Ecotoxicology and Environmental Safety 88 (2013) 142–147

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Ecotoxicology and Environmental Safety

0147-65

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Dissipation of fipronil granule formulation in sugarcane field soil

Kousik Mandal n, Balwinder Singh

Pesticide Residue Analysis Laboratory, Department of Entomology, Punjab Agricultural University, Ludhiana-141004, Punjab, India

a r t i c l e i n f o

Article history:

Received 20 February 2012

Received in revised form

3 November 2012

Accepted 5 November 2012Available online 26 November 2012

Keywords:

Soil

Fipronil

Metabolism

GLC

GC–MS

Residues

13/$ - see front matter & 2012 Elsevier Inc. A

x.doi.org/10.1016/j.ecoenv.2012.11.006

esponding author. Fax: þ911612412359.

ail address: kousik11@gmail.com (K. Mandal)

a b s t r a c t

The dissipation and persistence of fipronil in soil was studied following application of fipronil (Regent

0.3 G) @ 75 and 300 g a.i. ha�1. The limit of quantification for the analysis of fipronil and its metabolites

by gas liquid chromatography was 0.001 mg kg�1 for soil. The total residues of fipronil and its

metabolites in soil after 7 days of its application @ 75 and 300 g a.i. ha�1 were found to be 0.025 and

0.098 mg kg�1, respectively. These residues could not be detected after 210 and 240 days following the

application of fipronil at lower and higher dosages, respectively. In soil, fipronil was found to be the

main constituent, followed by its metabolites sulfone, sulfide, amide and desulfinyl. The presence of

sulfone in significant higher amount as compared to other metabolites in the soil clearly demonstrates

that oxidation process plays a major role in the metabolism of fipronil. The half-life values (T1/2) of

fipronil were calculated to be 50.2 and 43.0 days, respectively when applied @ 75 and 300 g a.i. ha�1.

& 2012 Elsevier Inc. All rights reserved.

1. Introduction

Fipronil, 5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile is a phe-nyl pyrazole insecticide first synthesized by Rhone Poulenc AgCompany (now Bayer Crop Science) in 1987, introduced for use in1993, and registered in the U.S. in 1996 (Tomlin, 2000; Ware,2000; Tingle et al., 2003). In India, fipronil is marketed under thetrade names of Regent, Termidor and Jump. The compound can beeffectively delivered to the target pests via soil, foliar, bait or seedtreatment and is widely used to control many species of soil andfoliar insects on various crops such as rice, vegetables and fruits(Collins and Callcott, 1998; Balanca and del Visscer, 1997; Tomlin,1994; Bobe et al., 1998a). It is also used for control of termites(sugarcane, corn) and click beetles infesting cereals, corn andsunflower (Mulrooney and Goli, 1999) and locust (Steinbauer andPeveling, 2011). Recently, Regent 0.3 G (fipronil) @ 10 kg per acrehas been found to be quite effective against early shoot borer andtermites in sugarcane (Mann et al., 2009; Sharma et al., 2007).

It has also been recommended for use where the insects havedeveloped resistance to conventional insecticides like pyrethroids,organophosphates and carbamates (Bobe et al., 1997). Fipronil isregistered for non-agricultural as well as agricultural use in manycountries. Its application rate 0.6–200 g a.i. ha�1 is lower than theconventional insecticides, thus making it popular from environmen-tal safety point of view (Connelly, 2001). Fipronil has been shown to

ll rights reserved.

.

interfere with the passage of chloride ions through the g-aminobu-tyric acid regulated chloride channel, thereby disrupting centralnervous system activity and, at sufficient doses, causing death (Coleet al., 1993).

When a pesticide is applied it enters a hostile environment andis subjected to a wide range of biological (enzymes), chemical(hydrolysis) and physical (photolysis) reactions, which maychange its chemical nature. These new chemical structures, calledmetabolites or degradation products, have different inherent prop-erties than the parent pesticide and the effects of these changesneed to be assessed both in terms of environmental and humansafety. Metabolism studies are conducted to provide a detailedunderstanding of the fate and behavior of the pesticide in theenvironment (Skidmore and Ambrus, 2004).

