Environ Monit Assess DOI 10.1007/s10661-013-3577-5
Simultaneous determination of insecticide fipronil and its metabolites in maize and soil by gas chromatography with electron capture detection Tielong Wang & Jiye Hu & Chaolun Liu
Received: 20 August 2013 / Accepted: 28 November 2013 # Springer Science+Business Media Dordrecht 2013
Abstract An integrated method for the simultaneous determination of insecticide fipronil and its three metabolites, desulfinyl, sulfide, and sulfone, in maize grain, maize stem, and soil was developed. This three-step method uses liquid–solid extraction with ultrasound or mechanical grinding, followed by liquid–liquid partitioning and florisil solid-phase extraction (SPE) for cleanup. The quantification was conducted by gas chromatography–electron capture detection in triplicate for each sample. The method was validated with five replicates at three fortification concentrations, 0.002, 0.01, and 0.1 mg kg−1, in each matrix and gave mean recoveries from 83 to 106 % with relative standard deviation ≤8.9 %. The limits of quantification (LOQ) were 0.002 mg kg−1 for the compounds in all matrixes. In the field study in Beijing and Shandong 2012, fipronil-coated maize seeds were planted and the proposed method was applied for checking the possible existence of four compounds in maize and soil samples, but none of them contained residues higher than the LOQs in both application rates. Moreover, the dissipation of fipronil in soil fits first-order kinetics with halflives 9.90 and 10.34 days in Beijing and Shandong, respectively. Combined with an adequate sample T. Wang : J. Hu (*) School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, China e-mail: [email protected] C. Liu College of Chemistry, Nankai University, Tianjin, People’s Republic of China
treatment, this technique offers good sensitivity and selectivity in the three complex matrixes. The results could provide guidance for the further research on pesticide distribution and safe use of fipronil as seed coat in cereals. Keywords Fipronil . Metabolites . Residue . GC–ECD . SPE . Maize
Introduction Maize (Zea mays) is an important grain crop for its heavy consumption, high nutritional value, economic value, and medical value. In modern agriculture, pesticides are normally applied in at least one phase of plant development (König et al. 2004), and as the pesticides were intensively and extensively used, their residues in food have posed a great threat to the consumers and environment (Amvrazi and Albanis 2009; Ozbey and Uygun 2007). Fipronil, (±)-5-amino-1-(2,6-dichloro-α,α,αtrifluoro-4-trifluromethyl sulphinyl pyrazole-3carbonitrile), is a halogen-substituted, thioethercontaining phenylpyrazole insecticide (Tingle et al. 2002), discovered in 1987 by the company RhonePoulenc Agro (now Bayer CropScience). Many insects (both beneficial and pests) are highly sensitive to fipronil and its major degradation products in larval and adult stages (Balança and de Visscher 1997). Fipronil may be found in pollen and nectar of the plants issued from treated seeds such as sunflowers and maize
Environ Monit Assess
(Aajoud et al. 2006), which is directly dangerous to nontarget pollinator insects such as honeybees (Kadar and Faucon 2006). Thus, the insecticide was recently allowed in agriculture, mostly as soil treatment or seed coat (Aajoud et al. 2003), resulting in a low-level existence of the pesticide in food and plant. Several studies indicated that fipronil may be converted to desulfinyl, sulfide, and sulfone metabolites, through photolysis, reduction, and oxidation processes, respectively (Bobé et al. 1998; Ying and Kookana 2001; Aajoud et al. 2003). Their structures of the four compounds are shown in Fig. 1. Fipronil is highly toxic to aquatic species, and its three main degradation products were reported as having similar or greater toxicity to aquatic invertebrates than the parent fipronil, and thus the environmental evaluation of degradation products is ecologically important (Schlenk et al. 2001; Maul et al. 2008; Gunasekara et al. 2007). In relation to the determination of fipronil, several works have been done with respect to the analysis for pollen and honey based on SPE or matrix solid-phase dispersion (MSPD) coupled with organic solvent extraction (Morzycka 2002; Kadar and Faucon 2006; Jiménez et al. 2007, 2008; Sánchez Brunete et al. 2008). Zhou et al. (2004) proposed a method for the determination of fipronil and its metabolites in soil and pakchoi by GC analysis after solvent extraction and SPE purification for vegetable. Bobé et al. (1998) proposed a gas chromatography method for the determination of fipronil residue in soil. Vı́lchez et al. (2001) developed a simple GC–MS method in combination with solid phase microextraction for determination of fipronil in water, soil, and human urine samples. However, the previous soil preparation methods showed their shortcomings, either not giving a satisfactory cleanup effect and thus showing a high limit of quantification or detecting the parent fipronil only without its metabolites. Fig. 1 Structures of fipronil and its three degradation products
To our knowledge, although some studies have been published on the environmental fate (Schlenk et al. 2001; Gunasekara et al. 2007; Raveton et al. 2007; Miranda and Mermut 2007; Lin et al. 2008; Mandal and Singh 2013) and analytical methods of fipronil, this is the first report on the trace and simultaneous analysis of fipronil and its metabolites in maize grain and stem using gas chromatography–electron capture detection (GC–ECD). Consequently, we developed a convenient analytical method which enables the simultaneous determination of fipronil and its main metabolites. This present study describes the analytical procedure and its use as a reliable tool for evaluating the dissipation of the insecticide in soil under field conditions and the persistence of the four toxic compounds in maize grain, stem, and soil. Until now, no residue limit has been established of fipronil in maize in China, and the results would be useful in establishing maximum residue limit (MRL) value. Besides, this work would facilitate the further study on the trace determination of fipronil and its metabolites, especially in cereals, and help the government to provide guidance on the proper and safe use of this insecticide.
