Pesticide Biochemistry and Physiology 115 (2014) 9–14
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Acute toxicity and sublethal effects of ﬁpronil on detoxiﬁcation enzymes in juvenile zebraﬁsh (Danio rerio) Haihua Wu a, Cuie Gao a, Yaping Guo b, Yuping Zhang c, Jianzhen Zhang a, Enbo Ma a,⇑ a
Institute of Applied Biology, Shanxi University, Taiyuan 030006, Shanxi, PR China College of Life Science, Shanxi University, Taiyuan 030006, Shanxi, PR China c Biology Department, Taiyuan Normal University, Taiyuan 030012, Shanxi, PR China b
a r t i c l e
i n f o
Article history: Received 30 January 2014 Accepted 27 July 2014 Available online 4 August 2014 Keywords: Fipronil Acute toxicity Sublethal effects Detoxiﬁcation enzymes Zebraﬁsh (Danio rerio)
a b s t r a c t The acute toxicity of ﬁpronil and its sublethal effects on detoxiﬁcation enzymes (carboxylesterases (CarEs), glutathione S-transferases (GSTs), and 7-ethoxycoumarin O-deethylase (ECOD)) in zebraﬁsh (Danio rerio) were investigated. The results indicated that the 24-h LC50 of ﬁpronil for zebraﬁsh was 220.4 lg/L (95% CI: 173.7–272.4 lg/L). Sublethal concentrations of ﬁpronil did not cause signiﬁcant changes in CarEs activities. In the liver and muscle tissues, GST activities at the tested concentrations did not signiﬁcantly differ from those in the control. In the brain and gill tissues, GST activities at a concentration of 4 lg/L were signiﬁcantly lower than those at a concentration of 2 lg/L. The results suggest that CarEs and GSTs were not suitable biomarkers for ﬁpronil effects in D. rerio. A signiﬁcant induction in the ECOD activities in the brain, gill, liver, and muscle tissues was observed compared with the control. Moreover, the dose-dependent responses of the ECOD activity were observed after treatment with sublethal concentrations of ﬁpronil in the range of 2–20 lg/L. The results suggested that ECOD could be a suitable biomarker of ﬁpronil effects in D. rerio. Ó 2014 Published by Elsevier Inc.
1. Introduction Fipronil is a phenylpyrazole insecticide developed by RhonePoulenc Agro , Fipronil’s mechanism of action is to block insect chloride channels that are controlled by gamma-aminobutyric acid (GABA) in the neurons of the central nervous system . Because ﬁpronil is used both commercially and in home applications, recent concerns for potential adverse public health effects have been raised . Fipronil is highly toxic to many non-target organisms, such as honeybees, ﬁsh, aquatic invertebrates, and upland game birds . Fipronil treatment is reportedly associated with thyroid disruption in rats . A very low dose of ﬁpronil (0.5 ng/ bee applied topically) impaired the olfactory learning of honeybees . Chandler et al.  reported that the fertility, reproduction, and development of an estuarine copepod were affected at ﬁpronil concentrations of 0.22 lg/L. A few studies have been conducted to determine the potential effect of ﬁpronil on aquatic organisms [8–11] and aquatic ecosystems [12,13]. Ngim and Crosby  found ﬁpronil to be toxic to crayﬁsh that live in rice ﬁelds at ppb levels: 14.3–19.5 lg/L. The exposure of Daphnia pulex to 30 lg/L was found to affect reproduction . Beggel et al.  found that ⇑ Corresponding author. Address: Institute of Applied Biology, 92 Wucheng Road, Shanxi University, Taiyuan 030006, Shanxi, PR China. Fax: +86 351 701 8871. E-mail address: [email protected]
(E. Ma). http://dx.doi.org/10.1016/j.pestbp.2014.07.010 0048-3575/Ó 2014 Published by Elsevier Inc.