Fipronil degrades to its major metabolities by reduction tosulfide MB45950 (Ramesh and Balsubramanian, 1999), oxidationto sulfone MB46136 (Bobe et al., 1998a), hydrolysis to amideRPA200766 (Bobe et al., 1998b; Ngim and Crosby, 2001) andphotolysis to desulfinyl MB46513 (Hainzl and Casida, 1996)(Fig. 1). The desulfinyl is extremely stable and actually moretoxic than the parent compound (USEPA, 1996). There are fourmain degradation products of fipronil and some of the metabo-lites are more toxic than the parent compound. The metaboliteMB 46513 is about 10 times more acutely toxic to mammals thanfipronil itself. The metabolite MB 46136 is highly toxic to birds,and the metabolites MB 46136 and MB 45950 are more toxic tofreshwater invertebrates than fipronil (Pesticide Action Network-UK (PAN), 2000).

The increasing number of pesticides used in agriculture hasacquired great importance due to the contamination of the

Fig. 1. Metabolites or degradation products of fipronil.

K. Mandal, B. Singh / Ecotoxicology and Environmental Safety 88 (2013) 142–147 143

environment. Degradation studies in soil are essential for evalua-tion of persistence of pesticides and their breakdown products.Data on the rate of pesticide degradation are extremely importantas they permit prediction of the potential risk associated withexposure.

Information on the nature and amount of pharmacologicalactive metabolites of fipronil in soil is necessary to ensure thesafety of the environment. Since, no information on the residues,metabolism, persistence and degradation of fipronil in soil isavailable under Indian conditions, therefore the present studieswere planned to know the fate of fipronil in soil.

2. Materials and methods

2.1. Chemicals and reagents

The technical grade analytical standards of fipronil MB-46030 (purity 97.5%),

sulfone MB-46136 (purity 99.7%), sulfide MB-45950 (purity 98.8%), desulfinyl MB-

46513 (purity 97.8%) and amide RPA-20076 (99.8%) were supplied by M/s Bayer

Crop Science India Ltd., Mumbai, India. Fipronil (Regent 0.3 G) formulation used

for field application was also obtained from M/s Bayer Crop Science India Ltd.,

Mumbai, India. Analysis of acetone extract of the formulation showed only

fipronil, and none of its metabolic products and no interfering peak was observed

under the retention time of the compound being estimated. Moreover, the

K. Mandal, B. Singh / Ecotoxicology and Environmental Safety 88 (2013) 142–147144

concentration of fipronil was found to be accurate with respect to its purity as

claimed by the manufacturers.

Solvents like acetone, dichloromethane and hexane were procured from

Merck, Darmstadt, Germany. Sodium chloride (ASC reagent gradeZ99.9%) was

also obtained from Merck, Darmstadt, Germany. Sodium sulfate anhydrous (AR

grade) and silica gel were from s. d. fine Chemicals and Qualigen, Mumbai,

respectively. All common solvents were redistilled in all-glass apparatus before

use. The suitability of the solvents and other chemicals was ensured by running

reagent blanks before actual analysis.

2.2. Preparation of standard solution

Standard stock solutions (1 mg/ml) each of fipronil and its metabolites were

prepared in acetone. The standard solutions required for plotting of calibration

curve (2.00, 1.50, 1.00, 0.50, 0.25 and 0.10 mg ml�1) were prepared from stock

solutions by serial dilution with acetone. All standard solutions were stored at

�4 1C before use.

2.3. Instrumentation

Analysis of the fipronil and its metabolites was carried out on gas liquid

chromatograph (GLC, Clarus 500) equipped with 63Ni electron capture detector

(ECD) supplied by M/S Perkin Elmer, Switzerland. A capillary column Rtx-5

(30 m�0.53 mm i.d. �0.25 mm film thickness of 5% phenylþ95% methyl poly-

siloxane) with splitless mode was used for estimation of fipronil and its

metabolites. Confirmation of fipronil and its metabolites was carried out on

a gas chromatograph (Shimadzu 2010) coupled with mass detector (Electron

Multiplier and Quader pole) equipped with capillary column (GCMS-QP 2010 plus,

Shimadzu, Rtx-5 Sil MS). The system software used was GCMS solution

version 2.5.

