), the China [2010-172] and Nanjing Agricultural University guidelines for the protection of animal welfare. Fig. 1. Chemical structure of fluralaner (A) and commercial product of BRAVECTO® (B).
2.2. Tested zebrafish, chemicals and solutions
and NexGard, respectively, and exhibit high activity to target organisms (Mita et al., 2015; Zhao and Casida, 2014). Hence, study of acute toxicity, bioconcentration and elimination of fluralaner, which could be considered as the representative isoxazoline insecticide, is very necessary in assessing its fate and potential toxic effects in aquatic organisms. Environmental contamination with insecticides is an important problem worldwide. Data on their bioconcentration and elimination are therefore valuable to allow the new insecticides to sell in the market. Meanwhile, insecticide could bring about toxicity, which could be caused by oxidative stress, including the increased production of reactive oxygen species (ROS), H2O2, lipid peroxidation, and associated change in antioxidant enzyme activities in fish (Liu et al., 2016). Cytochrome P450 enzyme (CYP) could metabolize and detoxify exogenous substrate, such as insecticides. Generally, the balance between ROS production and antioxidant enzymes activities exist under normal physiological conditions (Hilscherova et al., 2003). However, oxidative damage could occur and the expression level of CYP would be affected and involve the metabolism of xenobiotics, while the antioxidant enzymes are unable to clear the generation of ROS production in time (Lassen et al., 2008). Although fluralaner has high activity on pests and little effect on mammals (Walther et al., 2014b), the effects of fluralaner on nontargeted organisms such as fish, as well as its accumulation trend are still unknown. The objective of this study was to investigate the acute toxicity, bioconcentration, elimination level, and antioxidant response of fluralaner in zebrafish, Danio rerio, to help understand its ecological effects.
The zebrafish were purchased from the Aquarium Breeding Center of Nanjing and acclimated under laboratory conditions for at least 7 d with total mortality less than 5% to guarantee their health and quality. Dechlorinated tap water (DTW) was obtained by passing municipal tap water through a high-efficiency active carbon filtration system. During the acclimation period, air was blown through the water constantly to keep the dissolved oxygen (DO) level at 6.8 ± 0.5 mg L1, and artificial dry food approximately 1% of body weight was provided once a day, then uneaten food and faeces were siphoned daily from the test chambers shortly after feeding (0.5e1 h). During the whole experiment, the temperature and pH value of water were at 23 ± 1 C and 7.5 ± 0.5, respectively. Acetonitrile (HPLC grade) was purchased from J.T. Baker® (Avantor Performance Materials, Inc. Center Valley, PA). Bondesil Cleanert primary/secondary amine (PSA) with size of 40e60 mm was supplied by Agela Technologies (Tianjin, China). Deionized water was obtained from the Milli-Q SP Reagent Water system (Millipore, Bedford, MA); Sodium chloride (NaCl) and other reagents as AR grade were purchased from Sinopharm Chemical (Beijing, China). The fluralaner standard (purity99.0%) was obtained from the analytical laboratory of Nanjing Agricultural University and the BRAVECTO® chewable tablets (1400 mg fluralaner per tablet) were purchased from the Merck & Co., Inc. (Isando, South Africa). The stock standard solution (10,000 mg L1) of fluralaner was prepared with acetone for bioassay or acetonitrile for high performance liquid chromatography (HPLC) analysis and stored at 20 C. The working standard solution was diluted with the stock standard solution and acetonitrile, and stored at 4 C before use.