ﬁpronil could disrupt the normal regulation of the HPG axis and thus impair ﬁsh reproduction. Fipronil has been detected at P9 lg/L in surface waters downstream of treated ﬁelds in rice cultivation  and in surface water at concentrations up to 12.6 lg/L in residential areas . Due to the high risk on wildlife, China and the European Union banned its use in 2009 and 2013, respectively. The use of biomarkers in ﬁsh is considered as an effective strategy to obtain information about the state of the aquatic environment and the effect of pollutants on living resources. Biochemical biomarkers are increasingly used in the ecological risk assessments of aquatic ecosystems to identify the incidence of exposure to and effects caused by xenobiotics, such as pesticides. Their use has gained popularity because of their potential as rapid early warning systems for damaging effects at organismal and higher levels . Among the biomarkers available for use, detoxiﬁcation enzymes (esterases, especially carboxylesterases (CarEs), glutathione Stransferases (GSTs) and cytochrome P450s (P450s)) have been shown to be appropriate biomarkers in a large variety of polluted environments. Because ﬁsh are an important component of the food chain and easily obtained, various toxicity tests have been performed within this group to assess the risk of exposure to toxic chemicals. The physiological changes associated with ﬁsh exposed to low pesticide levels not only provide a means to understand the levels of
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environmental pollution in biological terms, but can also be used as a model for vertebrate toxicity, including humans. As an experimental ﬁsh model, zebraﬁsh (Danio rerio) has been proven to be amenable for toxicological analysis due to their rapid development, short generation period, easy maintenance and transparent embryos. Therefore, the OECD have also proposed zebraﬁsh as a test organism . To assess the environmental effects of ﬁpronil, this work focused on the acute toxicity and sublethal impacts of ﬁpronil on detoxiﬁcation enzymes of brain, gill, liver, and muscle tissues of zebraﬁsh. The aim of this study was to select an appropriate biomarker that may adequately reﬂect ﬁpronil contamination in aquatic systems. 2. Materials and methods 2.1. Chemicals Bicinchoninic acid solution (BCA), fast blue B salt (O-dianisidine, tetrazotized), a-naphthol, a-naphthyl acetate (a-NA), ﬁpronil (5-amino-(2,6-dichloro-a,a,a-triﬂuoro-ptolyl-)-4-triﬂuoromethylsulﬁnylpyrazole-3-carbonitrile phenylpyrazole, purity 97.6%), 7-ethoxycoumarin (7-EC), glutathione reductase, and b-nicotinamide adenine dinucleotide phosphate (reduced b-NADPH) were purchased from Sigma Chem. Co. (St. Louis, MO). Bovine serum albumin (BSA) was purchased from Bio-Rad Laboratories (Hercules, CA). Reduced glutathione (GSH) and oxidized glutathione (GSSG) were purchased from Bio. Basic Inc. (Calgary, Canada). Triton X-100 and 1-chloro-2, 4-dinitrobenzene (CDNB) were purchased from Sangon (Shanghai, China). 2.2. Experimental animals and acclimation Actively moving juveniles of zebraﬁsh (total length, 2.0 ± 1.0 cm, weight, 0.19 ± 0.09 g) were purchased from a commercial ﬁsh supplier (Shanxi Academy of Fishery Sciences, Taiyuan, China). The ﬁsh were acclimatized for 2 weeks to laboratory conditions in a 150-L glass aquarium before the experiments following the guideline of the OECD . During the acclimatization period, the ﬁsh were fed sludge-worms (Limnodrilus hoffmeisteri) twice a day at ﬁxed times. The feces and uneaten food were siphoned out once a day, and 50% of reconstituted water was exchanged daily. The water in the glass aquarium was aerated and heated. The water temperatures ranged from 23 to 25 °C, with a pH of 7.1 ± 0.5 and dissolved oxygen content of 7.3 ± 0.5 mg/L during the period of acclimation. The natural photoperiod of 13:11 L:D was maintained. To reduce the amount of excreted products in the test tanks, feeding was stopped 24 h prior to the commencement of acute bioassay tests. All animals were healthy; a mortality rate of less than 2% was observed in the stocks used during the acclimation period. All experiments were performed according to the Experimental Animal Management Law of China and approved by the Animal Ethics Committee of Shanxi University. 2.3. Acute bioassay tests To obtain the baseline ﬁpronil toxicity to select sublethal concentrations for enzyme responses in 24 h, static acute toxicity bioassays were performed for 24 h using juvenile zebraﬁsh exposed to six concentrations (100, 150, 200, 300, 400, and 500 lg/L) of ﬁpronil. The concentrations were designed based on the mortalities, which were within the range of 5–95%. Stock solutions of ﬁpronil were prepared by dissolving it in acetone. Test solutions of the chosen concentrations were delivered by adding 100 lL of stock solution to 1-L reconstituted water aerated for 24 h in a glass beaker to achieve the nominal concentrations stated
