Environmental Fate and Safety Management of Agrochemicals

Chapter 6

Quick Analysis of Fipronil and Its Metabolites in Gauze and Soil Samples

Downloaded by PENNSYLVANIA STATE UNIV on September 7, 2012 | http://pubs.acs.org Publication Date: March 8, 2005 | doi: 10.1021/bk-2005-0899.ch006

Sonia Campbell and Qing X. Li Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East West Road, Honolulu, HI 96822

This study focuses on the development of a quick method for the extraction and detection of fipronil residues and its main three metabolites in Hawaiian soil (Helemano series) and cotton gauze swipe samples. Pressurized fluid extraction was used for its ease of use and automated state, its reduction in organic solvent consumption, and time saving interests. Gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring mode was employed for the detection and quantification of the extracts. The extraction method was optimized for the Hawaiian soil for the simultaneous extraction of the four compounds, and was then applied to soil and cotton gauze samples collectedfromMaui, Hawaii, after a residential spray of fipronil.

Introduction Fipronil as an insecticide was first introduced by Rhone Poulenc Agro in 1993. It is also used for termite and fruit fly control in Australia and throughout the Pacific Region, but not registered yet in the U.S. for that use. Fipronil acts by blocking the chloride channels in the central nervous system (1) and is very effective in the case of flea control on pets (2,3) and wild animals (4). Its 62

© 2005 American Chemical Society In Environmental Fate and Safety Management of Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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activity for control of insects on crops such as sorghum and alfalfa has been evaluated and found comparable to other foliar insecticides (5). Fipronil is a choice candidate for pet and household treatment of pests because of its low mammalian toxicity (6). However, toxicity toward non-target species is becoming a concern. Anaphes Iole wasps (7), a biological control agent for the tarnished plant bug Lygus Lineolaris, and crayfish (8) in culture ponds located near fipronil treated ricefieldshave both shown to be highly sensitive to fipronil, thus, its residues should be monitored and managed adequately (9). Analytical methods for the detection of fipronil residues have been developed for such matrices as honeybees (10) using solid phase dispersion and gas chromatography (GC), and water and soil samples using solid phase microextraction (SPME) and GC-mass spectrometry (GC-MS) (11). Pressurized fluid extraction (PFE) is an extraction technique gaining in popularity due to its ease of use and automated state, its reduction in organic solvent consumption, and time saving. It is a prime extraction method for solid or semi solid samples (12). The objective of this work was to develop a quick and reliable method for the extraction and analysis of fipronil and its main three metabolites in the Hawaiian soil. The method was applied to soil and cotton gauze samples collected on the island of Maui, Hawaii, in March 2002 after a residential spray of fipronil.

Materials and Methods The Ottawa sand (20-30 mesh size) and extraction solvents (Optima grade) were purchased from Fisher Scientific (Pittsburgh, Pennsylvania). Fipronil ((±)-5amino-1 -(2,6-dichloro-a,a,a-tiifluoro-/7-tolyl)-4-trifluoromethylsulfinylpyra -3-carbonitrile) and the metabolites fipronil sulfide (A) [5-arnino-l-(2,6dichloro-α, a, a-trifluoro-/?-tolyl)- 4-trifluoromethylthiopyrazole-3-carbonitrile], fipronil sulfone (B) [5-amino-l-(2,6-dichloro-a, a, a-trifluoro-/7-tolyl)-4-trifluoromethylsulfonylpyrazole-3-carbonitrile], and desulfmyl fipronil (C) [5amino-l-(2,6-dichloro-ot, a, a-trifluoro-p-tolyl)- 4-trifluoromethylpyrazole-3carbonitrile] were the US EPA standards with respective purities of 99.4%, 99.8%, 99.7%, and 98.5%. A GC-MS system used was a series II 5890 GC interfaced with a 5989A MS from Hewlett Packard. The column was a DB5-MS (J&W Scientific, Folsom, California), 30 m in length, 0.25 mm i.d, and 0.25 μιη film thickness. The GCMS was operated in electron impact mode at 70 eV, with selected ion monitoring (SIM). The detailed operating conditions were injector temperature 250 °C, GCMS interface 280 °C, ion source 250 °C, the quadrupole 100 °C, and column 50 °C initially which was then ramped at 30 °C/min to 200 °C and held at 200 °C for