2.4. Field trial

2.4.1. Crop planting

The sugarcane (variety CoJ 88) was planted during the second week of

February, 2010 according to the recommended agronomic practices at University

Seed Farm, Ladhowal (3015802900 N, 7514701500 E), Punjab, India. There were three

replications for each treatment (i.e., control, recommended and four times the

recommended dosages) arranged in a randomized block design (RBD), and size

of the each plot was 500 m2. The soil under crop was of light texture with

low contents of organic matter. The other relevant properties of the soil were

organic carbon¼0.30%; pH¼8.0; sand¼78.0%; silt¼10.2%; clay¼11.8% and

EC¼0.30 dS m�1.

2.4.2. Application of the insecticide

Fipronil (Regent 0.3 G) was applied at recommended dose (75 g a.i. ha�1) and

four times the recommended dose (300 g a.i. ha�1) in the experimental plots, at

post-germination stage (45 days after planting). The insecticide was broadcasted

by mixing with sand to allow its uniform application.

2.4.3. Sampling

Soil samples were collected randomly from control and treated plots from

each treatment at 7, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240 and 270 days after

the application of insecticide. Soil samples were collected separately from 10 to 15

sites of each treated plot with the help of tube auger at a depth of about 10–

15 cm; the soil from each core was pooled and sieved, and extraneous matter,

including stones/pebbles, were removed. After thorough mixing, a subsample of

about 1 kg was taken from each pooled sample from each treatment plot and

transported to the laboratory. One part of the field sample was subjected to

extraction while another part was analysed to know the moisture content. The

results of fipronil residues in soil were expressed on dry weight basis.

2.5. Extraction and cleanup

A standardized analytical method as reported by Bhardwaj et al. (2012) was

followed for extraction and cleanup of fipronil and its metabolites. A representa-

tive 50 g sample of soil was extracted into 100 ml acetone in an Erlenmayer flask

by keeping it for 24 h. The extract was filtered into 1 l separatory funnel along

with rinsings of acetone. The filtrate in the separatory funnel was diluted with

600 ml brine solution (almost saturated sodium chloride solution), and partitioned

the contents two times into 100 ml dichloromethane and two times into 75 ml

hexane. Combined both the dichloromethane and hexane layers after passing

through anhydrous sodium sulfate and treated with 500 mg activated charcoal

powder for about 2–3 h at room temperature. The clear extract obtained was

filtered through Whatman filter paper No.1 and concentrated to near dryness

using rotary vacuum evaporator at o40 1C. The final volume was made upto 5 ml

using acetone.

2.6. Estimation by GLC

Analysis of fipronil and its metabolites was carried out using gas liquid

chromatograph (GLC) equipped with 63Ni ECD. The working conditions of GLC

were: injector temperature 290 1C, column initial hold temperature 200 1C for

5 min, followed by 270 1C for 3 min and detector temperature 320 1C. Carrier gas

(N2) flow was maintained at 30 ml min�1 with split ratio 1:10. Before use, the

column was primed with several injections of standard solution of fipronil till a

consistent response was obtained. Cleaned up samples were then injected into the

ECD mode of the detector. An injection volume of 2 ml was used in all experiments.

Under these operating conditions, the retention times of desulfinyl, sulfide,

fipronil, sulfone and amide were found to be 5.37, 7.75, 8.14, 11.15 and

15.93 min, respectively. The compounds in the sample were identified and

quantified by comparison of the retention times and peak heights of the sample

chromatograms with that of standards run under identical operating conditions.

2.7. Confirmation by GCMS

The confirmation of fipronil and its metabolites was done by gas chromato-

graph mass spectrometer (GC–MS) in single ion monitoring mode. Helium was

used as a carrier gas with a flow rate of 0.7 ml min�1. The GC–MS operating

conditions were: oven (program) initial temperature 150 1C and held for 2 min,

ramped 10 1C min�1 to 220 1C and held for 5 min, then again ramped 1 1C min�1

to 230 1C, held for 10 min; injector temperature was 280 1C and detector

temperature 290 1C. Injection volume was 1ml in splitless mode. Detector voltage

was maintained at 0.9 kV in SIM mode.

