Bull Environ Contam Toxicol (2015) 94:260–266 DOI 10.1007/s00128-014-1447-7
Determination of Residues of Fipronil and Its Metabolites in Cauliflower by Using Gas Chromatography-Tandem Mass Spectrometry Anil Duhan • Beena Kumari • Saroj Duhan
Received: 24 January 2014 / Accepted: 19 December 2014 / Published online: 1 January 2015 Ó Springer Science+Business Media New York 2015
Abstract Fipronil is a widely used insecticide with a welldescribed toxicological pathway. Recently it has been widely used in India to control vegetable pests. The present study has been carried out to observe the persistence pattern of fipronil and its metabolites—fipronil sulfone, fipronil sulfide, fipronil desulfinyl in cauliflower and soil so as to know the potential risk if any to consumers and environment. Fipronil was applied @ 56 g a.i. ha-1. Samples of cauliflower and soil were collected periodically; processed using QuEChERS method and analyzed by GCMS/MS. In cauliflower, residues of fipronil and its metabolites reached below detectable level before 30 days of application whereas in soil about 95 % of total fipronil residues got degraded within same time period. Washing and washing followed by cooking or boiling was found effective in reducing residues. A safe waiting period of 15 days is therefore suggested before consuming cauliflower. Keywords Cauliflower Fipronil Metabolites Residues GCMS/MS
A. Duhan (&) Agrochemicals Residues Testing Laboratory, Department of Agronomy, CCS Haryana Agricultural University, Hisar 125004, India e-mail: [email protected] B. Kumari Department of Entomology, CCS Haryana Agricultural University, Hisar 125004, India S. Duhan Department of Chemistry, Guru Jambheshwar University of Science and Technology, Hisar 125001, India
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India is the second largest producer of vegetables after China, but Indian vegetable’s export is very low because of high domestic requirement and other limitations like extensive crop devastation due to increased pest menace. The insect pests inflict crop losses to the tune of 40 % in vegetable production (Gaurav 2011; Sharma and Rao 2012). Cauliflower (Brassica oleracea var. Botrytis L.) is an important vegetable crop grown extensively in Asia. One of the major constraints in commercial growth of this crop is the heavy damage caused to its leaves and heads by large number of insect pests. The intensive insecticide application required to protect this crop against pests, are likely to leave residues on cauliflower heads and soil under crop cover, which may be hazardous to consumers (Raina and Raina 2008). Fipronil [±5 amino-1-(2,6-dichloro–a,a,a-trichlorop-tolyl)-4-trifluoromethylsulfinyl pyrazole-3-carbonitrile] is a phenyl pyrazole insecticide (Tomlin 2000). Due to its broad efficacy, it has been widely used for controlling major lepidopterous and orthopterous pests in vegetables and coleopterous larvae in soil. Its mode of action do not follow the common biochemical pathways of some classical insecticides like pyrethroids (sodium channel blockers), organophosphates and carbamates (cholinesterase inhibitors). Some insects have developed resistance against them (Aajoud et al. 2003). Fipronil (F) has a different mode of action as it prevent the inhibition of principal nerve transmitter amino butyric acid (GABA) receptors in insects (Cole et al. 1993), that is why it is also known as ‘new generation insecticide’. The chemical characteristics indicate that fipronil (F) and its three metabolites fipronil sulfone (FSO), fipronil sulfide (FS) and fipronil desulfinyl (FD) have different mobility and environmental fate. The degradation products of F are also very toxic. FD is 10 times, FSO 6.6 times and FS is 1.9 times more toxic than the parent compound (US
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EPA 1996). The metabolites FSO and FS are also more toxic to fresh water invertebrates (Pesticide Action Network-UK, PAN 2000). During application of pesticides on crop, less than 0.1 % of the pesticides applied for pest control reach their target pests. Thus, more than 99.9 % of pesticides used move into the environment where they adversely affect public health and beneficial biota, and contaminate soil, water and atmosphere of the ecosystem. Improved pesticide application technologies can improve pesticide use efficiency and protect public health and the environment. Now, the time has come when everyone is talking about residue free food as improved and strict guidelines from World Trade Organization and World Health Organization have demanded residue free food commodities in international market (WTO 2007; FAO/ WHO 2012). Most of the countries have taken serious concern and have implemented these guidelines in their foreign import and export policies. Therefore, the persistence behavior must be studied for different pesticides in crop and soil. In order to estimate the trace level residues, the role of modern analytical techniques and instruments like GCMS/MS with high sensitivity and accuracy cannot be neglected. Keeping this point in view, present study was carried out in order to assess the residues of F and its metabolites FSO, FS and FD in cauliflower and soil. Additionally, effect of processing like washing and washing followed by cooking or boiling have also been studied to know the role of these practices in reducing residues from the cauliflower head. Residues of F and its metabolites were also studied in wash water.