Materials and methods Chemicals and reagents Fipronil reference standard was purchased from Agroenvironmental Protection Institute, Ministry of Agriculture (Beijing, China) with certified purity of 98.8 %, and its metabolites, fipronil desulfinyl (desulfinyl, 10 mg L−1), fipronil sulfide (sulfide, 99.5 % purity), and fipronil sulfone (sulfone, 99.0 % purity), were obtained from DrEhrenstorfer GmbH (Augsburg, Germany). Fipronil formulation (5 %
Environ Monit Assess
fipronil flowable concentrate for seed treatment, FS) was supplied by Jiangxi Zhengbang Chemical Co., Ltd. (Nanchang, China). N-hexane for chromatography was procured from Thermo Fisher Scientific Inc. (Waltham MA 02454, USA). Acetonitrile (MeCN), sodium chloride, ethyl acetate, petroleum ether, and anhydrous magnesium sulfate were of analytical grade for pesticide residue analysis from the Beijing Reagent Company (Beijing, China). A centrifuge with 50-mL round-bottomed polypropylene centrifuge tubes and ultrasound cleaner (KQ5200 type) were from Conlin Co., Ltd. (Shanghai, China) and Kunshan Ultrasonic Instrument Co., Ltd. (Kunshan, China), respectively. Florisil (500 mg, 3 mL−1) cartridge was purchased from Agela Technologies (Tianjin, China). Individual stock standard solutions (1,000 mg L−1) of fipronil and its metabolites, sulfone and sulfide, were prepared in acetone. Fipronil desulfinyl was supplied as a solution (10 mg L−1, acetonitrile). A mixed working solution(1 mg L−1, mixing ratio, 1:1:1:1) in 25 mL of nhexane of four compounds was prepared and then diluted to obtain the concentrations 0.5, 0.1, 0.05, 0.01, and 0.001 mg L−1 using n-hexane. All the mixed standard solutions and stock solutions were maintained in amber bottles and stored at 4 °C and at −20 °C, respectively. Instrumentation A Shimadzu 2010 gas chromatograph system equipped with a 63Ni ECD was used for the determination of the parent and three metabolites of fipronil. The separation was performed on HP-5 quartz capillary column (30 m× 0.25 mm I.D., 0.25-μm film thickness) at a constant nitrogen (>99.999 %) flow rate of 20 cm s−1, using an oven-temperature program from 120 °C (1 min) to 250 °C (1 min) at a rate of 20 °C min−1, 250 to 260 °C at 3 °C min−1, and 260 to 280 °C at 20 °C min−1. The injection inlet was set at 280 °C with the split ratio 10: 1, ECD at 310 °C and makeup flow at 30 mL min−1. One microliter of sample was injected for analysis in all instances. Under the given conditions, the approximate retention times of the four compounds are 14.08 (desulfinyl), 15.77 (sulfide), 15.97 (fipronil), and 17.58 (sulfone)min, respectively (Fig. 2). Experimental design Field experiments, including the dissipation experiment in soil and terminal residue experiment as seed coating
Fig. 2 Representative GC–ECD chromatograms of fipronil and its three metabolites in standard solution (a, 0.01 mg L−1), maize sample (b), maize stem sample (c), and soil sample (d) fortified at 0.01 mg kg−1. Peak identification: 1 fipronil desulfinyl, 2 fipronil sulfide, 3 fipronil, 4 fipronil sulfone
formulation in maize (grain and stem), were conducted in 2012 at two sites: Beijing and Shandong Province, China. The experiment was designed according to “Guideline on pesticide residue trials” issued by the Institute of the Control of Agrochemicals, Ministry of Agriculture (ICAMA), the People’s Republic of China. All experiment treatments contained three replicate plots. Each plot was 30 m2, and a buffer area was used to separate the plots with different treatments. In order to investigate the terminal residue of fipronil and its three metabolites in maize grain and plant, maize seeds were coated with dust seed-coating agent containing 5 % a.i. fipronil at 150 g seeds per 1 g agent to give a fipronil coating of 0.222 mg a.i. fipronil per seed (the recommended dosage, low dosage level) and 100 g seeds per 1 g agent (high dosage level) to give a fipronil coating of 0.333 mg a.i. fipronil per seed, respectively. Control seeds were coated with seed adhesive only. The treated seeds were then planted (150 seeds per plot) with three replicates for each treatment in the field. The preharvest intervals were 70 days for green maize and 95 days for mature maize. Soil samples were collected at two time intervals in the vicinity of the seed (about 10 cm around the seed) at a depth of 0–15 cm, and meanwhile, maize grain and stem samples were collected and chopped immediately after harvesting. All the collected samples were mixed with the same plot, packed separately in polyethylene bags, labeled, and stored at −20 °C until analysis.
Environ Monit Assess
To investigate the dissipation trends of fipronil in soil, 5 % fipronil FS was sprayed at the dosage of 25 g a.i. ha−1 (the same to the high dosage level applied for maize) once in blank soil which no plants were grown in. A plot with the same size but no pesticide application was compared simultaneously. Two kilograms of representative soil samples were sampled within the depth of 0–10 cm randomly in each plot and at different intervals, i.e., 2 h and 1, 3, 5, 7, 14, 21, and 30 days after application. Immediately after sampling, the samples were put into polyethylene bags and transported to the laboratory, where they were thoroughly mixed and subsampled. The sample was kept deep-frozen (−20 °C) until analysis. Control samples were obtained from the control plots. Analytical procedure Extraction
flask. Then it was concentrated and reconstituted in 3 mL of petroleum ether for further SPE cleanup. Maize stem Approximately, 300 g of maize stem was chopped and homogenized, and 5 g of representative sample was weighed and extracted by shaking with 20 mL water and 20 mL methanol for 60 min in automatic shaker. The extract was filtered through Whatman no. 1 filter paper (12-cm Buchner funnel), and the residue was rinsed twice with additional 20 mL methanol/water (1:1, v:v). The filtrate combined was transferred to separating funnel containing 30 mL of 2 % aqueous sodium sulfate, followed by liquid–liquid partitioning with dichloromethane for three times at the volumes of 20, 20, and 20 mL sequentially. Organic layer was collected, concentrated, and then dissolved in 5 mL of petroleum ether and transferred to an SPE cartridge. Cleanup process
Soil An 8 g of the homogeneous air-dried soil sample was weighed into a 50-mL centrifuge tube. Two milliliters of distilled water and 8 mL acetonitrile were added into the tube sequentially. Then the screw cap was closed, and the tube vigorously shaken for 1 min, using a vortex mixer at a maximum speed. Next, the sample was subjected to ultrasonic extraction in an ultrasonic cleaner for 20 min with shaking at intervals avoiding agglomeration. Afterward, 1 g of sodium chloride and 1 g of anhydrous magnesium sulfate were added orderly. Then the sample was again vortexed for 1 min, followed by centrifugation at 4,000 rpm for 4 min. An aliquot of 4 mL of the supernatant (MeCN layer) was transferred to a 100-mL evaporating flask. Then, it was concentrated and reconstituted in 4 mL of petroleum ether and subjected to SPE cleanup.
Two milliliters of the dissolved sample was loaded into a florisil SPE cartridge which was preconditioned with 2 mL petroleum ether/ethyl acetate (85:15, v:v) for maize stem and soil, or (90:10, v:v) for maize grain, and 2 mL petroleum ether orderly. The column was washed by 2 mL petroleum ether and 2 mL petroleum ether/ethyl acetate (95:5, v:v) sequentially and then discarded. The compounds were eluted with 10 mL of petroleum ether/ethyl acetate (85:15, v:v) for maize stem and soil, or 8 mL of 90:10 (v:v), for maize grain. The eluate was evaporated with N2 and reconstituted to 2 mL with n-hexane for GC–ECD analysis.
Maize grain Approximately 300 g of maize sample was crushed and further homogenized, and a portion of 5 g sample was drawn in a 50-mL centrifuge tube. Two milliliters of distilled water and 10 mL acetonitrile were added into the tube sequentially. After 2 min of vigorous shaking, the sample was subjected to ultrasonic extraction in an ultrasonic cleaner for 20 min. Afterward, 1 g of sodium chloride and 1 g of anhydrous magnesium sulfate were added orderly. Then the sample was again shaken for 1 min, and the mixture was centrifuged at 4,000 rpm for 4 min. Six milliliters of the supernatant (MeCN layer) was transferred to a 100-mL evaporating
The method described for sample preparation was validated by a recovery investigation. Blank maize grain, stem, and soil samples were fortified with known amounts of fipronil and its metabolites and shaken for 1 min by hand, thoroughly mixed. The spiked samples were allowed to equilibrate for 1 h before extraction to allow the spiked solution to penetrate the matrix, processed according to the above procedure and then determined by the external standard method with single-point calibration. The fortified recovery experiment was set at three concentration levels (0.002, 0.01, 0.1 mg kg−1) with five replicates at each level.