2.8. Efficiency of the method

In the present investigations recovery experiments were carried out at

different levels to establish the reliability and validity of analytical method and

to know the efficiency of extraction and clean up procedures. To judge the

efficacies of extraction and cleanup, the recovery experiments were performed.

Samples of soil from control plots of sugarcane were spiked at levels of 0.001,

0.005, 0.010, 0.050 and 1.00 mg kg�1. These were extracted, cleaned and analyzed

following the method already described. The control samples from untreated plots

and reagent blanks were also processed in the same way so as to find out the

interferences, if any, due to the substrate and reagents, respectively. The mean per

cent recoveries of fipronil and its metabolites viz. sulfone, sulfide, amide and

desulfinyl from soil samples at the fortification level of 0.100 to 0.001 mg kg�1

ranged from 85.1 to 97.3 (Table 1). The average recovery values were found to be

more than 85%, thus, the results have been presented as such without applying

any correction factor.

3. Results

3.1. Limit of detection (LOD) and limit of quantification (LOQ)

Half-scale deflection was obtained for 0.1 ng fipronil, whichcould be easily identified from the baseline and 0.02 ng of thecompound produced 10% deflection. When 50 g of soil wasextracted, cleaned up and final volume made to 5 ml, 2 ml ofsample (eqivalent 20 mg soil) when injected did not produce anybackground interference. Thus, limit of quantification (LOQ) andlimit of detection (LOD) of fipronil in soil were found to be 0.001and 0.0003 mg kg�1, respectively.

3.2. Persistence and metabolism of fipronil in soil

Maximum residues of fipronil and its metabolites were foundto be 0.025 mg kg�1 in soil samples collected 7 days after theapplication of fipronil @ 75 g a.i. ha�1. These residues declined to0.022, 0.018, 0.015 and 0.013 mg kg�1in the samples of soilcollected after 15, 30, 45 and 60 days of application of theinsecticide. The residues were further declined to 0.002 mg kg�1

after 180 days of application. The residues were found to bebelow the determination limit (0.001 mg kg�1) in the samples ofsoil collected at 210 days after the application of fipronil atrecommended dosage (Table 2). All the four metaboilites wereformed in soil upto 90 days. Sulfone metabolite was detected upto

Table 1Recovery (%) of fipronil and its metabolites from fortified samples of soil.

Level of fortification (mg kg�1) Fipronil Metabolites

Sulfone Sulfide Desulfinyl Amide

0.001 a,85.773.11 87.071.25 91.372.84 87.173.04 89.173.27

0.005 85.172.47 89.373.86 90.373.72 89.072.93 87.971.76

0.010 93.971.28 87.071.72 87.072.17 89.172.62 89.573.11

0.050 93.672.67 93.573.40 91.871.06 92.372.77 94.472.00

0.100 94.772.50 91.272.32 95.772.08 87.773.37 97.371.53

a Mean7Standard deviation.

Table 2Residues of fipronil and its metabolites (mg kg�1) in soil following application at 75 g a.i. ha�1.

Days after treatment Fipronil Metabolites Total fipronil

Sulfone Sulfide Desulfinyl Amide

7 a,0.01370.011 b,(52.0) 0.00470.001 (16.0) 0.00170.001 (4.0) 0.00470.000 (16.0) 0.00370.001 (12.0) 0.02570.002

15 0.01070.001 (45.5) 0.00370.001 (13.6) 0.00470.001 (18.2) 0.00370.001 (13.6) 0.00270.001 (9.1) 0.02270.001

30 0.00670.001 (33.3) 0.00570.001 (27.8) 0.00570.002 (27.8) 0.00170.001 (5.6) 0.00170.000 (5.6) 0.01870.002

45 0.00570.001 (33.3) 0.00370.001 (20.0) 0.00370.001 (20.0) 0.00270.001 (13.3) 0.00270.001 (13.3) 0.01570.001

60 0.00370.001 (23.1) 0.00570.001 (38.5) 0.00370.001 (23.1) 0.00170.000 (7.7) 0.00170.000 (7.7) 0.01370.001