In Environmental Fate and Safety Management of Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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20 min. The ions monitored were 367 for fipronil, 351 for A, 383 + 213 for B, and 388 + 333 for C. A complete GC run took 26 min. PFE procedures for the soil were carried out in an Accelerated Solvent Extractor (ASE, Dionex Corporation, Salt Lake City, Utah). The extraction method was optimized by varying extraction temperature and number of extraction cycles. The extraction solvent was acetonitrile:methanol (1:1, v:v) and the pressure was kept constant at 1500 psi, which in previous studies pressure did not show a big influence on the extraction efficiency (13). The soil used for optimization was of the Helemano series (14), spiked in the laboratory by the organic solvent slurry method (15) at approximately 3 μg/g for each of the analytes. After extraction, the samples were filtered through sodium sulfate and 0.2 μπι nylon filter and concentrated with a gentle flow of nitrogen gas before GC-MS analysis. A portion of the spiked soil was kept in the dark at room temperature for 59 days in order to determine aging effects on extraction efficiency and the stability of the analytes in the soil. Each gauze was placed a 25 mL Nalgene tube with 10 mL of a mixture of acetone and acetonitrile (3:7, v:v). The tubes were capped and shaken for 60 min. The solvent extract of each sample was thenfilteredand reduced in volume prior to GC-MS analysis. Soil and gauze samples were collected in Kihei and at Camp Maluhia on the island of Maui, on March 21 and 22, 2003. The gauze samples were pre-wetted with acetone before the swipe; the soils were surface composite samples. The samples were were stored at 4 °C until they reached the laboratory where they were placed in a -20 °Cfreezeruntil analysis.

Results and Discussion Figure 1 presents the results of extraction optimization studies. It is known that fipronil is thermally labile, and thus, only extraction temperatures below 120 °C were investigated. Trials were run at 25, 50, 70, and 120 °C, with 1 or 2 extraction cycles that were 5 min for each cycle. The interest was to find a suitable condition for extraction of all four compounds simultaneously by one extraction procedure. Reproducibility of the extractions was good for all conditions examined. The standard deviations for the percentage of recoveries ranged from 2.0 for metabolite C at 70 °C and 1 cycle, to 12.2 for metabolite A at the same conditions.


65


For recovery of fipronil alone, the best extraction condition was 50 °C and 1 cycle with an average recovery of 70% ± 3 . At this condition, the recovery was 72% ± 3 for metabolite A, 63% ± 4 for B, and 67% ± 6 for C. The three metabolites showed a different optimum set of extraction parameters at 70 °C and 2 cycles, with improved recoveries of 94% ± 3 for metabolite A, 96% ± 10 for B, and 86% ± 2 for C. Fipronil recovery was then 60% ± 4.

A PFE condition of 70 °C and two 5-min extraction cycles appears to be a good choice, particularly when extraction time is a concern. When a limited amount of samples is available, one set of extractions can be performed with this procedure for the simultaneous analysis of fipronil and its metabolites. Table 1 shows the comparison of the average recoveries of the four analytes fortified in the soil and incubated for 1 day and 59 days. There was no significant difference between the 1-day and the 59-day periods. This indicates that the analytes do not degrade in the soil at the incubation conditions and are recoverable from the soil. However, the extraction method remains to be tested for long-term-aged soil. A representative GC-MS chromatogram of the four analyte standards and a mass spectrum of each are shown in Figure 2. The four analytes were well separated. The molecular ion peaks minus 1 (M - 1) for the metabolites A, Β and C are 420, 452, and 388, respectively. Fipronil did not give a molecular ion peak. +


66 Table 1. Average recoveries offiproniland its metabolites from fortified and aged soil


Analytes Fipronil Metabolite A Metabolite Β Metabolite C

2 months aging 75.8 % ± 10.6 91.6 % ± 9.9 90.5 % ± 11.5 75.6 % ± 11.6

/ day aging 60.3 % ± 4.2 93.1% ± 3.1 95.7 % ± 10.0 86.3 % ± 2.3

333

351

38δ|

255 77

420

213

JLmJL 367 369

213 77

L

I ι, i l l

420 .

