Science of the Total Environment 485–486 (2014) 415–420
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Enantioselective toxicity, bioaccumulation and degradation of the chiral insecticide fipronil in earthworms (Eisenia feotida) Han Qu, Peng Wang, Rui-xue Ma, Xing-xu Qiu, Peng Xu, Zhi-qiang Zhou, Dong-hui Liu ⁎ Department of Applied Chemistry, China Agricultural University, Beijing 100193, PR China
H I G H L I G H T S • • • •
R-fipronil was more toxic than S-fipronil to earthworms (Eisenia feotida). The bioaccumulation of fipronil in the earthworms was not significantly different from that of the enantiomers. The degradation of fipronil in earthworm was enantioselective: S-fipronil was biodegraded preferentially over R-fipronil. The degradation of fipronil in two types of soil (each with earthworms and without earthworms) was not enantioselective.
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Article history: Received 9 October 2013 Received in revised form 13 March 2014 Accepted 14 March 2014 Available online 16 April 2014 Editor: Kevin V. Thomas Keywords: Fipronil Enantioselectivity Toxicity Bioaccumulation Degradation
a b s t r a c t The enantioselective acute toxicity to earthworms of racemic fipronil and its individual enantiomers was studied. R-(−)-fipronil was approximately 1.5 times more toxic than the racemate and approximately 2 times more toxic than S-(+)-fipronil after 72 and 96 h of exposure, respectively. Assays of fipronil enantiomer bioaccumulation and degradation in earthworms were conducted. The bio-concentration factors (BCFs) were slightly different between the two enantiomers. The enantiomeric fraction (EF) values in earthworms in the bioaccumulation period were approximately 0.5, which indicated there was no enantioselective bioaccumulation. In contrast, the degradation of fipronil in earthworms was enantioselective: the t1/2 values for R- and S-fipronil were 3.3 and 2.5 days, respectively, in natural soil, and 2.1 and 1.4 days, respectively, in artificial soil. The results of soil analyses showed that the degradation of fipronil was not enantioselective, which suggested that the enantioselectivity of fipronil in earthworms results from the organism's metabolism. The study also demonstrated that the presence of earthworms could accelerate the degradation of fipronil in soil. © 2014 Elsevier B.V. All rights reserved.
1. Introduction It is estimated that approximately 30% of registered pesticides are chiral (Ulrich et al., 2009). As is ' known, the enantiomers of a chiral pesticide have identical physical and chemical properties but different biological and physiological properties in asymmetrical systems (Liu et al., 2005). In most cases, the great majority of chiral pesticides are manufactured and applied as their racemic mixtures (Garrison, 2011). The enantiomers of chiral pesticides usually differ with regard to their biological activity and toxicity and their processes of biodegradation and bioaccumulation, which may lead to differences in their environmental effects and fates. Therefore, it is of great importance to investigate the stereochemistry of chiral pesticides to enable accurate environmental risk assessments. ⁎ Corresponding author at: Department of Applied Chemistry, China Agricultural University, West Yuanmingyuan Road No 2, Beijing 100193, PR China. Tel.: + 86 10 62732937. E-mail address: [email protected] (D. Liu).
http://dx.doi.org/10.1016/j.scitotenv.2014.03.054 0048-9697/© 2014 Elsevier B.V. All rights reserved.
Synthetic insecticides of the phenylpyrazole class have been applied to control pests on various crops for more than 30 years (Schlenk et al., 2001). Fipronil, (R,S)-(5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-(trifluoromethylsulfinyl)-1-H-pyrazole-3-carbonitrile) is a chiral phenylpyrazole-class insecticide developed by Rhone-Poulenc Agro in 1987 (Bobe et al., 1997). It was first approved in 1996 in the United States of America for rice culture, turf grass management, and residential pest control (Konwick et al., 2005). The primary insecticidal action of fipronil involves blocking γ-aminobutyric acid (GABA) and chloride ion regulation channels, resulting in a loss of neuron signaling control and a disruption of normal central nervous system function (Hosie et al., 1995; Cole et al., 1993). The bioactivity and toxicity of fipronil is highly selective for arthropods, with inhibitory effects on invertebrates that are 500 times higher than those on mammalian targets due to differences in GABA receptor binding (Hainzl and Casida, 1996; Hainzl et al., 1998; Tomlin, 1999). It has been reported that the bioactivity of a given chiral pesticide is usually a result of the preferential reactivity of only one stereoisomer, while the other stereoisomer might exert toxic effects on non-target
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organisms. The S-fipronil has a longer duration of control for ticks but a lower level of emesis for dogs than the R-enantiomer (Huber, 2002). The toxicity of the S-enantiomer to Ceriodaphnia dubia is approximately 3 times higher than that of the R-enantiomer, while the R-fipronil was shown to have significantly more androgen and progesterone activity than its antipode (Author, 2004). Previous studies have indicated that the two enantiomers of fipronil may perform differently with regard to their accumulation and degradation in the environment (Jones et al., 2007). A number of studies have determined the enantioselective environmental behaviors of fipronil, such as metabolism (Lu et al., 2010), sorption and degradation (Doran et al., 2009), and transformation (Konwick et al., 2005; Konwick et al., 2006; Jones et al., 2007). With the wide application of fipronil all over the world, the environmental load and ecological risks have been increasing. However, the potential enantioselective residues may lead to an inaccurate environmental risk evaluation under traditional consideration. One non-target organism, the earthworm (Eisenia feotida), plays an important role in environmental safety assessments, especially in the accumulation, degradation and remediation of pollutants in soil. It is reported that the bioaccumulation behavior of benalaxyl in earthworm tissue was enantioselective (Xu et al., 2009). The enantioselective bio-behaviors of alpha-cypermethrin and metalaxyl enantiomers in earthworms have also been