90 0.00270.001 (22.2) 0.00470.001 (44.4) 0.00170.000 (11.1) 0.00170.000 (11.1) 0.00170.001 (11.1) 0.00970.002

120 0.00170.001 (14.3) 0.00470.001 (57.1) 0.00170.001 (14.3) 0.00170.000 (14.3) BDL 0.00770.001

150 0.00170.000 (20.0) 0.00370.001 (60.0) 0.00170.001 (20.0) BDL BDL 0.00570.001

180 cBDL 0.00270.001 (100.0) BDL BDL BDL 0.00270.001

210 BDL BDL BDL BDL BDL BDL

a Mean7Standard deviation.b (%) Dissipation of individual compounds.c BDL¼Below determination limit of 0.001 mg kg�1.

Table 3Residues of fipronil and its metabolites (mg kg�1) in soil following application @ 300 g a.i. ha�1.

Days after treatment Fipronil Metabolites Total fipronil

Sulfone Sulfide Desulfinyl Amide

7 a,0.05970.004 b,(60.2) 0.01370.001 (13.3) 0.00670.001 (6.1) 0.01570.002 (15.3) 0.00570.001 (5.1) 0.09870.005

15 0.03970.004 (39.8) 0.00970.001 (9.2) 0.00970.001 (9.2) 0.00370.001 (3.1) 0.00370.001 (3.1) 0.06370.003

30 0.02670.002 (59.1) 0.00770.002 (15.9) 0.00670.001 (13.6) 0.00370.001 (6.8) 0.00270.001 (4.6) 0.04470.003

45 0.01470.002 (35.9) 0.01470.001 (35.9) 0.00470.001 (10.3) 0.00270.001 (5.1) 0.00570.001 (12.8) 0.03970.003

60 0.00870.001 (22.2) 0.01670.001 (44.4) 0.00570.002 (13.9) 0.00370.001 (8.3) 0.00470.001 (11.1) 0.03670.001

90 0.00670.002 (20.0) 0.01270.002 (40.0) 0.00370.001 (10.0) 0.00470.001 (13.3) 0.00570.002 (16.7) 0.03070.004

120 0.00570.001 (17.9) 0.01670.002 (57.1) 0.00170.001 (3.6) 0.00270.001 (7.1) 0.00470.001 (14.3) 0.02870.001

150 0.00370.001 (17.7) 0.01070.002 (58.8) 0.00170.001 (5.9) 0.00170.000 (5.9) 0.00270.001 (11.8) 0.01770.001

180 0.00270.001 (28.6) 0.00570.001 (71.4) BDL BDL BDL 0.00770.001

210 0.00170.000 (33.3) 0.00270.001 (66.7) BDL BDL BDL 0.00370.001

240 cBDL BDL BDL BDL BDL BDL

a Mean7Standard deviation.b % dissipation of individual compounds.c BDL¼Below determination limit of 0.001 mg kg�1.

K. Mandal, B. Singh / Ecotoxicology and Environmental Safety 88 (2013) 142–147 145

180 days in soil. But no metabolites were found 210 days after theapplication of the insecticide.

The total residues of fipronil and its metabolites in soil after7 days of its application @ 300 g a.i. ha�1 were found to be0.098 mg kg�1. The corresponding levels were found to be 0.063,0.044, 0.039 and 0.036 mg kg�1 in the samples of soil collectedafter 15, 30, 45 and 60 days, respectively after application. All thefour metabolites (sulfone, sulfide, amide and desulfinyl) werefound in soil samples upto 150 days. Sulfone metabolite wasfound upto 210 days in soil. No residues of fipronil or itsmetabolites were detected in the samples collected 240 daysafter application of fipronil (Table 3). The residues of fipronil weremainly present in the form of parent compound. Among meta-bolites, the amount of sulfone was found to be maximum

followed by sulfide, amide and desulfinyl. Therefore, the degrada-tion process of fipronil in soil seems to occur through oxidation,reduction, photolysis and hydrolysis. The presence of sulfone inhigher amount as compared to other metabolites in the soilclearly demonstrates that oxidation process plays a major rolein the metabolism of fipronil.