Recovery studies
Environ Monit Assess
compounds and the cartridge sorbent occurred, especially for fipronil and its sulfone metabolite. Due to the difference of polarity, in contrast to desulfinyl and sulfide, the more retained compounds were fipronil and sulfone in the cartridge, and the maximum elution for the former ones in soil and maize stem was observed at the third fraction and for the latter ones at the fifth fraction and were not detected in the following ones, respectively. So, 10 mL of petroleum ether/ethyl acetate (85:15, v:v) was needed to recover all the four compounds for maize stem and soil samples and 8 mL of petroleum ether/ethyl acetate (90:10, v:v) for maize samples.
Results and discussion Optimization of extraction and cleanup In order to get a cleaner final extract with less interference in maize stem, different acetonitrile–water and methanol–water mixtures were tested as extracting solvent. No significant improvement in selectivity was observed in relation to the use of acetonitrile with its ratio volume from 50 to 100 %. On the other hand, in the optimization of methanol–water ratio, on the premise of qualified recoveries, results showed that methanol– water (1:1, v:v) was the best extraction solvent for a minimum of interference, pigment especially. To optimize the cleanup procedure, the effect of the mixed ratio and volume of the elution solvent was studied. In our study, for the three different matrixes, maize matrix in petroleum ether showed less affinity to florisil sorbent, resulting in more impurities eluted. Thus, petroleum ether/ethyl acetate (90:10, v:v) was chosen as the optimized elution solvent which contained less percentage of ethyl acetate compared with petroleum ether/ethyl acetate (85:15, v:v) used for maize stem and soil. To optimize the minimum elution volume, fractions of 2 mL of the elution solvent were collected and analyzed individually to check in which volume the elution of the four compounds was completed. The results of this study showed that different interaction between the
Linearity The calibration graphs of four compounds obtained by plotting concentration versus average peak area (each sample injected in triplicate) were linear over the range of 0.001–0.5 mg L−1. A linear relation could be observed between detector response (y) and analyte concentration (x). The results showed good linearity with the correlation coefficients (γ) ranging from 0.9970 to 0.9988. LODs and LOQs The limits of detection (LODs) of the metabolites and parent fipronil were 0.0005 and 0.0003 mg L −1 ,
Table 1 Mean recoveries of fipronil and its three metabolites in soil, maize grain, and maize stem at three different levels and relative standard deviations (RSD) (n=5) Compound
Spike level (mg kg−1)
Maize grain Recovery (%)
Fipronil desulfinyl
Fipronil sulfide
Fipronil
Fipronil sulfone
Maize stem RSD (%)
Recovery (%)
Soil RSD (%)
Recovery (%)
RSD (%)
0.002
103
3.4
91
2.3
96
1.5
0.01
103
3.0
99
4.9
101
3.0
0.1
103
4.6
88
2.3
101
3.9
0.002
99
3.3
99
8.1
103
3.5
0.01
101
0.9
106
4.6
98
1.8
0.1
97
4.5
82
1.4
100
4.2
0.002
98
3.7
99
5.9
102
0.63
0.01
100
3.4
97
7.5
97
4.8
0.1
100
8.9
101
102
5.9
1
0.002
89
9.2
83
4.3
91
0.71
0.01
100
1.8
91
8.7
99
5.3
0.1
83
2.6
106
2.8
91
4.1
Environ Monit Assess Table 2 Regression equation, correlation coefficient, and half-life of fipronil in soil in Beijing and Shandong Sample location
Regression equation
Correlation coefficient (r)
Beijing
Ct =0.0428e−0.07t
0.9872
9.90
Shandong
Ct =0.0253e−0.067t
0.9536
10.34
respectively, with a signal-to-noise ratio of 3:1. The limits of quantification (LOQs) defined as the minimum fortified level of recovery in maize grain, stem, and soil were all 0.002 mg kg−1. Despite the great structural similarity among the compounds, the LOD values showed that fipronil presents a little lower sensitivity than its metabolites in ECD. The LOQ values obtained are suitable for the determination of residues of these compounds in maize, considering that the values are well below the tolerance level of 0.02 and 0.005 mg kg−1 established in the USA and European Union, respectively, showing the method’s advantage for the determination of the pesticide in cereal and soil. Quantitative analysis of fipronil and its metabolites The technique GC–ECD demonstrated high performance for the detection and quantification of the four compounds, allowing an analysis with good sensitivity. Figure 2 shows the representative chromatograms of the four compounds in standard solution (a, 0.01 mg L−1), and three sample matrixes spiked all at 0.01 mg kg−1 (b, c, and d). The selectivity of the method can be evaluated Fig. 3 Dissipation pattern of fipronil in soil
Half-life (days)
based on these chromatograms, and no interferences from the three matrixes were observed in the retention times of the compounds. In our study, the use of SPE greatly facilitated the effective cleanup with less sample handing, solvent consumption, and interference matrix especially at trace level.
Method performance The method accuracy was evaluated by the fortified recovery at three concentration levels with five replicates. The average recoveries of fipronil and its three metabolites, desulfinyl, sulfide, and sulfone, in maize grain were 83–103 % with the relative standard deviations (RSDs) less than 9.2 %, in stem were 83–106 % with RSDs less than 8.7 %, and in soil were 91–102 % with the RSDs less than 5.9 % (Table 1). The results showed that the analyses by GC–ECD gave good recoveries at different concentrations with low RSDs (reproducibility), indicating that good performance of extraction, cleanup, and chromatographic parameters for residue determination in cereals and soil.
Environ Monit Assess
Dissipation of fipronil in soil The dissipation data of fipronil in soil under field conditions in Beijing and Shandong Province is shown in Table 2 and Fig. 3. The initial residues of fipronil were found to be 0.046 and 0.035 mg kg−1 in soil collected in Beijing and Shandong on 0 day (2 h after treatment) after the application at 25 g a.i. ha−1, respectively. Halflife (t 1/2) period of fipronil alone in Beijing and Shandong were calculated to be 9.90 and 10.34 days, respectively. Similar result of the insecticide has been reported by Saini et al. (2013) with half-life period of 10.81 days for fipronil in soil. Terminal residue of fipronil and its metabolites in maize grain, stem, and soil The terminal residues of fipronil and its three metabolites in maize, stem, and soil at harvest time were detected. It was found that the residues of the four compounds in maize, stem, and soil from two sites were all lower than the LOQ values (0.002 mg kg−1), so the terminal residues of fipronil in maize were below the available MRLs (defined as the sum of amounts of parent fipronil and its sulfone metabolite, calculated as fipronil) established by USA (0.02 mg kg−1 in maize and 0.30 mg kg−1 in maize stover) and European Union (0.005 mg kg−1 in maize) (Zhuang 2010), indicating the safe use of 5 % fipronil FS under recommended dosage. For the degradation in plants, growth dilution factor might have played a significant role (Tewary et al. 2005), especially for the decline in maize plant as the pesticide acted as seed coat.