Please cite this article in press as: Jia, Z.-Q., et al., Acute toxicity, bioconcentration, elimination and antioxidant effects of fluralaner in zebrafish, Danio rerio, Environmental Pollution (2017), http://dx.doi.org/10.1016/j.envpol.2017.09.032
Z.-Q. Jia et al. / Environmental Pollution xxx (2017) 1e8
2.3. Acute toxicity experiment The 120 h toxicity experiment was conducted using a modified method outlined by the OECD (Code: 203e17.07.92) and Standard for Environmental Safety Evaluation of Chemical Pesticides (SECP) of China. Zebrafish were randomly exposed to a range of concentrations of fluralaner (0.625, 1.25, 2.50, 5.00 and 10.00 mg L1) selected based on the solubility of fluralaner and pre-assay. Fluralaner-free water with other accessory solvent was used as control. BRAVECTO® chewable tablets were easily dissolved in water at theoretical concentration of active ingredient (a.i.) up to 100 mg L1. Each test concentration was replicated at least three times. Each aquarium contained 10 L DTW with exposure solution and 10 zebrafish. All exposures were conducted at 23 ± 1 C with a 12: 12 h (light: dark) photoperiod. The exposure solution was fully renewed for every 48 h, and water-quality parameters, including pH, DO, and temperature were monitored every day. During the whole experiment, zebrafish were not fed and dead zebrafish were removed in a timely manner. 2.4. Bioconcentration and elimination experiment Bioconcentration of fluralaner in zebrafish was examined by exposing ~1000 zebrafish to fluralaner in 12 different glass aquaria. Each aquarium contained 20 L DTW, 80 zebrafish, and the proper amount of fluralaner (2.00 and 0.20 mg L1). Experiments for each concentration were replicated three times. After 0 h, 12 h, 24 h, 48 h, 96 h, 144 h (6d), 192 h (8d), 240 h (10d), 288 h (12d) and 360 h (15d) of exposure, seven fish and 1 mL water from each aquarium were collected and stored at 20 C before undergoing sample preparation. After 15d, the remaining zebrafish in exposure solution were transferred into the clean DTW for elimination test at 17d, 19d and 21d. The test solution was re-prepared each 48 h to maintain a stable concentration of fluralaner in water (Fig. S1). The corresponding water samples were collected from the aquaria during the bioconcentration period (0, 0.5, 1, 2, 4, 5, 6, 8, 10, 12, and 15d), and elimination period (17, 19 and 21d), respectively and stored at 20 C before sample preparation. 2.4.1. Water sample preparation The quantitative determination of fluralaner in water involved extraction with acetonitrile and NaCl after optimization (data not shown). In brief, 800 mL water sample was mixed with 900 mL acetonitrile and 100 mg NaCl, and then vortexed at 1500 rpm for 1.5 min in Silence Shake HYQ-3110 vortex (Crystal Technology & Industries, Inc., Addison, TX). After the above mixture was centrifuged at 6100 g for 5 min, the supernatant was removed and filtered by 0.22 mm nylon filter (Jin-long Material Co. Ltd., Tianjin, China) and stored at 4 C before HPLC analysis. 2.4.2. Zebrafish sample preparation and recovery experiment On account of the characteristic of fluralaner and the complexity of fish matrix, the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method was applied to extract fluralaner from zebrafish with some modifications (Anastassiades et al., 2003; Zhao et al., 2011). A three-step experimental procedure was set up as follows: Step 1. Sample preparation - Total amount of 2.0 g whole zebrafish body was rinsed with DTW, blotted dry on absorbent paper, killed and cut into small pieces with stainless steel scissors, and thoroughly homogenized quickly at room temperature (RT) in mortar and pestle. Finally the sample was moved into a 15 mL plastic tube. Step 2. Sample extraction and cleanup - 8 mL acetonitrile was added into the 15 mL plastic tube, mixed with the fresh zebrafish sample in shaker and kept at RT for 1h; then samples were vortexed
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at 1500 rpm for 1.5 min after 2.00 g NaCl was added. Samples were centrifuged at 3000 g for 5 min by Allegra TM 25R Centrifuge (Beckman Coulter, Inc., Brea, CA). A 1.00 mL aliquot of the supernatant was transferred to a 2 mL plastic tube containing 50.00 mg PSA, vortexed at 1500 rpm for 2 min and then centrifuged at 6100 g for 5 min in Centrifuge 5424 R (Eppendorf, Hauppauge, NY). Step 3. Supernatant collection - Supernatant (~600 mL) was filtered with 0.22 mm nylon filter and analyzed by HPLC. 2.4.3. HPLC analysis and analytical method validation The full-wave length scan of fluralaner on diode array detector (DAD) was conducted (Fig. S2) and 264 nm was chosen as the optimal wave-length. Fluralaner extracted from water and fish samples was determined on Agilent 1220 Infinity LC (Agilent Technologies, Santa Clara, CA) fitted with quaternary pump at wavelength of 264 nm. Separation was carried out using a ZORBAX Eclipse XDB-C18 column (250 mm 4.6 mm, 5 mm) (Agilent Technologies). The optimized mobile phase A (0.05% trifluoroacetic acid in water) and B (acetonitrile) was pumped at a flow rate of 1.0 mL min1 with gradient elution mode at RT. The mobile phase composition was changed as follows: 50% B (t ¼ 0 min), 90% B (t ¼ 10min), 60% B (t ¼ 12 min) and 50% B (t ¼ 15min) (GarcíaReynaga et al., 2013). The injection volume was 20 mL. Negative controls in recovery experiments revealed no background interference. The amount of fluralaner was calculated with an external standard calibration curve. For the higher concentration (>1 mg L1), the solution should be dissolved with acetonitrile. The retention time of fluralaner was 10.72 min. 2.5. Bioconcentration factor (BCF) The BCF of fluralaner in zebrafish was calculated using the following equation (Opperhuizen, 1991; Van der Oost et al., 2003): BCF ¼ Cf/Cw Where Cf and Cw is the average concentration of fluralaner in zebrafish (mg kg1) and water (mg L1), respectively. Considerations: low bioconcentration (BCF