4. Discussion

4.1. Persistence and metabolism of fipronil in soil

The above findings revealed that higher rate of application offipronil resulted in higher residues. As with the other insecticides,

K. Mandal, B. Singh / Ecotoxicology and Environmental Safety 88 (2013) 142–147146

the residues of fipronil on soil declined with time and fairly highrate of dissipation was observed. Total fipronil derived residuesobserved on 7-day soil samples when applied at 75 g a.i. ha�1

were found to be 0.025 mg kg�1. It constituted the parentcompound fipronil (52.0%), fipronil sulfone (16.0%), fipronil sul-fide (4.0%), fipronil desulfinyl (16.0%) and fipronil amide (12.0%).The parent compound and its metabolites decline gradually. Therate of persistence of fipronil sulfone and fipronil sulfide wereobserved to be quite high in comparison to the parent compoundfipronil or its metabolites fipronil desulfinyl and fipronil amide. Itwas further observed that after 150 days fipronil sulfone con-stituted 60.0% of the total fipronil derived residues.

Similarly, the observed total fipronil derived residues on soilwere found to be 0.098 mg kg�1 on 7-day after application at300 g a.i. ha�1. It constituted fipronil (60.2%), fipronil sulfone(13.3%), fipronil sulfide (6.1%), fipronil desulfinyl (15.3%) andfipronil amide (5.1%). The parent compounds and its metabolitesdeclined gradually. Metabolites fipronil sulfone and fipronil sulfidedissipated at a faster rate in comparison to fipronil or fipronildesulfinyl and fipronil amide. It was further observed that fipronilsulfone constituted 71.4% of the total fipronil derived residuesafter 180 days of the application. The findings are in contrast tothose of Fenet et al. (2001) who studied the fate of fipronil in soilunder tropical field conditions. Two different plots were treatedwith a formulation of fipronil at doses of 5 and 10 g a.i. ha�1,respectively. Soil at depths of 0–5 and 5–20 cm were sampled forup to 2 months after treatment. In soil, a rapid initial decrease offipronil was observed with a rapid formation of the sulfone andthe photodegradate; the amide and the sulfide were not detected.

However, the studies are in agreement with those of Kharbadeand Dethe, (2001) who evaluated the residues of fipronil insugarcane field soil. Fipronil 0.3 G was applied in soil at the dosesof 100, 200 and 400 g a.i. ha�1, 5 weeks after planting of sugarcane.Fipronil 5SC (soluble concentrate) was applied three times @ 100,200 and 400 g a.i. ha�1 at an interval of 15 days by initiating thefirst spray at about 2 months after plantation of sugarcane. The pre-harvest interval from soil application of granules and last spray wasabout 9.5 and 8.5 months, respectively. Residues of fipronil in soilwere found to be below the detectable limit of 0.01 mg kg�1.

Pei et al. (2004) studied the dynamics of fipronil residues invegetable field ecosystem. These results showed that degradationreaction occurred in soil was governed mainly by photodegrada-tion and oxidation accompanying with production of the meta-bolites, desulfinyl and sulfone. Reduction and hydrolysis playedlittle role in the degradation process. This might be revealed tothe low moisture conditions in vegetable fields. The fact that thequantity of desulfinyl was higher than sulfone strongly demon-strated that under the tested conditions, photodegradation playedthe major role in degradation of fipronil, while oxidation might be

Fig. 2. Semi-logarithm graph showing dissipatio

the second important factor and reduction and hydrolysis mightonly account for little effect.

Studies were conducted in rice fields using a fipronil granularformulation @ 40 g a.i. ha�1 just after transplanting, whichcorresponded to 20 kg ha�1 of formulated product. Soil analysisshowed that the amount of fipronil declined from 0.032 too0.009 mg kg�1 from one day to 14 days after application andafter 28 days, no analyte was left (Hadjmohammadi et al., 2006).Similarly, Dutta et al. (2008) studied the persistence of fiproniland its metabolites in soil following its application @ 0.05 and0.10 kg ha�1 in cabbage field. Initial concentration of fipronil insoil was 1.24 and 2.05 mg g�1, which dissipated to 0.15 and0.24 mg g�1 in 15 days, respectively, at low and high dose.Fipronil was found to get metabolized to sulfone and desulfinyljust after one day of its application. The concentration of fipronilsulfone was more as compared to desulfinyl. Fipronil sulfide wasfound in negligible amount in soil. The study showed that thefipronil desulfinyl and fipronil sulfone were reached their max-imum on 3rd day in soil samples.