Conclusion A simple and practical GC–ECD method in combination with SPE for the simultaneous determination of the insecticide fipronil and its metabolites in maize grain, stem, and soil is presented. SPE proved to be an adequate way for the separation of the analytes from matrix interferences. The method has been validated for the three matrixes with a high level sensitivity and good selectivity and meets the current requirements with less cost to determine fipronil and its metabolites in maize and soil. The fipronil dissipation rates in soil and terminal residues in maize grain and stem were also studied to evaluate consumer and environmental safety and the
proper use of the pesticide. The results showed that the half-lives in soil were 9.90 and 10.34 days in Beijing and Shandong, respectively, under the field conditions. According to the results of the terminal residue study, when fipronil was used under the experimental design, the maximum residues in maize at intervals of 70 and 95 days after the pesticide application were far below the available MRLs, which were considered safe for consumption. This work would aid in the establishment of MRL value and the safe and proper use of fipronil in maize in China.
References Aajoud, A., Ravanel, P., & Tissut, M. (2003). Fipronil metabolism and dissipation in a simplified aquatic ecosystem. Journal of Agricultural and Food Chemistry, 51, 1347–1352. Aajoud, A., Raveton, M., Aouadi, H., Tissut, M., & Ravanel, P. (2006). Uptake and xylem transport of fipronil in sunflower. Journal of Agricultural and Food Chemistry, 54, 5055–5060. Amvrazi, E. G., & Albanis, T. A. (2009). Pesticide residue assessment in different types of olive oil and preliminary exposure assessment of Greek consumers to the pesticide residues detected. Food Chemistry, 113, 253–261. Balança, G., & de Visscher, M. N. (1997). Effects of very low doses of fipronil on grasshoppers and non-target insects following field trials for grasshopper control. Crop Protection, 16, 553–564. Bobé, A., Cooper, J. F., Coste, C. M., & Muller, M. A. (1998). Behaviour of fipronil in soil under Sahelian Plain field conditions. Pesticide Science, 52, 275–281. Gunasekara, A. S., Truong, T., Goh, K. S., Spurlock, F., & Tjeerdema, R. S. (2007). Environmental fate and toxicology of fipronil. Journal of Pesticide Science, 32, 189–199. Jiménez, J. J., Bernal, J. L., del Nozal, M. J., Martín, M. T., & Mayo, R. (2007). Comparative study of sample preparation procedures to determine fipronil in pollen by gas chromatography with mass spectrometric and electron-capture detection. Journal of Chromatography A, 1146, 8–16. Jiménez, J. J., Bernal, J. L., del Nozal, M. J., Martín, M. T., & Mayo, R. (2008). Sample preparation methods to analyze fipronil in honey by gas chromatography with electroncapture and mass spectrometric detection. Journal of Chromatography A, 1187, 40–45. Kadar, A., & Faucon, J. P. (2006). Determination of traces of fipronil and its metabolites in pollen by liquid chromatography with electrospray ionization-tandem mass spectrometry. Journal of Agricultural and Food Chemistry, 54, 9741–9746. König, A., Cockburn, A., Crevel, R. W. R., Debruyne, E., Grafstroem, R., Hammerling, U., et al. (2004). Assessment of the safety of foods derived from genetically modified (GM) crops. Food and Chemical Toxicology, 42, 1047–1088. Lin, K., Haver, D., Oki, L., & Gan, J. (2008). Transformation and sorption of fipronil in urban stream sediments. Journal of Agricultural and Food Chemistry, 56, 8594–8600.
Environ Monit Assess Mandal, K., & Singh, B. (2013). Persistence of fipronil and its metabolites in sandy loam and clay loam soils under laboratory conditions. Chemosphere, 91, 1596–1603. Maul, J. D., Brennan, A. A., Harwood, A. D., & Lydy, M. J. (2008). Effect of sediment-associated pyrethroids, fipronil, and metabolites on Chironomus tentans growth rate, body mass, condition index, immobilization, and survival. Environmental Toxicology and Chemistry, 27, 2582–2590. Miranda, M. C. S., & Mermut, A. R. (2007). Sorption of fipronil and its sulfide derivative by soils and goethite. Geoderma, 140, 1–7. Morzycka, B. (2002). Simple method for the determination of trace levels of pesticides in honeybees using matrix solidphase dispersion and gas chromatography. Journal of Chromatography A, 982, 267–273. Ozbey, A., & Uygun, U. (2007). Behaviour of some organophosphorus pesticide residues in peppermint tea during the infusion process. Food Chemistry, 104, 237–241. Raveton, M., Aajoud, A., Willison, J., Cherifi, M., Tissut, M., & Ravanel, P. (2007). Soil distribution of fipronil and its metabolites originating from a seed-coated formulation. Chemosphere, 69, 1124–1129. Saini, S., Rani, M., & Kumari, B. (2013). Persistence of fipronil and its metabolites in soil under field conditions. Environmental Monitoring and Assessment. doi:10.1007/ s10661-013-3356-3. Sánchez Brunete, C., Miguel, E., Albero, B., & Tadeo, J. L. (2008). Determination of fipronil residues in honey and
pollen by gas chromatography. Spanish Journal of Agricultural Research, 6, 7–14. Schlenk, D., Huggett, D. B., Allgood, J., Bennett, E., Rimoldi, J., Beeler, A. B., et al. (2001). Toxicity of fipronil and its degradation products to Procambarus sp.: field and laboratory studies. Archives of Environmental Contamination and Toxicology, 41, 325–332. Tewary, D. K., Kumar, V., Ravindranath, S. D., & Shanker, A. (2005). Dissipation behavior of bifenthrin residues in tea and its brew. Food Control, 16, 231–237. Tingle, C. C. D., Rother, J. A., Dewhurst, C. F., Lauer, S., & King, W. J. (2002). Fipronil: environmental fate, ecotoxicology and human health concerns. Reviews of Environmental Contamination and Toxicology, 176, 1–66. Vı́lchez, J. L., Prieto, A., Araujo, L., & Navalón, A. (2001). Determination of fipronil by solid-phase micro extraction and gas chromatography–mass spectrometry. Journal of Chromatography A, 919, 215–221. Ying, G. G., & Kookana, R. S. (2001). Sorption of fipronil and its metabolites on soils from South Australia. Journal of Environmental Science and Health. Part. B, 36, 545–558. Zhou, P., Lu, Y., Liu, B. F., & Gan, J. J. (2004). Dynamics of fipronil residue in vegetable-field ecosystem. Chemosphere, 57, 1691–1696. Zhuang, W. J. (2010). The global regulations on maximum residue limits (MRLs) for pesticides in food stuffs and feedstuffs. Beijing: Chemical Industry Press.