studied (Diao et al., 2011; Xu et al., 2011). Owing to the limited information concerning the toxicity and bio-behavior of fipronil in earthworms, further research on enantiomeric effects in the earthworm needs to be conducted to enable environmental risk assessment. In this study, the acute toxicity of R-(−)-, S-(+)- and rac-fipronil were determined using the filter paper contact test. In addition, the enantioselective bioaccumulation and degradation of fipronil in earthworms was also studied. The aims of this work were to determine the toxicity of racemate and single-enantiomer fipronil to earthworms and to study the potential enantioselective environmental behaviors of fipronil in earthworms and, thus, evaluate the remediation effects of earthworms for fipronil pollutants in soil. 2. Materials and methods 2.1. Chemicals The racemic insecticide fipronil (96.5% purity) was provided by the China Ministry of Agriculture Institute for Control of Agrochemicals. The two enantiomers of fipronil were prepared by high-performance liquid chromatography HPLC (Agilent Technologies) using a chiral column with a cellulose tri-(3,5-dimethylphenyl-carbamate) chiral stationary phase, and the resulting enantiomeric purities of R-(−)and S-(+)-fipronil were 99.5% and 99.4%, respectively. Analytical grade reagents were redistilled and filtered through a 0.45-μm filter membrane prior to use. Ultrapure water was obtained using a Milli-Q water purification system (Millipore, Billerica Massachusetts, USA). All other chemicals and solvents were analytical grade and were purchased from Beijing Chemical Reagent Co., China. 2.2. Earthworms Mature earthworms (E. feotida) were purchased from a suburban farm and then maintained in a wooden breeding box containing a mixture of soil and cattle manure for 1 week to acclimate to the laboratory. The worms were active and healthy for the experiments. 2.3. Soil In accordance with OECD guideline 317 (OECD, 2010), the tests were performed with both artificial soil and natural soil. The natural soil was collected from an orchard in Beijing, China, that had not received fipronil application for at least the prior 10 years. The soil was sieved,
air-dried at room temperature, and kept in the dark before use. The physicochemical properties of the soil were as follows: organic matter (OM), 3.96%; clay, 2.48%; sand, 36.25%; silt, 61.27%; and pH 6.07. The artificial soil was composed of 10% sphagnum peat (pH 5.5 to 6.0, no visible plant remains, finely ground, and dried to measure moisture content), 20% kaolin clay (kaolinite content preferably above 30%), and 70% industrial sand (more than 50% of the particles between 50 and 200 μm), with a pH value of 6.0 ± 0.5 adjusted by calcium carbonate. Water content was adjusted to 35%. 2.4. Acute toxicity test In accordance with OECD guideline 207 (OECD, 1984), a paper contact toxicity assay was used to determine the acute toxicity of rac-fipronil and the R-(−)- and S-(+)-enantiomers to earthworms. The test substances were prepared in acetone. A series of known concentrations, 7.87, 15.74, 23.61, 31.48, 39.35, 47.22, 55.09, 62.96, 70.83, 78.70, 86.57, and 94.44 mg cm− 2 for S-fipronil and 7.87, 15.74, 23.61, 31.48, 39.35, 47.22, 55.09, 62.96, 70.83, and 78.70 mg cm− 2 for rac-fipronil and R-fipronil were spiked on filter papers. Solvent was volatilized under a stream of compressed air, and 1 mL of deionized water was added to each vial. After evacuating the gut contents for 3 h, the earthworms were rinsed by deionized water and dried by blotting-paper before introducing to the vials. Controls were set with the solvent alone. Ten replicates for each treatment were performed. The earthworms were placed on the filter papers at 20 ± 2 °C. Mortality was observed after incubation for 72 and 96 h, and LC50 values were calculated using SPSS Version 18.0 (SPSS Inc., Chicago, USA) 2.5. Bioaccumulation and degradation experiment 2.5.1. Bioaccumulation Based on OECD 317, the bioaccumulations of both fipronil enantiomers in the natural soil and artificial soil were investigated. For the natural soil, 250 g was weighed in a beaker, and rac-fipronil (12.5 mg in 10 mL of acetone) was slowly added; then the beaker was put aside for 24 h to evaporate the acetone. Deionized water was added to the contaminated soil to maintain water content of 20–30%, and the soil was fully stirred. After the gut contents were evacuated, the earthworms (20 g) were transferred into the beaker and placed in an incubator at 20 ± 2 °C. Five grams of earthworms was sampled after exposure times of 0, 0.5, 1, 2, 3, 7, 12, 15, and 18 days and frozen at − 20 °C before analysis. Every treated beaker was used for one sample point, and each sampling was conducted in triplicate. The bioaccumulation experiment in the artificial soil was the same as in the natural soil test, except the sampling points were at 0, 0.5, 1, 2, 4, 7, 10, and 14 days. 2.5.2. Degradation To evaluate the degradation of the two enantiomers, the earthworms were exposed to fipronil for 15 days. (The fipronil concentration and the amount of earthworms and soil were the same as in the bioaccumulation period). Then, the earthworms were collected and transferred to 250 g of the uncontaminated soil (as 0 days for purification phase). For the natural soil, at each of the sampling points of 0, 0.5, 1, 2, 3, 5, and 7 days, 10 g of earthworms and 20 g of soil were sampled and stored at −20 °C. Similarly, these were sampled at 0, 1, 2, 3, 5, and 7 days in artificial soil. The degradation of fipronil in the natural soil (25 mg kg−1, without earthworm) was also studied. The experiment (natural soil, 25 mg kg−1 fipronil, with 20 g of earthworms) was designed to investigate the influence of earthworms on pollution remediation. The sample consisted of 20 g of soil, collected at each sampling point: 0, 1, 2, 3, 7, 12, 15, and 18 days.
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Fig. 1. The typical chromatograms of chiral separation of standard fipronil (a) and in the earthworms in the degradation period on the 3th day (b).
of 20 °C. The chiral recognition of the two enantiomers was confirmed by CD detector.
The beakers were weighed daily, and the loss of water was compensated by addition of deionized water. During the uptake and elimination periods, 2 g of dried and crushed cow dung was spread on the soil surface each week to feed the earthworms.