383

77

213

255

.1

m/z

•ipronil

m/z

Β

6.0

8.0

10.0 12.0

14.0 16.0

18.0 20.0 22.0

454 335 452

24.0

26.0

Time (min) Figure 2. GC-MS chromatogram and mass spectra of fipronil and its metabolites. Fipronil 420 = (M-0-l) , Metabolite A 420 = (M-l) , Metabolite Β 452 = (M-l)\ Metabolite C 388 = (M-l)\ +

+

Table 2 shows the results for the field samples collected from Maui in March 2002. Five of the gauze samples showed presence of fipronil with amounts varying from 6.23 to 27.4 μg per gauze, two of those also containing the sulfone metabolite (B) in amounts of 13.7 and 14.3 μg per gauze. This metabolite is an oxidative product of fipronil. The soil sample labeled 032202-02 was a control which was collected outside of the area of fipronil use, and as such did not show fipronil or the metabolites. It


67 is interesting to note though that this sample contained significant amounts of the pesticide DDT and its metabolites. The soil sample collected within the area of the spray showed presence of fipronil at 16.1 μg/g and all the three metabolites from 0.54 to 1.70 μg/g.


In conculsion, this study offered a quick procedure to determine fipronil and its metabolites in fortified soil samples. It was applied successfully to native soil and cotton gauze swipe samples. Table 2. Quantification of soil and gauze samples (results expressed in μg per gauze or in μg/g of soil) Sample name 032102-01 032102-02 032102-03 032102-04 032102-05 032102-06 032102-07 032102-08 032202-01 032202-02

Sample type gauze gauze gauze gauze gauze gauze gauze gauze soil soil

Fipronil ND ND 7.15 6.23 7.58 27.4 7.27 ND 16.1 ND

A fago^g/g) fag ND ND ND ND ND ND ND ND 0.54 ND

Β C orMg/g) fag or iig/g) ND ND ND ND ND ND ND 13.7 ND ND ND 14.3 ND ND ND ND 0.59 1.70 ND ND

ND: not detected

Acknowledgements This study was supported, in part, by a grant from the State of Hawaii Department of Agriculture - Pesticides Branch, and by a contractual agreement with the State of Hawaii Department of Health - Office of Hazard Evaluation and Emergency Response.

References 1. Bloomquist, J.R. Annu Rev Entomol. 1996, 41, 163-190. 2. Payne, P.A., Dryden, M.W., Smith, V., Ridley, R.K. Vet Parasitol. 2001, 120,331-340. 3. Cardiergues, M.C., Caubet, C., Franc, M . The Veterinary Record. 2001, 149, 704-706.


68 4. 5. 6. 7. 8.


9. 10. 11. 12. 13. 14.

15.

Metzger, M.E., Rust, M.K. J Med Entomol. 2002, 39, 152-161. Al-Deeb, M.A., Wilde, G.E., Zhu, K.Y. J Econ Entomol. 2001, 94, 1353-1360. Hovda, L.R., Hooser, S.B. Small Animal Practice. 2002, 32, 455-467. Williams, L., Price, L.D., Manrique, V. Bio Control. 2003, 26, 217-223. Schlenk, D., Huggett, D.B., Allgood, J., Bennett, E., Rimoldi, J., Beeler, A.B., Block, D., Holder, A.W., Hovinga, R., Bedient, P. Arch environ Contamin Toxicol. 2001, 41, 325-332. Chaton, P.F., Ravanel, P., Tissut, M., Meyran, J.C. Ecotoxicol Environ Safety. 2002, 52, 8-12. Morzycka, B.J Chromatogr A. 2002, 982, 267-273. Vilchez, J.L., Prieto, Α., Araujo, L., Navalon, A. J Chromatogr A. 2001, 919, 215-221. Schantz, M., Nichols, J.J., Wise, S.A. Anal Chem. 1997, 69, 4210-4219. Zhu, Y., Yanagihara, K., Guo, F., Li, Q.X. J Agric Food Chem. 2000, 48, 4097-4102. USDASCS (U S. Department of Agriculture Soil Conservation Service). UHAES (University of Hawaii Agricultural Experiment Station). 1972, Soil Survey of Islands of Kauai, Oahu, Maui, Molokai and Lanai, State of Hawaii; U.S. Government Printing Office: Washington, DC. David, M.D., Campbell, S., Li, Q.X. Anal. Chem. 2000, 72, 3665-3670.


Environmental Fate and Safety Management of Agrochemicals

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