Simultaneous determination of insecticide fipronil and its metabolites in maize and soil by gas chromatography with electron capture detection Tielong Wang & Jiye Hu & Chaolun Liu
Received: 20 August 2013 / Accepted: 28 November 2013 # Springer Science+Business Media Dordrecht 2013
Abstract An integrated method for the simultaneous determination of insecticide fipronil and its three metabolites, desulfinyl, sulfide, and sulfone, in maize grain, maize stem, and soil was developed. This three-step method uses liquid–solid extraction with ultrasound or mechanical grinding, followed by liquid–liquid partitioning and florisil solid-phase extraction (SPE) for cleanup. The quantification was conducted by gas chromatography–electron capture detection in triplicate for each sample. The method was validated with five replicates at three fortification concentrations, 0.002, 0.01, and 0.1 mg kg−1, in each matrix and gave mean recoveries from 83 to 106 % with relative standard deviation ≤8.9 %. The limits of quantification (LOQ) were 0.002 mg kg−1 for the compounds in all matrixes. In the field study in Beijing and Shandong 2012, fipronil-coated maize seeds were planted and the proposed method was applied for checking the possible existence of four compounds in maize and soil samples, but none of them contained residues higher than the LOQs in both application rates. Moreover, the dissipation of fipronil in soil fits first-order kinetics with halflives 9.90 and 10.34 days in Beijing and Shandong, respectively. Combined with an adequate sample T. Wang : J. Hu (*) School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, China e-mail: [email protected] C. Liu College of Chemistry, Nankai University, Tianjin, People’s Republic of China
treatment, this technique offers good sensitivity and selectivity in the three complex matrixes. The results could provide guidance for the further research on pesticide distribution and safe use of fipronil as seed coat in cereals. Keywords Fipronil . Metabolites . Residue . GC–ECD . SPE . Maize
Introduction Maize (Zea mays) is an important grain crop for its heavy consumption, high nutritional value, economic value, and medical value. In modern agriculture, pesticides are normally applied in at least one phase of plant development (König et al. 2004), and as the pesticides were intensively and extensively used, their residues in food have posed a great threat to the consumers and environment (Amvrazi and Albanis 2009; Ozbey and Uygun 2007). Fipronil, (±)-5-amino-1-(2,6-dichloro-α,α,αtrifluoro-4-trifluromethyl sulphinyl pyrazole-3carbonitrile), is a halogen-substituted, thioethercontaining phenylpyrazole insecticide (Tingle et al. 2002), discovered in 1987 by the company RhonePoulenc Agro (now Bayer CropScience). Many insects (both beneficial and pests) are highly sensitive to fipronil and its major degradation products in larval and adult stages (Balança and de Visscher 1997). Fipronil may be found in pollen and nectar of the plants issued from treated seeds such as sunflowers and maize
Environ Monit Assess
(Aajoud et al. 2006), which is directly dangerous to nontarget pollinator insects such as honeybees (Kadar and Faucon 2006). Thus, the insecticide was recently allowed in agriculture, mostly as soil treatment or seed coat (Aajoud et al. 2003), resulting in a low-level existence of the pesticide in food and plant. Several studies indicated that fipronil may be converted to desulfinyl, sulfide, and sulfone metabolites, through photolysis, reduction, and oxidation processes, respectively (Bobé et al. 1998; Ying and Kookana 2001; Aajoud et al. 2003). Their structures of the four compounds are shown in Fig. 1. Fipronil is highly toxic to aquatic species, and its three main degradation products were reported as having similar or greater toxicity to aquatic invertebrates than the parent fipronil, and thus the environmental evaluation of degradation products is ecologically important (Schlenk et al. 2001; Maul et al. 2008; Gunasekara et al. 2007). In relation to the determination of fipronil, several works have been done with respect to the analysis for pollen and honey based on SPE or matrix solid-phase dispersion (MSPD) coupled with organic solvent extraction (Morzycka 2002; Kadar and Faucon 2006; Jiménez et al. 2007, 2008; Sánchez Brunete et al. 2008). Zhou et al. (2004) proposed a method for the determination of fipronil and its metabolites in soil and pakchoi by GC analysis after solvent extraction and SPE purification for vegetable. Bobé et al. (1998) proposed a gas chromatography method for the determination of fipronil residue in soil. Vı́lchez et al. (2001) developed a simple GC–MS method in combination with solid phase microextraction for determination of fipronil in water, soil, and human urine samples. However, the previous soil preparation methods showed their shortcomings, either not giving a satisfactory cleanup effect and thus showing a high limit of quantification or detecting the parent fipronil only without its metabolites. Fig. 1 Structures of fipronil and its three degradation products
To our knowledge, although some studies have been published on the environmental fate (Schlenk et al. 2001; Gunasekara et al. 2007; Raveton et al. 2007; Miranda and Mermut 2007; Lin et al. 2008; Mandal and Singh 2013) and analytical methods of fipronil, this is the first report on the trace and simultaneous analysis of fipronil and its metabolites in maize grain and stem using gas chromatography–electron capture detection (GC–ECD). Consequently, we developed a convenient analytical method which enables the simultaneous determination of fipronil and its main metabolites. This present study describes the analytical procedure and its use as a reliable tool for evaluating the dissipation of the insecticide in soil under field conditions and the persistence of the four toxic compounds in maize grain, stem, and soil. Until now, no residue limit has been established of fipronil in maize in China, and the results would be useful in establishing maximum residue limit (MRL) value. Besides, this work would facilitate the further study on the trace determination of fipronil and its metabolites, especially in cereals, and help the government to provide guidance on the proper and safe use of this insecticide.