2.7. Statistical analysis The statistical significance (P b 0.05) of acute toxicity was analyzed using SPSS Version 18.0. Bioaccumulation and degradation were analyzed using GraphPad Prism 5 for Windows (GraphPad Software, San Diego, USA). The enantiomeric fraction (EF) was used to assess the enantioselectivity of fipronil in earthworms and soil, which was expressed as the equation:
2.6. Chemical analysis For the pretreatment, 20 g of soil was added into a 50-mL polypropylene centrifuge tube and 8 mL of deionized water was added. Then, the tubes were shaken for 3 min. After that, activated carbon (0.03 g), neutral alumina (0.2 g), and acetone (30 mL) were added, vortexmixed for 5 min, and centrifuged at 4000 rpm for 5 min. The extraction was repeated following the same steps. The organic phase was combined and evaporated to dryness using a vacuum rotary evaporator at 35 °C, and the residue was diluted to 1.0 mL with n-hexane/2-propanol (95:5, v/v). The earthworms were homogenized using a Waring (Torrington CT) stainless steel blender, and 5 g was weighed for extraction in a 100-mL polypropylene centrifuge tube. After adding 15 mL of acetonitrile, the tube was vortexed for 3 min and then centrifuged at 4000 rpm for 5 min. The extraction was repeated twice and the organic phase was combined and extracted three times using n-hexane to remove lipids. Then, the acetonitrile was evaporated to dryness and dissolved in 1 mL of methanol for purification. An SPE procedure was applied for clean up with a C18 cartridge. The sample was loaded and eluted with 5 mL of methanol/water (1/5, v/v). The eluent was evaporated to dryness under a stream of nitrogen and dissolved in 1.0 mL of 2-propanol for HPLC analysis. All analyses were performed using an Agilent 1200 series HPLC system (Agilent Technology, Beijing, China), equipped with Quat Pump G1311A, degasser G1322A, injection valve G1328A, DAD G1315, circular dichroism detector CD2095, and column heater and cooler AT-930. Fipronil enantiomers were separated on an (R, R) Whelk-O1 chiral column (4.6 × 250 mm I.D, Regis, Illinois, USA) with the following operation conditions: a flow rate of 1.0 mL min−1, mobile phase of n-hexane / 2-propanol (95:5, v/v), detection wavelength of 225 nm, injection volume of 20 μL, and column temperature
EF ¼ peak area of ð−Þ=½ð−Þ þ ðþÞ The EF values range from 0 to 1, with EF = 0.5 representing the racemic mixture. Bio-concentration factor (BCF) is a function of the relative sorptive capacities in the organism and the surrounding soil, and it is defined as BCF ¼ Ce =Cs where Ce is the concentration of fipronil in earthworm tissue (g kg−1 dry weight), and Cs is the concentration of fipronil in soil (g kg−1 dry weight). The BCF was used to express the bioaccumulation of fipronil in the earthworms. 3. Results and discussion 3.1. Chiral analysis and method validation The two enantiomers were completely separated on the (R,R) Whelk-O1 chiral column, as shown in Fig. 1. The absolute configuration of fipronil enantiomers was assigned by comparing the CD signals with those reported in the literature (Liu et al., 2008). The first eluted enantiomer was S-(+)-fipronil, and the second was R-(−)-fipronil (Liu et al., 2008). The average recovery of both enantiomers at levels between 0.5 and 50 mg kg−1 ranged from 87.67% to 101.08% in the earthworms and from
Table 1 72 h and 96 h LC50 values of rac-fipronil and the enantiomers. Chemicals
72-h-LC50(mg cm−2)
R2
Pa
96-h-LC50(mg cm−2)
R2
Pa
rac-fipronil R-fipronil S-fipronil
0.044 [0.041–0.052]b 0.029 [0.020–0.036]b 0.050 [0.041–0.058]b
0.924 0.885 0.932
0.006 0.012 0.001
0.019 [0.013–0.026]b 0.015 [0.011–0.022]b 0.036 [0.026–0.040]b
0.894 0.931 0.868
0.003 0.005 0.008
a b
The probability by t test. P value smaller than 0.05 indicates that the correlation of linear equation is significant. 95% confidence interval.
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90.0% to 96.0% in the soil, with SD ≤ 10% (n = 3) for both the earthworms and the soil. The limit of detection for both enantiomers was 0.01 mg L−1. 3.2. Acute toxicity test The mortality in the controls did not exceed 10% after exposure for 96 h. Table 1 lists the LC50 values of rac-fipronil and the R/Senantiomers. The acute toxicity rates observed among the racemate and single enantiomers were significantly different. The LC50 values of rac-fipronil were 0.044 and 0.019 mg cm−2 at 72 h and 96 h, respectively, while they were 0.029 and 0.015 mg cm−2 at 72 h and 96 h, respectively, for the R-(−)-enantiomer. The S-(+)-enantiomer showed the least acute toxicity to earthworms: the LC50 values were 0.050 and 0.036 mg cm−2 at 72 h and 96 h, respectively. It has been reported that S-(+)-fipronil exhibits higher toxicity to Procambarus clarkii and C. dubia (Konwick et al., 2005; Liu et al., 2008; Li et al., 2009). In this work, in contrast, the R-(−)-fipronil was observed to be more toxic to
Fig. 2. The accumulation of fipronil enantiomers in earthworm in the natural soil (a) and the artificial soil (b). Bars are standard deviations.
Fig. 3. Degradation curves of fipronil enantiomers in earthworm in natural soil (a) and artificial soil (b). Bars are standard deviations.
Fig. 4. Enantiomeric fractions (EFs) of fipronil enantiomers in earthworms during the accumulation period (a), the degradation period (b) and in the natural soil with and without earthworms (c). Bars are standard deviations.