4.2. Degradation dynamics of total fipronil residues on soil

The degradation kinetics of the fipronil and its metabolites insoil were determined by plotting residue concentration againsttime, and the maximum squares of correlation coefficients foundwere used to determine the equations of best fit curves. For all thesamples studied, exponential relations were found to apply,corresponding to first order rate equation. Confirmation of thefirst order kinetics was further made graphically from thelinearity of the plots of logC against time (C¼residues�1000;Fig. 2). Total fipronil residues were not followed the first orderkinetics. But these residues were followed the pseudo first orderkinetics (R2) with 0.960 and 0.909 for recommended dose andfour times the recommeded dose, respectively. Half-life (T1/2) offipronil calculated as per Hoskins (1961) was observed to be 50.2and 43.0 days, respectively when applied at 75 and 300 ga.i. ha�1.

Similarly, the dissipation of fipronil followed pseudo first orderkinetics (r2

¼0.85–0.95) for the various formulations applied in afield (Ngim and Crosby, 2001). The residues of fipronil dissipatedalmost completely in 90 days following application of fipronil(Regent 0.3 G) @ 56 and 112 g a.i. ha�1 in cotton field. Kineticstudies revealed that dissipation of fipronil followed first orderkinetics with half-life period of 23.35 and 24.31 days at singleanddouble dose, respectively (Chopra et al., 2011). The half-life offipronil and its metabolites in soil indicate that they are persis-tent; degradation typically ranges from 111 to 350 days depend-ing upon soil conditions (Gunasekara et al., 2007).

n kinetics of total fipronil residues on soil.

Table 4The molecular mass, retention time, mass spectra of fipronil and its metabolites.

Fipronil and its metabolites Structure Code no. Molecular mass Retention time (Rt) (m/z)

Desulfinyl C12H4Cl2F6N4 MB-46513 389.1 11.8 69, 179, 213, 281, 333, 369, 388

Sulfide C12H4 Cl2 F6 N4S MB-45950 421.1 15.8 69, 213, 228, 255, 351, 420

Fipronil C12H4Cl2F6N4OS MB-46030 437.1 16.4 351, 367

Sulfone C12H4 Cl2 F6 N4O2S MB-46136 453.1 19.7 69, 213, 255, 355, 383

Amide C12H6 Cl2 F6 N4O2S RPA-20076 455.1 24.1 44, 69, 255, 368, 385

K. Mandal, B. Singh / Ecotoxicology and Environmental Safety 88 (2013) 142–147 147

4.3. Confirmation by GCMS

The residues were confirmed on electron ionization (EI) mode.The compounds were identified based on m/z ratio of total ionchromatograph (TIC) and fragmentations of selective ions mon-itoring (SIM) compared with fragmentations of different massnumbers obtained with standard fipronil and its metabolites(Table 4). The compounds were identified both in total scan andSIM mode based on m/z ratio. The sensitivity of the instrumentincreased in SIM mode as the most abundant fragment ions,characteristics of the analyte, were counted. The mass spectra ofstandard fipronil and its metabolites showed the most abundantions as its base peak.

5. Conclusions

Fipronil, a novel phenyl pyrazole has an outstanding long terminsecticidal activity and effective against early shoot borer andtermite on sugarcane. In soil, fipronil was found to be the mainconstituent, followed by its metabolites sulfone, sulfide, amideand desulfinyl. These residues declined to below the determina-tion limit (0.001 mg kg�1) after 210 and 240 days followingapplication of fipronil at 75 and 300 g a.i. ha�1, respectively.The presence of sulfone in higher amount as compared to othermetabolites in the soil clearly demonstrates that oxidation pro-cess plays a major role in the metabolism of fipronil.

Acknowledgments

The authors are thankful to the Professor and Head, Depart-ment of Entomology, Punjab Agricultural University, Ludhiana forproviding the necessary research facilities.

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