Materials and methods Chemicals and reagents Fipronil reference standard was purchased from Agroenvironmental Protection Institute, Ministry of Agriculture (Beijing, China) with certified purity of 98.8 %, and its metabolites, fipronil desulfinyl (desulfinyl, 10 mg L−1), fipronil sulfide (sulfide, 99.5 % purity), and fipronil sulfone (sulfone, 99.0 % purity), were obtained from DrEhrenstorfer GmbH (Augsburg, Germany). Fipronil formulation (5 %
Environ Monit Assess
fipronil flowable concentrate for seed treatment, FS) was supplied by Jiangxi Zhengbang Chemical Co., Ltd. (Nanchang, China). N-hexane for chromatography was procured from Thermo Fisher Scientific Inc. (Waltham MA 02454, USA). Acetonitrile (MeCN), sodium chloride, ethyl acetate, petroleum ether, and anhydrous magnesium sulfate were of analytical grade for pesticide residue analysis from the Beijing Reagent Company (Beijing, China). A centrifuge with 50-mL round-bottomed polypropylene centrifuge tubes and ultrasound cleaner (KQ5200 type) were from Conlin Co., Ltd. (Shanghai, China) and Kunshan Ultrasonic Instrument Co., Ltd. (Kunshan, China), respectively. Florisil (500 mg, 3 mL−1) cartridge was purchased from Agela Technologies (Tianjin, China). Individual stock standard solutions (1,000 mg L−1) of fipronil and its metabolites, sulfone and sulfide, were prepared in acetone. Fipronil desulfinyl was supplied as a solution (10 mg L−1, acetonitrile). A mixed working solution(1 mg L−1, mixing ratio, 1:1:1:1) in 25 mL of nhexane of four compounds was prepared and then diluted to obtain the concentrations 0.5, 0.1, 0.05, 0.01, and 0.001 mg L−1 using n-hexane. All the mixed standard solutions and stock solutions were maintained in amber bottles and stored at 4 °C and at −20 °C, respectively. Instrumentation A Shimadzu 2010 gas chromatograph system equipped with a 63Ni ECD was used for the determination of the parent and three metabolites of fipronil. The separation was performed on HP-5 quartz capillary column (30 m× 0.25 mm I.D., 0.25-μm film thickness) at a constant nitrogen (>99.999 %) flow rate of 20 cm s−1, using an oven-temperature program from 120 °C (1 min) to 250 °C (1 min) at a rate of 20 °C min−1, 250 to 260 °C at 3 °C min−1, and 260 to 280 °C at 20 °C min−1. The injection inlet was set at 280 °C with the split ratio 10: 1, ECD at 310 °C and makeup flow at 30 mL min−1. One microliter of sample was injected for analysis in all instances. Under the given conditions, the approximate retention times of the four compounds are 14.08 (desulfinyl), 15.77 (sulfide), 15.97 (fipronil), and 17.58 (sulfone)min, respectively (Fig. 2). Experimental design Field experiments, including the dissipation experiment in soil and terminal residue experiment as seed coating
Fig. 2 Representative GC–ECD chromatograms of fipronil and its three metabolites in standard solution (a, 0.01 mg L−1), maize sample (b), maize stem sample (c), and soil sample (d) fortified at 0.01 mg kg−1. Peak identification: 1 fipronil desulfinyl, 2 fipronil sulfide, 3 fipronil, 4 fipronil sulfone
formulation in maize (grain and stem), were conducted in 2012 at two sites: Beijing and Shandong Province, China. The experiment was designed according to “Guideline on pesticide residue trials” issued by the Institute of the Control of Agrochemicals, Ministry of Agriculture (ICAMA), the People’s Republic of China. All experiment treatments contained three replicate plots. Each plot was 30 m2, and a buffer area was used to separate the plots with different treatments. In order to investigate the terminal residue of fipronil and its three metabolites in maize grain and plant, maize seeds were coated with dust seed-coating agent containing 5 % a.i. fipronil at 150 g seeds per 1 g agent to give a fipronil coating of 0.222 mg a.i. fipronil per seed (the recommended dosage, low dosage level) and 100 g seeds per 1 g agent (high dosage level) to give a fipronil coating of 0.333 mg a.i. fipronil per seed, respectively. Control seeds were coated with seed adhesive only. The treated seeds were then planted (150 seeds per plot) with three replicates for each treatment in the field. The preharvest intervals were 70 days for green maize and 95 days for mature maize. Soil samples were collected at two time intervals in the vicinity of the seed (about 10 cm around the seed) at a depth of 0–15 cm, and meanwhile, maize grain and stem samples were collected and chopped immediately after harvesting. All the collected samples were mixed with the same plot, packed separately in polyethylene bags, labeled, and stored at −20 °C until analysis.
Environ Monit Assess
To investigate the dissipation trends of fipronil in soil, 5 % fipronil FS was sprayed at the dosage of 25 g a.i. ha−1 (the same to the high dosage level applied for maize) once in blank soil which no plants were grown in. A plot with the same size but no pesticide application was compared simultaneously. Two kilograms of representative soil samples were sampled within the depth of 0–10 cm randomly in each plot and at different intervals, i.e., 2 h and 1, 3, 5, 7, 14, 21, and 30 days after application. Immediately after sampling, the samples were put into polyethylene bags and transported to the laboratory, where they were thoroughly mixed and subsampled. The sample was kept deep-frozen (−20 °C) until analysis. Control samples were obtained from the control plots. Analytical procedure Extraction
flask. Then it was concentrated and reconstituted in 3 mL of petroleum ether for further SPE cleanup. Maize stem Approximately, 300 g of maize stem was chopped and homogenized, and 5 g of representative sample was weighed and extracted by shaking with 20 mL water and 20 mL methanol for 60 min in automatic shaker. The extract was filtered through Whatman no. 1 filter paper (12-cm Buchner funnel), and the residue was rinsed twice with additional 20 mL methanol/water (1:1, v:v). The filtrate combined was transferred to separating funnel containing 30 mL of 2 % aqueous sodium sulfate, followed by liquid–liquid partitioning with dichloromethane for three times at the volumes of 20, 20, and 20 mL sequentially. Organic layer was collected, concentrated, and then dissolved in 5 mL of petroleum ether and transferred to an SPE cartridge. Cleanup process
Soil An 8 g of the homogeneous air-dried soil sample was weighed into a 50-mL centrifuge tube. Two milliliters of distilled water and 8 mL acetonitrile were added into the tube sequentially. Then the screw cap was closed, and the tube vigorously shaken for 1 min, using a vortex mixer at a maximum speed. Next, the sample was subjected to ultrasonic extraction in an ultrasonic cleaner for 20 min with shaking at intervals avoiding agglomeration. Afterward, 1 g of sodium chloride and 1 g of anhydrous magnesium sulfate were added orderly. Then the sample was again vortexed for 1 min, followed by centrifugation at 4,000 rpm for 4 min. An aliquot of 4 mL of the supernatant (MeCN layer) was transferred to a 100-mL evaporating flask. Then, it was concentrated and reconstituted in 4 mL of petroleum ether and subjected to SPE cleanup.
Two milliliters of the dissolved sample was loaded into a florisil SPE cartridge which was preconditioned with 2 mL petroleum ether/ethyl acetate (85:15, v:v) for maize stem and soil, or (90:10, v:v) for maize grain, and 2 mL petroleum ether orderly. The column was washed by 2 mL petroleum ether and 2 mL petroleum ether/ethyl acetate (95:5, v:v) sequentially and then discarded. The compounds were eluted with 10 mL of petroleum ether/ethyl acetate (85:15, v:v) for maize stem and soil, or 8 mL of 90:10 (v:v), for maize grain. The eluate was evaporated with N2 and reconstituted to 2 mL with n-hexane for GC–ECD analysis.
Maize grain Approximately 300 g of maize sample was crushed and further homogenized, and a portion of 5 g sample was drawn in a 50-mL centrifuge tube. Two milliliters of distilled water and 10 mL acetonitrile were added into the tube sequentially. After 2 min of vigorous shaking, the sample was subjected to ultrasonic extraction in an ultrasonic cleaner for 20 min. Afterward, 1 g of sodium chloride and 1 g of anhydrous magnesium sulfate were added orderly. Then the sample was again shaken for 1 min, and the mixture was centrifuged at 4,000 rpm for 4 min. Six milliliters of the supernatant (MeCN layer) was transferred to a 100-mL evaporating
The method described for sample preparation was validated by a recovery investigation. Blank maize grain, stem, and soil samples were fortified with known amounts of fipronil and its metabolites and shaken for 1 min by hand, thoroughly mixed. The spiked samples were allowed to equilibrate for 1 h before extraction to allow the spiked solution to penetrate the matrix, processed according to the above procedure and then determined by the external standard method with single-point calibration. The fortified recovery experiment was set at three concentration levels (0.002, 0.01, 0.1 mg kg−1) with five replicates at each level.