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Fig. 5. The concentrations of fipronil enantiomers in the natural soil with and without earthworms. * and ** represent statistically significant differences at P b 0.05 in 95% confidence intervals. Bars are standard deviations.
earthworm than its antipode. Hence, the toxicity of fipronil to other organisms on the enantiomeric level needs to be evaluated, and its enantioselectivity should be taken into consideration for environmental safety assessment. 3.3. Bioaccumulation and degradation of fipronil in earthworms 3.3.1. Bioaccumulation The earthworms were active and the weight loss was less than 15% at the end of the bioaccumulation and degradation phases. The two enantiomers of fipronil in earthworms and soil were measured in natural and artificial soil conditions. The concentration of each enantiomer in earthworms showed similar trends in both natural and artificial soils: there was no obvious enantioselectivity observed, with EF values of approximately 0.5 (Fig. 4a). During the accumulation period, fipronil concentration increased to a maximum level at the 15th day in earthworms in the natural soil and at the 10th day in the artificial soil, after which it remained at a relatively steady level (Fig. 2). Fig. 2 shows the concentration curves of bioaccumulation in earthworms in natural and artificial soil. For the accumulation experiment, the concentrations of the enantiomers in the corresponding soils were also measured, and BCFs were calculated. The BCFs of R- and S-fipronil in the artificial soil were 1.27 and 1.24, respectively, and were 1.33 and 1.32, respectively, in the natural soil. There was no significant difference between the two enantiomers, indicating that the uptake of fipronil in the earthworms was not enantioselective. The BCF values also inferred that the bio-enrichment ability of the earthworms for accumulating fipronil in soil was limited. 3.3.2. Degradation The degradation of fipronil in the earthworms in both soils followed first-order kinetics. The chromatogram of fipronil enantiomers in the earthworms during the degradation period is shown in Fig. 1b, and the degradation curve of R-(−)- and S-(+)-fipronil in the earthworms in the natural soil is shown in Fig. 3a. At the end of the bioaccumulation period, the fipronil concentrations in earthworms in the natural soil were higher than those in the artificial soil, resulting in the different concentrations at the beginning of degradation. The half-lives of R-(−)- and S-(+)-fipronil were 3.3 and 2.5 days, respectively. After 7 days, approximately 76% of the R-(−)-fipronil and 85%
of S-(+)-fipronil were degraded, with an EF value of 0.64 (Fig. 4b). The results demonstrated a preferential degradation of the Senantiomer by the earthworms. The results were similar for the artificial soil, as shown in Fig. 3b. Approximately 74% of R-(−)-fipronil and 85% of S-(+)-fipronil were degraded by the earthworms in the artificial soil after 7 days, with an EF value of 0.69 (Fig. 4b). The rates of degradation of the two enantiomers by earthworms were found to be slightly different between the artificial soil and the natural soil. The half-life of R-(−)-fipronil in the artificial soil was 2.1 days, 1.5 times shorter than that in the natural soil, and the half-life of the S-(+)- form was 1.4 days, approximately 1.7 times faster than that in the natural soil. The metabolic rate of the earthworms in the natural soil experiment was slower than that of the worms in the artificial soil. The reasons might lie in the strong influence of the organic matter on the elimination of a given chemical. The high organic content in the natural soil most likely had a high adsorptive capacity and thus led to a decrease in fipronil elimination by the earthworms; this condition existed not only in the surroundings of the organism's body but also in the gut (Amorim et al., 2002). For investigating the influences of earthworms on pollution remediation, analysis of the degradation of fipronil in the natural soil without earthworms was also conducted as a control experiment. The concentration changes in the soils are shown in Fig. 5. Fipronil was found to dissipate more quickly in the soil in which earthworms were present, indicating that earthworms helped to accelerate the degradation rate and thus promoted fipronil pollution remediation. No significant enantioselectivity was found in the soil with or without earthworms (Fig. 4c). Therefore, the enantioselectivity for fipronil in earthworms results from the organisms' metabolism. 4. Conclusions In this study, the enantioselective acute toxicity of fipronil to earthworms and the bioaccumulation and degradation of fipronil in both earthworms and soils were examined under laboratory conditions. The filter paper contact acute toxicity test showed that R-(−)-fipronil was approximately twice as toxic to earthworms as was S-(+)-fipronil. Fortunately, the toxicity of the racemate and both enantiomers to earthworms was low. The bioaccumulation of fipronil in the earthworms was not significantly enantioselective, but the less toxic S-(+)-fipronil was
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degraded preferentially by the earthworms. The results also suggested that the earthworms could promote fipronil degradation in soil and play a part in pollution remediation. These experimental data provide additional background information on the potential toxicity of fipronil and on the metabolic processes and enantioselective behavior of earthworms. Acknowledgments This work was supported by the National Natural Science Foundation of China (contract grant numbers 21307155 and 21277171), the Foundation for the Author of National Excellent Doctoral Dissertation of PR China, the New-Star of Science and Technology program, and the Beijing Metropolis Beijing Nova program. References Amorim MJ, Sousa JP, Nogueira AJA, Soares A. Bioavailability and toxicokinetics of 14Clindane (gamma-HCH) in the enchytracid Enchytracus albidus in two soil types: the aging effect. Arch Environ Contam Toxicol 2002;43:221–8. Author A. On the issue of enantioselectivity of chiral pesticides: a green chemistry opportunity. Green Chem 2004;6:77–8. Bobe A, Coste CM, Cooper JF. Factors influencing the adsorption of fipronil on soils. J Agric Food Chem 1997;45:4861–5. Cole LM, Nicholson RA, Casida JE. Action of phenylpyrazole insecticides at the GABA-gated chloride channel. Pestic Biochem Physiol 1993;46:47–54. Diao JL, Xu P, Liu DH, Lu YL, Zhou ZQ. Enantiomer-specific toxicity and bioaccumulation of alpha-cypermethrin to earthworm Eisenia fetida. J Hazard Mater 2011;192:1072–8. Doran G, Eberbach P, Helliwell S. Sorption and degradation of fipronil flooded anaerobic rice soils. J Agric Food Chem 2009;57:10296–301. Garrison AW. An introduction to pesticide chirality and the consequences of stereoselectivity. Chiral Pesticides: Stereoselectivity and Its Consequences. Washington DC: American Chemical Society; 2011. Hainzl D, Casida JE. Fipronil insecticide: novel photochemical desulfinylation with retention of neurotoxicity. Proc Natl Acad Sci 1996;93:12764–7. Hainzl D, Cole LM, Casida JE. Mechanisms for selective toxicity of fipronil insecticide and its sulfone metabolite and desulfinyl photoproduct. Chem Res Toxicol 1998;11: 1529–35.