above. The same procedure was used to treat ﬁsh with corresponding concentrations of acetone in water as controls. Water quality for all treatments ranged as follows: Ca2+ (2.2 ± 0.2 mmol/ L), mg2+ (0.6 ± 0.15 mmol/L), Na+ (0.78 ± 0.03 mmol/L), K+ (0.079 ± 0.002 mmol/L), pH (7.1 ± 0.5), dissolved oxygen (7.3 ± 0.5 mg/L), and conductivity (8.5 ± 1.4 lScm 1). Twelve ﬁsh (6 females and 6 males) were used at each test concentration and in the control per replicate and were placed in a glass tank (30 cm 10 cm 10 cm). The acute toxicity experiments were performed in triplicate. The ﬁsh were not fed during the acute toxicity test, the solutions were not aerated and heated, and glass tanks were maintained at a constant room temperature of 25 ± 1 °C. Fish were considered dead if movement was not visible. The average mortality values were calculated using the formula described by Abbott . The median lethal concentration (24-h LC50) and 95% conﬁdence intervals (CIs) were determined with a probit analysis  using a computer-based program. 2.4. Sub-acute short-term test Sub-lethal test concentrations of 1/10, 1/50, 1/100 LC50-24 h (20, 4, 2 lg/L) were used to assess the biochemical effects of ﬁpronil on zebraﬁsh. Forty-eight ﬁsh (24 females and 24 males) were used at each test concentration and in the control and were placed in a glass tank (30 cm 10 cm 10 cm). The test was carried out in three replicates for each concentration, and each replicate consisted of 16 ﬁsh, including equal numbers of female and males. The treatment procedure was carried out as mentioned in Section 2.3. 2.5. Tissue samples After 24 h of exposure to ﬁpronil, the ﬁsh were washed twice using distilled water, dried with ﬁlter paper and decapitated after being euthanized on ice. Tissues were quickly removed in the following order: gill, liver, brain and muscle. Tissue samples from 8 individuals for each replicate were placed in a pre-chilled Eppendorf tube for the CarE and GST activity assays. The samples from the remaining 8 individuals were placed in another new tube for the cytochrome P450-dependent O-deethylation (ECOD) activity assay. The samples were immediately frozen and stored in liquid nitrogen. For the CarE and GST activity assay, frozen tissue samples were weighed and homogenized in ice-cold 0.1 M phosphate buffer (pH 7.5) containing 0.3% (v/v) Triton X-100 at a ratio of 1:10 (w/v). All homogenates were centrifuged at 15,000g for 20 min at 4 °C. The supernatants were transferred to fresh tubes to serve as enzyme sources. The frozen samples for measuring the ECOD were weighed and homogenized in 150 mM phosphate buffer (pH 7.4) containing 50 mM sucrose (1:5 w/v). The homogenate was centrifuged at 10,000g for 20 min at 4 °C. The resulting supernatants were transferred to new tubes to serve as the enzyme sources. 2.6. Biochemical assays 2.6.1. Determination of protein contents The protein contents of the enzyme preparations were determined according to Smith et al.  using BSA as a standard. Measurements were performed with a SpectraMAX190 microplate reader and the SOFTmax software (Molecular Devices, Sunnyvale, CA) at 560 nm. 2.6.2. CarE activity assay The CarE activities in different tissues were determined based on the method of Zhu and He  with some modiﬁcation. Brieﬂy, 15 lL of appropriately diluted enzyme preparation was incubated in a ﬁnal reaction volume of 150 lL in 0.1 M phosphate buffer
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(pH 7.5) containing 0.27 mM a-NA and 10 4 M eserine at 37 °C for 30 min. The reactions were stopped by adding 50 lL of fast blue BSDS solution. After 15 min, the absorbance was determined at 600 nm using a microplate reader.
ﬁpronil for zebraﬁsh was determined to be 220.4 lg/L (95% CI: 173.7–272.4 lg/L).
2.6.3. GST activity assay The GST activities in different tissues were determined based on the method of Zhu et al.  with some modiﬁcation using CDNB as substrate. Brieﬂy, 10 lL of appropriately diluted enzyme preparation was mixed with 188 lL of 10.35 mM reduced glutathione in 0.1 M phosphate buffer (pH 7.5) and 2 lL of 200 mM CDNB in acetone in a microplate well. The change in absorbance was immediately recorded at 340 nm over 10 s intervals for 1 min using the microplate reader. All assays were corrected for non-enzymatic conjugation using the same mixtures, substituting 10 lL of 0.1 M phosphate buffer (pH 7.5) containing 0.3% (v/v) Triton X-100 for the enzyme preparation. The amount of glutathione conjugate formed was calculated using an extinction coefﬁcient of 9.6 mM 1 cm 1 for CDNB.