Recovery studies
Environ Monit Assess
compounds and the cartridge sorbent occurred, especially for fipronil and its sulfone metabolite. Due to the difference of polarity, in contrast to desulfinyl and sulfide, the more retained compounds were fipronil and sulfone in the cartridge, and the maximum elution for the former ones in soil and maize stem was observed at the third fraction and for the latter ones at the fifth fraction and were not detected in the following ones, respectively. So, 10 mL of petroleum ether/ethyl acetate (85:15, v:v) was needed to recover all the four compounds for maize stem and soil samples and 8 mL of petroleum ether/ethyl acetate (90:10, v:v) for maize samples.
Results and discussion Optimization of extraction and cleanup In order to get a cleaner final extract with less interference in maize stem, different acetonitrile–water and methanol–water mixtures were tested as extracting solvent. No significant improvement in selectivity was observed in relation to the use of acetonitrile with its ratio volume from 50 to 100 %. On the other hand, in the optimization of methanol–water ratio, on the premise of qualified recoveries, results showed that methanol– water (1:1, v:v) was the best extraction solvent for a minimum of interference, pigment especially. To optimize the cleanup procedure, the effect of the mixed ratio and volume of the elution solvent was studied. In our study, for the three different matrixes, maize matrix in petroleum ether showed less affinity to florisil sorbent, resulting in more impurities eluted. Thus, petroleum ether/ethyl acetate (90:10, v:v) was chosen as the optimized elution solvent which contained less percentage of ethyl acetate compared with petroleum ether/ethyl acetate (85:15, v:v) used for maize stem and soil. To optimize the minimum elution volume, fractions of 2 mL of the elution solvent were collected and analyzed individually to check in which volume the elution of the four compounds was completed. The results of this study showed that different interaction between the
Linearity The calibration graphs of four compounds obtained by plotting concentration versus average peak area (each sample injected in triplicate) were linear over the range of 0.001–0.5 mg L−1. A linear relation could be observed between detector response (y) and analyte concentration (x). The results showed good linearity with the correlation coefficients (γ) ranging from 0.9970 to 0.9988. LODs and LOQs The limits of detection (LODs) of the metabolites and parent fipronil were 0.0005 and 0.0003 mg L −1 ,
Table 1 Mean recoveries of fipronil and its three metabolites in soil, maize grain, and maize stem at three different levels and relative standard deviations (RSD) (n=5) Compound
Spike level (mg kg−1)
Maize grain Recovery (%)
Fipronil desulfinyl
Fipronil sulfide
Fipronil
Fipronil sulfone
Maize stem RSD (%)
Recovery (%)
Soil RSD (%)
Recovery (%)
RSD (%)
0.002
103
3.4
91
2.3
96
1.5
0.01
103
3.0
99
4.9
101
3.0
0.1
103
4.6
88
2.3
101
3.9
0.002
99
3.3
99
8.1
103
3.5
0.01
101
0.9
106
4.6
98
1.8
0.1
97
4.5
82
1.4
100
4.2
0.002
98
3.7
99
5.9
102
0.63
0.01
100
3.4
97
7.5
97
4.8
0.1
100
8.9
101
102
5.9
1
0.002
89
9.2
83
4.3
91
0.71
0.01
100
1.8
91
8.7
99
5.3
0.1
83
2.6
106
2.8
91
4.1
Environ Monit Assess Table 2 Regression equation, correlation coefficient, and half-life of fipronil in soil in Beijing and Shandong Sample location
Regression equation
Correlation coefficient (r)
Beijing
Ct =0.0428e−0.07t
0.9872
9.90
Shandong
Ct =0.0253e−0.067t
0.9536
10.34
respectively, with a signal-to-noise ratio of 3:1. The limits of quantification (LOQs) defined as the minimum fortified level of recovery in maize grain, stem, and soil were all 0.002 mg kg−1. Despite the great structural similarity among the compounds, the LOD values showed that fipronil presents a little lower sensitivity than its metabolites in ECD. The LOQ values obtained are suitable for the determination of residues of these compounds in maize, considering that the values are well below the tolerance level of 0.02 and 0.005 mg kg−1 established in the USA and European Union, respectively, showing the method’s advantage for the determination of the pesticide in cereal and soil. Quantitative analysis of fipronil and its metabolites The technique GC–ECD demonstrated high performance for the detection and quantification of the four compounds, allowing an analysis with good sensitivity. Figure 2 shows the representative chromatograms of the four compounds in standard solution (a, 0.01 mg L−1), and three sample matrixes spiked all at 0.01 mg kg−1 (b, c, and d). The selectivity of the method can be evaluated Fig. 3 Dissipation pattern of fipronil in soil
Half-life (days)
based on these chromatograms, and no interferences from the three matrixes were observed in the retention times of the compounds. In our study, the use of SPE greatly facilitated the effective cleanup with less sample handing, solvent consumption, and interference matrix especially at trace level.
Method performance The method accuracy was evaluated by the fortified recovery at three concentration levels with five replicates. The average recoveries of fipronil and its three metabolites, desulfinyl, sulfide, and sulfone, in maize grain were 83–103 % with the relative standard deviations (RSDs) less than 9.2 %, in stem were 83–106 % with RSDs less than 8.7 %, and in soil were 91–102 % with the RSDs less than 5.9 % (Table 1). The results showed that the analyses by GC–ECD gave good recoveries at different concentrations with low RSDs (reproducibility), indicating that good performance of extraction, cleanup, and chromatographic parameters for residue determination in cereals and soil.
Environ Monit Assess
Dissipation of fipronil in soil The dissipation data of fipronil in soil under field conditions in Beijing and Shandong Province is shown in Table 2 and Fig. 3. The initial residues of fipronil were found to be 0.046 and 0.035 mg kg−1 in soil collected in Beijing and Shandong on 0 day (2 h after treatment) after the application at 25 g a.i. ha−1, respectively. Halflife (t 1/2) period of fipronil alone in Beijing and Shandong were calculated to be 9.90 and 10.34 days, respectively. Similar result of the insecticide has been reported by Saini et al. (2013) with half-life period of 10.81 days for fipronil in soil. Terminal residue of fipronil and its metabolites in maize grain, stem, and soil The terminal residues of fipronil and its three metabolites in maize, stem, and soil at harvest time were detected. It was found that the residues of the four compounds in maize, stem, and soil from two sites were all lower than the LOQ values (0.002 mg kg−1), so the terminal residues of fipronil in maize were below the available MRLs (defined as the sum of amounts of parent fipronil and its sulfone metabolite, calculated as fipronil) established by USA (0.02 mg kg−1 in maize and 0.30 mg kg−1 in maize stover) and European Union (0.005 mg kg−1 in maize) (Zhuang 2010), indicating the safe use of 5 % fipronil FS under recommended dosage. For the degradation in plants, growth dilution factor might have played a significant role (Tewary et al. 2005), especially for the decline in maize plant as the pesticide acted as seed coat.