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Enantioselective toxicity, bioaccumulation and degradation of the chiral insecticide fipronil in earthworms (Eisenia feotida) Han Qu, Peng Wang, Rui-xue Ma, Xing-xu Qiu, Peng Xu, Zhi-qiang Zhou, Dong-hui Liu ⁎ Department of Applied Chemistry, China Agricultural University, Beijing 100193, PR China
H I G H L I G H T S • • • •
R-fipronil was more toxic than S-fipronil to earthworms (Eisenia feotida). The bioaccumulation of fipronil in the earthworms was not significantly different from that of the enantiomers. The degradation of fipronil in earthworm was enantioselective: S-fipronil was biodegraded preferentially over R-fipronil. The degradation of fipronil in two types of soil (each with earthworms and without earthworms) was not enantioselective.
a r t i c l e
i n f o
Article history: Received 9 October 2013 Received in revised form 13 March 2014 Accepted 14 March 2014 Available online 16 April 2014 Editor: Kevin V. Thomas Keywords: Fipronil Enantioselectivity Toxicity Bioaccumulation Degradation
a b s t r a c t The enantioselective acute toxicity to earthworms of racemic fipronil and its individual enantiomers was studied. R-(−)-fipronil was approximately 1.5 times more toxic than the racemate and approximately 2 times more toxic than S-(+)-fipronil after 72 and 96 h of exposure, respectively. Assays of fipronil enantiomer bioaccumulation and degradation in earthworms were conducted. The bio-concentration factors (BCFs) were slightly different between the two enantiomers. The enantiomeric fraction (EF) values in earthworms in the bioaccumulation period were approximately 0.5, which indicated there was no enantioselective bioaccumulation. In contrast, the degradation of fipronil in earthworms was enantioselective: the t1/2 values for R- and S-fipronil were 3.3 and 2.5 days, respectively, in natural soil, and 2.1 and 1.4 days, respectively, in artificial soil. The results of soil analyses showed that the degradation of fipronil was not enantioselective, which suggested that the enantioselectivity of fipronil in earthworms results from the organism's metabolism. The study also demonstrated that the presence of earthworms could accelerate the degradation of fipronil in soil. © 2014 Elsevier B.V. All rights reserved.
1. Introduction It is estimated that approximately 30% of registered pesticides are chiral (Ulrich et al., 2009). As is ' known, the enantiomers of a chiral pesticide have identical physical and chemical properties but different biological and physiological properties in asymmetrical systems (Liu et al., 2005). In most cases, the great majority of chiral pesticides are manufactured and applied as their racemic mixtures (Garrison, 2011). The enantiomers of chiral pesticides usually differ with regard to their biological activity and toxicity and their processes of biodegradation and bioaccumulation, which may lead to differences in their environmental effects and fates. Therefore, it is of great importance to investigate the stereochemistry of chiral pesticides to enable accurate environmental risk assessments. ⁎ Corresponding author at: Department of Applied Chemistry, China Agricultural University, West Yuanmingyuan Road No 2, Beijing 100193, PR China. Tel.: + 86 10 62732937. E-mail address: [email protected] (D. Liu).
http://dx.doi.org/10.1016/j.scitotenv.2014.03.054 0048-9697/© 2014 Elsevier B.V. All rights reserved.
Synthetic insecticides of the phenylpyrazole class have been applied to control pests on various crops for more than 30 years (Schlenk et al., 2001). Fipronil, (R,S)-(5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-(trifluoromethylsulfinyl)-1-H-pyrazole-3-carbonitrile) is a chiral phenylpyrazole-class insecticide developed by Rhone-Poulenc Agro in 1987 (Bobe et al., 1997). It was first approved in 1996 in the United States of America for rice culture, turf grass management, and residential pest control (Konwick et al., 2005). The primary insecticidal action of fipronil involves blocking γ-aminobutyric acid (GABA) and chloride ion regulation channels, resulting in a loss of neuron signaling control and a disruption of normal central nervous system function (Hosie et al., 1995; Cole et al., 1993). The bioactivity and toxicity of fipronil is highly selective for arthropods, with inhibitory effects on invertebrates that are 500 times higher than those on mammalian targets due to differences in GABA receptor binding (Hainzl and Casida, 1996; Hainzl et al., 1998; Tomlin, 1999). It has been reported that the bioactivity of a given chiral pesticide is usually a result of the preferential reactivity of only one stereoisomer, while the other stereoisomer might exert toxic effects on non-target
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organisms. The S-fipronil has a longer duration of control for ticks but a lower level of emesis for dogs than the R-enantiomer (Huber, 2002). The toxicity of the S-enantiomer to Ceriodaphnia dubia is approximately 3 times higher than that of the R-enantiomer, while the R-fipronil was shown to have significantly more androgen and progesterone activity than its antipode (Author, 2004). Previous studies have indicated that the two enantiomers of fipronil may perform differently with regard to their accumulation and degradation in the environment (Jones et al., 2007). A number of studies have determined the enantioselective environmental behaviors of fipronil, such as metabolism (Lu et al., 2010), sorption and degradation (Doran et al., 2009), and transformation (Konwick et al., 2005; Konwick et al., 2006; Jones et al., 2007). With the wide application of fipronil all over the world, the environmental load and ecological risks have been increasing. However, the potential enantioselective residues may lead to an inaccurate environmental risk evaluation under traditional consideration. One non-target organism, the earthworm (Eisenia feotida), plays an important role in environmental safety assessments, especially in the accumulation, degradation and remediation of pollutants in soil. It is reported that the bioaccumulation behavior of benalaxyl in earthworm tissue was enantioselective (Xu et al., 2009). The enantioselective bio-behaviors of alpha-cypermethrin and metalaxyl enantiomers in earthworms have also been studied (Diao et al., 2011; Xu et al., 