Fig. 1 shows the effects of ﬁpronil on the CarE activities in various tissues. Fipronil did not cause signiﬁcant changes in the CarE activities in the brain, gill, liver, and muscle tissues exposed to different sublethal concentrations (P = 0.15, 0.68, 0.93, 0.12, respectively).
2.6.4. Cytochrome P450 monooxygenase activity assay The ECOD activity was determined using 7-EC as a substrate and the method of Ulrich and Weber  as modiﬁed by de Sousa et al.  and later Stumpf and Nauen . The 80-lL reaction mixture containing 50 mM 7-EC, 62.5 mM reduced b-NADPH, and 40 lL of enzyme preparation was added to each microplate well. The microplate was incubated for 30 min at 30 °C while shaking at 240 rpm using an HZQ-F100 Oscillated Culture Box (Harbin Donglian Electronic & Technology Development Co. Ltd, Harbin, China). The O-deethylation of 7-EC produces ﬂuorescent umbelliferone (7-hydroxycoumarin). b-NADPH was removed by oxidizing it to non-ﬂuorescent NADP+ using the method of Chauret et al.  because the absorption of light and emission of ﬂuorescence by b-NADPH is similar to that of umbelliferone. Following incubation, 10 lL of 100 mM oxidized glutathione and 1.0 U glutathione reductase were added to each microplate well containing the reaction mixture and incubated for 15 min at 37 °C. The reaction was stopped with 120 lL of 50% (v/v) acetonitrile in 50 mM TRIZMA-base buffer (pH 10). The ﬂuorescence of umbelliferone was monitored with a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA) at an excitation wavelength of 390 nm and emission wavelength of 465 nm. The enzyme activity of 7-EC-O-deethylation was determined based on an umbelliferone standard curve. 2.7. Data analysis All assays were performed in triplicate. The mean and standard deviation values were determined for all biochemical parameters, and the results are expressed as the mean ± SD (n = 3) from triplicate assays. The data were analyzed employing normality tests of one-way variance (ANOVA). Fisher’s least signiﬁcant difference (LSD) multiple comparisons were then used to separate the means among the treatments for the same tissue. Two-way ANOVA was used to distinguish the interaction effects of concentrations and tissues. All statistical analyses were performed using the Statistical Product and Service Solutions (SPSS Inc.) software. P-values below 0.05 were considered signiﬁcant. 3. Results 3.1. LC50-24 h of ﬁpronil on zebraﬁsh Based on these results (Table 1), the regression equation of ﬁpronil to zebraﬁsh was y = 3.97x 4.33 (R2 = 0.97), where y is the probit value and x is the log concentration. The 24-h LC50 of
3.2. Effects of sublethal concentrations of ﬁpronil on CarE activity
3.3. Effects of sublethal concentrations of ﬁpronil on GST activity Fig. 2 depicts the changes in the GST activities in various tissues exposed to different sublethal concentrations of ﬁpronil. In the studied tissues, the GST activities did not signiﬁcantly differ between the tested concentrations and the control (P = 0.12, 0.07, respectively). 3.4. Effects of sublethal concentrations of ﬁpronil on ECOD activity The effects of ﬁpronil treatment on the ECOD activities in the various tissues of zebraﬁsh are shown in Fig. 3. After ﬁpronil treatment, the ECOD activities in all tissues signiﬁcantly increased compared with the control (P = 0.001 in the brain tissue, P < 0.001 in the gill, liver and muscle tissues). The respective increases in the ECOD activity in response to 2, 4, and 20 lg/L ﬁpronil were 40.6%, 77.8%, and 125.6% (brain); 25.6%, 42.1%, and 101.6% (gill); 26.2%, 72.1%, and 125.2% (liver); and 22.9%, 28.1%, and 53.1% (muscle) compared with the control. The two-way ANOVA results for the concentration and tissue effects are shown in Table 2. The concentration and tissue effects on ECOD activity were statistically signiﬁcant (P < 0.05). The interaction between the concentration and tissue was statistically signiﬁcant (P < 0.05). 