Conclusion A simple and practical GC–ECD method in combination with SPE for the simultaneous determination of the insecticide fipronil and its metabolites in maize grain, stem, and soil is presented. SPE proved to be an adequate way for the separation of the analytes from matrix interferences. The method has been validated for the three matrixes with a high level sensitivity and good selectivity and meets the current requirements with less cost to determine fipronil and its metabolites in maize and soil. The fipronil dissipation rates in soil and terminal residues in maize grain and stem were also studied to evaluate consumer and environmental safety and the
proper use of the pesticide. The results showed that the half-lives in soil were 9.90 and 10.34 days in Beijing and Shandong, respectively, under the field conditions. According to the results of the terminal residue study, when fipronil was used under the experimental design, the maximum residues in maize at intervals of 70 and 95 days after the pesticide application were far below the available MRLs, which were considered safe for consumption. This work would aid in the establishment of MRL value and the safe and proper use of fipronil in maize in China.
References Aajoud, A., Ravanel, P., & Tissut, M. (2003). Fipronil metabolism and dissipation in a simplified aquatic ecosystem. Journal of Agricultural and Food Chemistry, 51, 1347–1352. Aajoud, A., Raveton, M., Aouadi, H., Tissut, M., & Ravanel, P. (2006). Uptake and xylem transport of fipronil in sunflower. Journal of Agricultural and Food Chemistry, 54, 5055–5060. Amvrazi, E. G., & Albanis, T. A. (2009). Pesticide residue assessment in different types of olive oil and preliminary exposure assessment of Greek consumers to the pesticide residues detected. Food Chemistry, 113, 253–261. Balança, G., & de Visscher, M. N. (1997). Effects of very low doses of fipronil on grasshoppers and non-target insects following field trials for grasshopper control. Crop Protection, 16, 553–564. Bobé, A., Cooper, J. F., Coste, C. M., & Muller, M. A. (1998). Behaviour of fipronil in soil under Sahelian Plain field conditions. Pesticide Science, 52, 275–281. Gunasekara, A. S., Truong, T., Goh, K. S., Spurlock, F., & Tjeerdema, R. S. (2007). Environmental fate and toxicology of fipronil. Journal of Pesticide Science, 32, 189–199. Jiménez, J. J., Bernal, J. L., del Nozal, M. J., Martín, M. T., & Mayo, R. (2007). Comparative study of sample preparation procedures to determine fipronil in pollen by gas chromatography with mass spectrometric and electron-capture detection. Journal of Chromatography A, 1146, 8–16. Jiménez, J. J., Bernal, J. L., del Nozal, M. J., Martín, M. T., & Mayo, R. (2008). Sample preparation methods to analyze fipronil in honey by gas chromatography with electroncapture and mass spectrometric detection. Journal of Chromatography A, 1187, 40–45. Kadar, A., & Faucon, J. P. (2006). Determination of traces of fipronil and its metabolites in pollen by liquid chromatography with electrospray ionization-tandem mass spectrometry. Journal of Agricultural and Food Chemistry, 54, 9741–9746. König, A., Cockburn, A., Crevel, R. W. R., Debruyne, E., Grafstroem, R., Hammerling, U., et al. (2004). Assessment of the safety of foods derived from genetically modified (GM) crops. Food and Chemical Toxicology, 42, 1047–1088. Lin, K., Haver, D., Oki, L., & Gan, J. (2008). Transformation and sorption of fipronil in urban stream sediments. Journal of Agricultural and Food Chemistry, 56, 8594–8600.
Environ Monit Assess Mandal, K., & Singh, B. (2013). Persistence of fipronil and its metabolites in sandy loam and clay loam soils under laboratory conditions. Chemosphere, 91, 1596–1603. Maul, J. D., Brennan, A. A., Harwood, A. D., & Lydy, M. J. (2008). Effect of sediment-associated pyrethroids, fipronil, and metabolites on Chironomus tentans growth rate, body mass, condition index, immobilization, and survival. Environmental Toxicology and Chemistry, 27, 2582–2590. Miranda, M. C. S., & Mermut, A. R. (2007). Sorption of fipronil and its sulfide derivative by soils and goethite. Geoderma, 140, 1–7. Morzycka, B. (2002). Simple method for the determination of trace levels of pesticides in honeybees using matrix solidphase dispersion and gas chromatography. Journal of Chromatography A, 982, 267–273. Ozbey, A., & Uygun, U. (2007). Behaviour of some organophosphorus pesticide residues in peppermint tea during the infusion process. Food Chemistry, 104, 237–241. Raveton, M., Aajoud, A., Willison, J., Cherifi, M., Tissut, M., & Ravanel, P. (2007). Soil distribution of fipronil and its metabolites originating from a seed-coated formulation. Chemosphere, 69, 1124–1129. Saini, S., Rani, M., & Kumari, B. (2013). Persistence of fipronil and its metabolites in soil under field conditions. Environmental Monitoring and Assessment. doi:10.1007/ s10661-013-3356-3. Sánchez Brunete, C., Miguel, E., Albero, B., & Tadeo, J. L. (2008). Determination of fipronil residues in honey and
pollen by gas chromatography. Spanish Journal of Agricultural Research, 6, 7–14. Schlenk, D., Huggett, D. B., Allgood, J., Bennett, E., Rimoldi, J., Beeler, A. B., et al. (2001). Toxicity of fipronil and its degradation products to Procambarus sp.: field and laboratory studies. Archives of Environmental Contamination and Toxicology, 41, 325–332. Tewary, D. K., Kumar, V., Ravindranath, S. D., & Shanker, A. (2005). Dissipation behavior of bifenthrin residues in tea and its brew. Food Control, 16, 231–237. Tingle, C. C. D., Rother, J. A., Dewhurst, C. F., Lauer, S., & King, W. J. (2002). Fipronil: environmental fate, ecotoxicology and human health concerns. Reviews of Environmental Contamination and Toxicology, 176, 1–66. Vı́lchez, J. L., Prieto, A., Araujo, L., & Navalón, A. (2001). Determination of fipronil by solid-phase micro extraction and gas chromatography–mass spectrometry. Journal of Chromatography A, 919, 215–221. Ying, G. G., & Kookana, R. S. (2001). Sorption of fipronil and its metabolites on soils from South Australia. Journal of Environmental Science and Health. Part. B, 36, 545–558. Zhou, P., Lu, Y., Liu, B. F., & Gan, J. J. (2004). Dynamics of fipronil residue in vegetable-field ecosystem. Chemosphere, 57, 1691–1696. Zhuang, W. J. (2010). The global regulations on maximum residue limits (MRLs) for pesticides in food stuffs and feedstuffs. Beijing: Chemical Industry Press.