2011). Owing to the limited information concerning the toxicity and bio-behavior of fipronil in earthworms, further research on enantiomeric effects in the earthworm needs to be conducted to enable environmental risk assessment. In this study, the acute toxicity of R-(−)-, S-(+)- and rac-fipronil were determined using the filter paper contact test. In addition, the enantioselective bioaccumulation and degradation of fipronil in earthworms was also studied. The aims of this work were to determine the toxicity of racemate and single-enantiomer fipronil to earthworms and to study the potential enantioselective environmental behaviors of fipronil in earthworms and, thus, evaluate the remediation effects of earthworms for fipronil pollutants in soil. 2. Materials and methods 2.1. Chemicals The racemic insecticide fipronil (96.5% purity) was provided by the China Ministry of Agriculture Institute for Control of Agrochemicals. The two enantiomers of fipronil were prepared by high-performance liquid chromatography HPLC (Agilent Technologies) using a chiral column with a cellulose tri-(3,5-dimethylphenyl-carbamate) chiral stationary phase, and the resulting enantiomeric purities of R-(−)and S-(+)-fipronil were 99.5% and 99.4%, respectively. Analytical grade reagents were redistilled and filtered through a 0.45-μm filter membrane prior to use. Ultrapure water was obtained using a Milli-Q water purification system (Millipore, Billerica Massachusetts, USA). All other chemicals and solvents were analytical grade and were purchased from Beijing Chemical Reagent Co., China. 2.2. Earthworms Mature earthworms (E. feotida) were purchased from a suburban farm and then maintained in a wooden breeding box containing a mixture of soil and cattle manure for 1 week to acclimate to the laboratory. The worms were active and healthy for the experiments. 2.3. Soil In accordance with OECD guideline 317 (OECD, 2010), the tests were performed with both artificial soil and natural soil. The natural soil was collected from an orchard in Beijing, China, that had not received fipronil application for at least the prior 10 years. The soil was sieved,
air-dried at room temperature, and kept in the dark before use. The physicochemical properties of the soil were as follows: organic matter (OM), 3.96%; clay, 2.48%; sand, 36.25%; silt, 61.27%; and pH 6.07. The artificial soil was composed of 10% sphagnum peat (pH 5.5 to 6.0, no visible plant remains, finely ground, and dried to measure moisture content), 20% kaolin clay (kaolinite content preferably above 30%), and 70% industrial sand (more than 50% of the particles between 50 and 200 μm), with a pH value of 6.0 ± 0.5 adjusted by calcium carbonate. Water content was adjusted to 35%. 2.4. Acute toxicity test In accordance with OECD guideline 207 (OECD, 1984), a paper contact toxicity assay was used to determine the acute toxicity of rac-fipronil and the R-(−)- and S-(+)-enantiomers to earthworms. The test substances were prepared in acetone. A series of known concentrations, 7.87, 15.74, 23.61, 31.48, 39.35, 47.22, 55.09, 62.96, 70.83, 78.70, 86.57, and 94.44 mg cm− 2 for S-fipronil and 7.87, 15.74, 23.61, 31.48, 39.35, 47.22, 55.09, 62.96, 70.83, and 78.70 mg cm− 2 for rac-fipronil and R-fipronil were spiked on filter papers. Solvent was volatilized under a stream of compressed air, and 1 mL of deionized water was added to each vial. After evacuating the gut contents for 3 h, the earthworms were rinsed by deionized water and dried by blotting-paper before introducing to the vials. Controls were set with the solvent alone. Ten replicates for each treatment were performed. The earthworms were placed on the filter papers at 20 ± 2 °C. Mortality was observed after incubation for 72 and 96 h, and LC50 values were calculated using SPSS Version 18.0 (SPSS Inc., Chicago, USA) 2.5. Bioaccumulation and degradation experiment 2.5.1. Bioaccumulation Based on OECD 317, the bioaccumulations of both fipronil enantiomers in the natural soil and artificial soil were investigated. For the natural soil, 250 g was weighed in a beaker, and rac-fipronil (12.5 mg in 10 mL of acetone) was slowly added; then the beaker was put aside for 24 h to evaporate the acetone. Deionized water was added to the contaminated soil to maintain water content of 20–30%, and the soil was fully stirred. After the gut contents were evacuated, the earthworms (20 g) were transferred into the beaker and placed in an incubator at 20 ± 2 °C. Five grams of earthworms was sampled after exposure times of 0, 0.5, 1, 2, 3, 7, 12, 15, and 18 days and frozen at − 20 °C before analysis. Every treated beaker was used for one sample point, and each sampling was conducted in triplicate. The bioaccumulation experiment in the artificial soil was the same as in the natural soil test, except the sampling points were at 0, 0.5, 1, 2, 4, 7, 10, and 14 days. 2.5.2. Degradation To evaluate the degradation of the two enantiomers, the earthworms were exposed to fipronil for 15 days. (The fipronil concentration and the amount of earthworms and soil were the same as in the bioaccumulation period). Then, the earthworms were collected and transferred to 250 g of the uncontaminated soil (as 0 days for purification phase). For the natural soil, at each of the sampling points of 0, 0.5, 1, 2, 3, 5, and 7 days, 10 g of earthworms and 20 g of soil were sampled and stored at −20 °C. Similarly, these were sampled at 0, 1, 2, 3, 5, and 7 days in artificial soil. The degradation of fipronil in the natural soil (25 mg kg−1, without earthworm) was also studied. The experiment (natural soil, 25 mg kg−1 fipronil, with 20 g of earthworms) was designed to investigate the influence of earthworms on pollution remediation. The sample consisted of 20 g of soil, collected at each sampling point: 0, 1, 2, 3, 7, 12, 15, and 18 days.
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Fig. 1. The typical chromatograms of chiral separation of standard fipronil (a) and in the earthworms in the degradation period on the 3th day (b).
of 20 °C. The chiral recognition of the two enantiomers was confirmed by CD detector.
The beakers were weighed daily, and the loss of water was compensated by addition of deionized water. During the uptake and elimination periods, 2 g of dried and crushed cow dung was spread on the soil surface each week to feed the earthworms.