4. Discussion In this study, we obtained the 24-h LC50 value (220.4 lg/L) of ﬁpronil for zebraﬁsh, which was higher than the 96-h LC50 value (1.81 lg/L) reported by Wang et al. . The differences might be related to the exposure time, ﬁsh size, and water chemistry. According to the LC50 value obtained from this study, ﬁpronil was more toxic than diazinon (24-h LC50: 3.59 104 lg/L) and was less toxic than deltamethrin (24-h LC50: 56 lg/L) for the same species . Comparing the LC50 value with those reported in the literature showed that D. rerio was less sensitive to ﬁpronil than Americamysis bahia (24-h EC50: 0.14 lg/L)  and more susceptible than the fathead minnow (24-h LC50: 398.29 lg/L) . The value obtained here was close to that of Daphnia magna (24-h LC50: 240 lg/L) . These results conﬁrm that the toxicity of ﬁpronil varies depending on the animal taxon. Sancho et al.  argued that the different activities of xenobiotic-metabolizing enzymes in the two ﬁsh (Poecilia reticulata and D. rerio) could be used to explain the toxicity difference. Küster and Altenburger  noted that the increased sensitivity of zebraﬁsh compared to other ﬁsh species could be due to an inherently low abundance of enzymes of the CYP P450 isoenzymatic family. Whether the differences of ﬁpronil toxicity among the aquatic organisms is caused by xenobiotic-metabolizing enzymes needs to be conﬁrmed in the further study. In addition, the toxicity difference may be due to the size of the species tested. Carboxylesterases are a class of enzymes that hydrolyze estercontaining compounds to the corresponding alcohol and acid (hydrolysis products). These enzymes are important in the metabolism and subsequent detoxiﬁcation of many xenobiotic
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Table 1 Acute lethal test of ﬁpronil to zebraﬁsh after 24 h exposure. Log concentration
No. of tested ﬁshes
Probit value of mortality
LC50 (95% CI) lg/L
100 150 200 300 400 500
2 2.18 2.30 2.48 2.60 2.70
12 12 12 12 12 12
5.56 27.78 52.78 66.67 80.56 91.67
3.41 4.41 5.07 5.43 5.86 6.39
Data are the average of the triplicate experiments.
CarE activity (µmol/min/mg protein)
.5 control 2µg/L 4µg/L 20µg/L
ECOD activity (pmol/min/mg protein)
Concentration of ﬁpronil (lg/L)
700 control 2µg/L 4µg/L 20µg/L
Table 2 Two-way ANOVA analysis of the ECOD activities in D. rerio exposed to ﬁpronil. Source
15 1 3 3 9
294.47 12114.5 268.496 1076.24 42.53
0.000 0.000 0.000 0.000 0.000
Fig. 3. Effects of ﬁpronil on the ECOD activities in the various tissues of zebraﬁsh. Data represent the means ± SD of triplicate assays. Means within bars followed by the same letter are not signiﬁcantly different (P > 0.05, ANOVA).
Corrected model Intercept Concentration Tissue Concentration tissue
Fig. 1. Effects of ﬁpronil on the CarE activities in the various tissues of zebraﬁsh. Data represent the means ± SD of triplicate assays. Means within bars are not signiﬁcantly different among the treatments and the control (P > 0.05, ANOVA).
GST activity (nmol/min/mg protein)
control 2µg/L 4µg/L 20µg/L
Tissue Fig. 2. Effects of ﬁpronil on the GST activities in the various tissues of zebraﬁsh. Data represent the means ± SD of triplicate assays. Means within bars followed by the same letter are not signiﬁcantly different (P > 0.05, ANOVA).
and endogenous compounds . The CarEs activity has been documented to be higher and exhibited a greater sensitivity to inhibition by pesticides in some aquatic invertebrates . Moreover, CarEs determination proved to be a good indicator of exposure to pesticides, as recently reviewed [36,37]. However, ﬁpronil did not cause signiﬁcant changes in the CarE activities in our results. This ﬁnding suggested that CarEs is not an appropriate biomarker to detect ﬁpronil exposure in D. rerio.