2.7. Statistical analysis The statistical significance (P b 0.05) of acute toxicity was analyzed using SPSS Version 18.0. Bioaccumulation and degradation were analyzed using GraphPad Prism 5 for Windows (GraphPad Software, San Diego, USA). The enantiomeric fraction (EF) was used to assess the enantioselectivity of fipronil in earthworms and soil, which was expressed as the equation:
2.6. Chemical analysis For the pretreatment, 20 g of soil was added into a 50-mL polypropylene centrifuge tube and 8 mL of deionized water was added. Then, the tubes were shaken for 3 min. After that, activated carbon (0.03 g), neutral alumina (0.2 g), and acetone (30 mL) were added, vortexmixed for 5 min, and centrifuged at 4000 rpm for 5 min. The extraction was repeated following the same steps. The organic phase was combined and evaporated to dryness using a vacuum rotary evaporator at 35 °C, and the residue was diluted to 1.0 mL with n-hexane/2-propanol (95:5, v/v). The earthworms were homogenized using a Waring (Torrington CT) stainless steel blender, and 5 g was weighed for extraction in a 100-mL polypropylene centrifuge tube. After adding 15 mL of acetonitrile, the tube was vortexed for 3 min and then centrifuged at 4000 rpm for 5 min. The extraction was repeated twice and the organic phase was combined and extracted three times using n-hexane to remove lipids. Then, the acetonitrile was evaporated to dryness and dissolved in 1 mL of methanol for purification. An SPE procedure was applied for clean up with a C18 cartridge. The sample was loaded and eluted with 5 mL of methanol/water (1/5, v/v). The eluent was evaporated to dryness under a stream of nitrogen and dissolved in 1.0 mL of 2-propanol for HPLC analysis. All analyses were performed using an Agilent 1200 series HPLC system (Agilent Technology, Beijing, China), equipped with Quat Pump G1311A, degasser G1322A, injection valve G1328A, DAD G1315, circular dichroism detector CD2095, and column heater and cooler AT-930. Fipronil enantiomers were separated on an (R, R) Whelk-O1 chiral column (4.6 × 250 mm I.D, Regis, Illinois, USA) with the following operation conditions: a flow rate of 1.0 mL min−1, mobile phase of n-hexane / 2-propanol (95:5, v/v), detection wavelength of 225 nm, injection volume of 20 μL, and column temperature
EF ¼ peak area of ð−Þ=½ð−Þ þ ðþÞ The EF values range from 0 to 1, with EF = 0.5 representing the racemic mixture. Bio-concentration factor (BCF) is a function of the relative sorptive capacities in the organism and the surrounding soil, and it is defined as BCF ¼ Ce =Cs where Ce is the concentration of fipronil in earthworm tissue (g kg−1 dry weight), and Cs is the concentration of fipronil in soil (g kg−1 dry weight). The BCF was used to express the bioaccumulation of fipronil in the earthworms. 3. Results and discussion 3.1. Chiral analysis and method validation The two enantiomers were completely separated on the (R,R) Whelk-O1 chiral column, as shown in Fig. 1. The absolute configuration of fipronil enantiomers was assigned by comparing the CD signals with those reported in the literature (Liu et al., 2008). The first eluted enantiomer was S-(+)-fipronil, and the second was R-(−)-fipronil (Liu et al., 2008). The average recovery of both enantiomers at levels between 0.5 and 50 mg kg−1 ranged from 87.67% to 101.08% in the earthworms and from
Table 1 72 h and 96 h LC50 values of rac-fipronil and the enantiomers. Chemicals
72-h-LC50(mg cm−2)
R2
Pa
96-h-LC50(mg cm−2)
R2
Pa
rac-fipronil R-fipronil S-fipronil
0.044 [0.041–0.052]b 0.029 [0.020–0.036]b 0.050 [0.041–0.058]b
0.924 0.885 0.932
0.006 0.012 0.001
0.019 [0.013–0.026]b 0.015 [0.011–0.022]b 0.036 [0.026–0.040]b
0.894 0.931 0.868
0.003 0.005 0.008
a b
The probability by t test. P value smaller than 0.05 indicates that the correlation of linear equation is significant. 95% confidence interval.
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90.0% to 96.0% in the soil, with SD ≤ 10% (n = 3) for both the earthworms and the soil. The limit of detection for both enantiomers was 0.01 mg L−1. 3.2. Acute toxicity test The mortality in the controls did not exceed 10% after exposure for 96 h. Table 1 lists the LC50 values of rac-fipronil and the R/Senantiomers. The acute toxicity rates observed among the racemate and single enantiomers were significantly different. The LC50 values of rac-fipronil were 0.044 and 0.019 mg cm−2 at 72 h and 96 h, respectively, while they were 0.029 and 0.015 mg cm−2 at 72 h and 96 h, respectively, for the R-(−)-enantiomer. The S-(+)-enantiomer showed the least acute toxicity to earthworms: the LC50 values were 0.050 and 0.036 mg cm−2 at 72 h and 96 h, respectively. It has been reported that S-(+)-fipronil exhibits higher toxicity to Procambarus clarkii and C. dubia (Konwick et al., 2005; Liu et al., 2008; Li et al., 2009). In this work, in contrast, the R-(−)-fipronil was observed to be more toxic to
Fig. 2. The accumulation of fipronil enantiomers in earthworm in the natural soil (a) and the artificial soil (b). Bars are standard deviations.
Fig. 3. Degradation curves of fipronil enantiomers in earthworm in natural soil (a) and artificial soil (b). Bars are standard deviations.
Fig. 4. Enantiomeric fractions (EFs) of fipronil enantiomers in earthworms during the accumulation period (a), the degradation period (b) and in the natural soil with and without earthworms (c). Bars are standard deviations.
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Fig. 5. The concentrations of fipronil enantiomers in the natural soil with and without earthworms. * and ** represent statistically significant differences at P b 0.05 in 95% confidence intervals. Bars are standard deviations.
earthworm than its antipode. Hence, the toxicity of fipronil to other organisms on the enantiomeric level needs to be evaluated, and its enantioselectivity should be taken into consideration for environmental safety assessment. 3.3. Bioaccumulation and degradation of fipronil in earthworms 3.3.1. Bioaccumulation The earthworms were active and the weight loss was less than 15% at the end of the bioaccumulation and degradation phases. The two enantiomers of fipronil in earthworms and soil were measured in natural and artificial soil conditions. The concentration of each enantiomer in earthworms showed similar trends in both natural and artificial soils: there was no obvious enantioselectivity observed, with EF values of approximately 0.5 (Fig. 4a). During the accumulation period, fipronil concentration increased to a maximum level at the 15th day in earthworms in the natural soil and at the 10th day in the artificial soil, after which it remained at a relatively steady level (Fig. 2). Fig. 2 shows the concentration curves of bioaccumulation in earthworms in natural and artificial soil. For the accumulation experiment, the concentrations of the enantiomers in the corresponding soils were also measured, and BCFs were calculated. The BCFs of R- and S-fipronil in the artificial soil were 1.27 and 1.24, respectively, and were 1.33 and 1.32, respectively, in the natural soil. There was no significant difference between the two enantiomers, indicating that the uptake of fipronil in the earthworms was not enantioselective. The BCF values also inferred that the bio-enrichment ability of the earthworms for accumulating fipronil in soil was limited. 3.3.2. Degradation The degradation of fipronil in the earthworms in both soils followed first-order kinetics. The chromatogram of fipronil enantiomers in the earthworms during the degradation period is shown in Fig. 1b, and the degradation curve of R-(−)- and S-(+)-fipronil in the earthworms in the natural soil is shown in Fig. 3a. At the end of the bioaccumulation period, the fipronil concentrations in earthworms in the natural soil were higher than those in the artificial soil, resulting in the different concentrations at the beginning of degradation. The half-lives of R-(−)- and S-(+)-fipronil were 3.3 and 2.5 days, respectively. After 7 days, approximately 76% of the R-(−)-fipronil and 85%
of S-(+)-fipronil were degraded, with an EF value of 0.64 (Fig. 4b). The results demonstrated a preferential degradation of the Senantiomer by the earthworms. The results were similar for the artificial soil, as shown in Fig. 3b. Approximately 74% of R-(−)-fipronil and 85% of S-(+)-fipronil were degraded by the earthworms in the artificial soil after 7 days, with an EF value of 0.69 (Fig. 4b). The rates of degradation of the two enantiomers by earthworms were found to be slightly different between the artificial soil and the natural soil. The half-life of R-(−)-fipronil in the artificial soil was 2.1 days, 1.5 times shorter than that in the natural soil, and the half-life of the S-(+)- form was 1.4 days, approximately 1.7 times faster than that in the natural soil. The metabolic rate of the earthworms in the natural soil experiment was slower than that of the worms in the artificial soil. The reasons might lie in the strong influence of the organic matter on the elimination of a given chemical. The high organic content in the natural soil most likely had a high adsorptive capacity and thus led to a decrease in fipronil elimination by the earthworms; this condition existed not only in the surroundings of the organism's body but also in the gut (Amorim et al., 2002). For investigating the influences of earthworms on pollution remediation, analysis of the degradation of fipronil in the natural soil without earthworms was also conducted as a control experiment. The concentration changes in the soils are shown in Fig. 5. Fipronil was found to dissipate more quickly in the soil in which earthworms were present, indicating that earthworms helped to accelerate the degradation rate and thus promoted fipronil pollution remediation. No significant enantioselectivity was found in the soil with or without earthworms (Fig. 4c). Therefore, the enantioselectivity for fipronil in earthworms results from the organisms' metabolism. 4. Conclusions In this study, the enantioselective acute toxicity of fipronil to earthworms and the bioaccumulation and degradation of fipronil in both earthworms and soils were examined under laboratory conditions. The filter paper contact acute toxicity test showed that R-(−)-fipronil was approximately twice as toxic to earthworms as was S-(+)-fipronil. Fortunately, the toxicity of the racemate and both enantiomers to earthworms was low. The bioaccumulation of fipronil in the earthworms was not significantly enantioselective, but the less toxic S-(+)-fipronil was
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degraded preferentially by the earthworms. The results also suggested that the earthworms could promote fipronil degradation in soil and play a part in pollution remediation. These experimental data provide additional background information on the potential toxicity of fipronil and on the metabolic processes and enantioselective behavior of earthworms. Acknowledgments This work was supported by the National Natural Science Foundation of China (contract grant numbers 21307155 and 21277171), the Foundation for the Author of National Excellent Doctoral Dissertation of PR China, the New-Star of Science and Technology program, and the Beijing Metropolis Beijing Nova program. References Amorim MJ, Sousa JP, Nogueira AJA, Soares A. Bioavailability and toxicokinetics of 14Clindane (gamma-HCH) in the enchytracid Enchytracus albidus in two soil types: the aging effect. Arch Environ Contam Toxicol 2002;43:221–8. Author A. On the issue of enantioselectivity of chiral pesticides: a green chemistry opportunity. Green Chem 2004;6:77–8. Bobe A, Coste CM, Cooper JF. Factors influencing the adsorption of fipronil on soils. J Agric Food Chem 1997;45:4861–5. Cole LM, Nicholson RA, Casida JE. Action of phenylpyrazole insecticides at the GABA-gated chloride channel. Pestic Biochem Physiol 1993;46:47–54. Diao JL, Xu P, Liu DH, Lu YL, Zhou ZQ. Enantiomer-specific toxicity and bioaccumulation of alpha-cypermethrin to earthworm Eisenia fetida. J Hazard Mater 2011;192:1072–8. Doran G, Eberbach P, Helliwell S. Sorption and degradation of fipronil flooded anaerobic rice soils. J Agric Food Chem 2009;57:10296–301. Garrison AW. An introduction to pesticide chirality and the consequences of stereoselectivity. Chiral Pesticides: Stereoselectivity and Its Consequences. Washington DC: American Chemical Society; 2011. Hainzl D, Casida JE. Fipronil insecticide: novel photochemical desulfinylation with retention of neurotoxicity. Proc Natl Acad Sci 1996;93:12764–7. Hainzl D, Cole LM, Casida JE. Mechanisms for selective toxicity of fipronil insecticide and its sulfone metabolite and desulfinyl photoproduct. Chem Res Toxicol 1998;11: 1529–35.
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