GST represents an important family of enzymes because of their role as catalysts for the conjugation of various electrophilic compounds with tripeptide glutathione . They are enzymes involved in the detoxiﬁcation of various xenobiotic chemicals . GSTs have been used as a biomarker in studies of both vertebrates and invertebrates [40,41]. However, the GST activities did not signiﬁcantly differ between the tested concentrations and the control in the tissues examined in this study. The lack of dose responses in GST suggested that GST is not a sensitive biomarker of ﬁpronil exposure or effect in D. rerio. Cytochrome P450 enzymes are a widely distributed protein superfamily and are responsible for the Phase I biotransformation of endogenous compounds and xenobiotics . Certain environmental pollutants are potent inducers of cytochrome P450 . The induction of cytochrome P450-dependent O-deethylation is extensively used as an indicator of exposure and response to organic pollutants in ﬁsh and other vertebrates. In this study, various concentrations of ﬁpronil dose-dependently induced ECOD activity. These results suggested that ECOD could be used as a
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biomarker of ﬁpronil effects in D. rerio. Anderson and Zhu  reported that atrazine could induce ECOD activity in Chironomus tentans. The exposure of marine snails to ﬂuoranthene for 5 days resulted in an increase in ECOD activity measured in isolated live digestive cells . TCB together with stress has been shown to signiﬁcantly elevate the liver ECOD activity in rainbow trout . However, Raffali et al.  reported that imidazole had no apparent effect on the ECOD activity. The response of ECOD, such as induction, inhibition and inactivation, may depend on the chemical stressor. The induction in ECOD activities suggested the participation of ECOD enzymes in ﬁpronil transformation in the tested tissues. The high values of ECOD activities might result in greater amounts of ﬁpronil toxic metabolites. The sulfone metabolite (P450-dependent metabolite of ﬁpronil) and ﬁpronil desulﬁnyl, a product of photodegradation, were reported to be more toxic to insects, mammals, ﬁsh and birds than the parent compound itself . The ﬁpronil-sulfone and ﬁpronil-desulﬁnyl metabolites are 6.6 and 1.9 times more toxic to freshwater invertebrates, respectively, than ﬁpronil . The combined toxicity of the parent compound and its toxic metabolites increase the risk of ﬁpronil to exposed organisms. 5. Conclusions The 24-h LC50 of ﬁpronil for zebraﬁsh was determined to be 220.4 lg/L (95% CI: 173.7–272.4 lg/L). The effects of sublethal concentrations of ﬁpronil on detoxiﬁcation enzymes were investigated in the brain (central nervous system), gills (respiratory organ), liver (detoxiﬁcation organ), and muscle (locomotor tissue). Sublethal concentrations of ﬁpronil did not cause signiﬁcant changes in the CarE and GST activities compared with the control. Fipronil dosedependently induced ECOD activities. Thus, ECOD could be selected as a suitable biomarker to reﬂect ﬁpronil effects in D. rerio. Further work is needed to study P450 molecular changes under ﬁpronil stress and to address the ECOD induction mechanisms by ﬁpronil. Acknowledgments This work was supported by the National Natural Science Foundation of China – China (31172161 and 31201548), Natural Science Foundation of Shanxi Province – China (2013011028-3), and Research Fund for the Doctoral Program of Higher Education of China – China (2013M530180). We thank Shifeng Wang, Hongde Zhang, and Wei Xing for their assistance. References  A. Bobé, J.F. Cooper, C.M. Coste, M.A. Muller, Behaviour of ﬁpronil in soil under Sahelian plain ﬁeld conditions, Pestic. Sci. 52 (1998) 275–281.  J.R. Bloomquist, Chloride channels as tools for developing selective insecticides, Arch. Insect Biochem. Physiol. 54 (2003) 145–156.  C.C. Tingle, J.A. Rother, C.F. Dewhurst, S. Lauer, W.J. King, Fipronil: environmental fate, ecotoxicology, and human health concerns, Rev. Environ. Contam. Toxicol. 176 (2003) 1–66.  US Environmental Protection Agency, New Pesticide Fact Sheet, PB-96-181516. US EPA Ofﬁce of Prevention, Pesticides, and Toxic Substances. EPA737-F-96005, 1996.  J. Leqhait, V. Gayrard, N. Picard-Hagen, M. Camp, E. Perdu, P.L. Toutain, C. Viquié, Fipronil-induced disruption of thyroid function in rats is mediated by increased total and free thyroxine clearances concomitantly to increased activity of hepatic enzymes, Toxicology 255 (2009) 38–44.  A.K.E. Hassani, M. Dacher, M. Gauthier, C. Armengaud, Effects of sublethal doses of ﬁpronil on the behavior of the honeybee (Apis mellifera), Pharmacol. Biochem. Behav. 82 (2005) 30–39.  G.T. Chandler, T.L. Cary, D.C. Volz, S.S. Walse, J.L. Ferry, S.L. Klosterhaus, Fipronil effects on copepod development, fertility, and reproduction: a rapid life-cycle assay in 96-well microplate format, Environ. Toxicol. Chem. 23 (2004) 117–124.  J.D. Stark, R.I. Vargas, Toxicity and hazard assessment of ﬁpronil to Daphnia pulex, Ecotoxicol. Environ. Saf. 62 (2005) 11–16.
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