Materials and Methods The analytical standards of F (purity 97.5 %), FSO, FS and FD and formulation (Regent 80 WG, used for field application) were supplied by M/s Bayer Crop Science Limited, Mumbai, India. Solvents like acetone, dichloromethane, hexane and other chemicals were procured from Merck, Darmstadt, Germany. All the solvents were glass distilled before use so as to remove traces of non-volatile impurities, water and acids like acetic acid. Cauliflower crop was raised during October 2012 in University Research Farm, Department of Entomology, CCS Haryana Agricultural University Hisar, Haryana, India under recommended agronomical practices. There were three replications for each treatment, recommended dose of 56 g a.i. ha-1 and a control. In control plots, no insecticide was applied. The experiment was laid out in randomized block design having size of each plot as 23.5 m 9 6.8 m. Periodic sampling of cauliflower was done on 0 (2 h), 1, 3, 7, 10, 15 and 30 days after spray. In order to assess the effect of household processing, one part
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of the sample was subjected to washing for one min under tap water. Second part of the sample was first washed and then cooked. For this, 20 g of sample was taken in a 250 mL conical flask and cooked till softness (10–15 min) after adding 15 mL water to it. Soil samples were drawn in triplicate from top 15 cm of soil profile on 0 (2 h), 1, 3, 5, 7, 10, 15, 20 and 30 days of treatments at 6–8 different places from each plot in polythene bags and brought to the laboratory for further analysis. The samples of cauliflower heads were extracted and cleaned up by QuEChERS method with slight modification using acetone as extraction solvent (Pizzutti et al. 2007, 2009) in place of acetonitrile. The speed and user friendliness of the acetone extraction with comparable recoveries to acetonitrile motivated us for performing modification in above method. The samples were chopped into small pieces and after quartering, a representative 20 g sample was macerated with 5–10 g anhydrous sodium sulphate in mixer blender (Remi make, Mumbai, India). The macerated samples were taken in centrifuge tubes and added 20 mL acetone and rotary shaked at 50 r.p.m on a roto-spin for 1 h. The extracts were filtered though a Buchner funnel and concentrated to 10 mL on rotary evaporator. The acetone extract obtained in previous step was taken in centrifuge tube along with 4 g of anhydrous MgSO4 and 1 g of NaCl. The extract was centrifuged at 10,000 r.p.m. for 10 min. After centrifugation, 5 mL of supernatant was taken out and concentrated to 1 mL under a stream of nitrogen and subjected to clean-up by dispersive solidphase extraction using 75 mg primary secondary amine sorbent and 150 mg anhydrous MgSO4. The extract was again centrifuged for 5 min at 5,000 r.p.m. The supernatant was taken, concentrated to dryness and final volume of 2 mL was prepared in n-hexane for analysis by GCMS/MS. Similarly, samples of cauliflower after washing and washing followed by cooking or boiling were also processed. For extraction of F and its metabolites FSO, FS and FD in wash water, 250 mL of water was taken in a separatory funnel and 15 g of NaCl was added to it. The aqueous layer was partitioned with n-hexane thrice 50, 30 and 20 mL. The organic phases were collected, mixed together and concentrated up to 10 mL. Clean-up was done in similar manner as performed in cauliflower samples. Soil samples were extracted and cleaned-up as per method of Kumari et al. (2008). The samples were air dried, ground and sieved (2 mm) before use. About 20 g of soil was mixed with activated charcoal and florisil (0.3 g each) along with 10 g of anhydrous sodium sulphate before its compact packing in a glass column (60 cm 9 22 mm i.d.) in between two layers of anhydrous sodium sulphate. Residues were eluted with 125 mL of hexane: acetone (9:1 v/v) at flow rate of 2–3 mL min-1.
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Analysis of the F and its three metabolites were carried out at Agrochemicals Residues Testing Laboratory, Department of Agronomy, CCS Haryana Agricultural Universirty, Hisar, Haryana, India using GCMS/MS (Agilent 7890A series). The instrument was tuned properly before injection of standard of F and its metabolites. The operating parameters were: injection port temperature: 280°C. Column: HP-5 (30 m 9 0.32 mm i.d. 9 0.25 lm film thickness) containing 5 % diphenyl and 95 % dimethyl polysiloxane. Oven temperature ramping was: 70°C (2 min) ? @ 25°C min-1 ? 150°C (0 min) ? @ 15°C min-1 ? 200°C (0 min) ? @ 8°C min-1 ? 280°C (2 min). Detector: Mass 7000 GCMS/MS; detector parameters were: source temperature, 230°C; emission current, 35 lA; energy, -70 eV; repeller voltage, 11 V; ion body, 12 V; extractor, -7.2 V; ion focus, -7.4 V; quadrupole one (MS1) temperature, 150°C; quadrupole two (MS2) temperature, 150°C. Gas flow rates: helium (carrier gas), 1 mL min-1 though column and 2.25 mL min-1 as collision flow/quench flow, nitrogen (collision cell), 1.15 mL min-1. Other parameters: split ratio, 1:10; vacuum (high pressure), 2.23 9 10-05 torr; rough vacuum, 1.51 9 10?02 torr; injection volume, 2 lL. Under these
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conditions retention time of F was 26.3 min; FD, 23.1 min; FS, 26.1 min and FSO, 28.3 min (Fig. 1). The confirmation and quantification of F and its three metabolites was achieved by developing a programming in SCAN, product ion and finally multiple reaction monitoring (MRM). Characteristic ions with relatively high intensity and strong anti-turbulence were selected as monitoring and quantitative ions [Table 1; Fig. 1(2–5)].
Results and Discussion Different known concentrations of F, FSO, FS and FD were injected and measured the peak area resulting from the elution of the compound and its metabolites. Calibration curves plotted for concentration of the standard injected versus area for the standards of F, FSO, FS and FD were found linear within dynamic range of 0.001–1 lg mL-1. LOD and LOQ for F and its three metabolites were observed to be 0.001 and 0.003 lg mL-1 respectively. Recovery experiments were carried out to check the validity of the method in cauliflower, soil and water by fortifying the control samples of each matrix @ 0.25, 0.5
Fig. 1 1 F and its metabolites eluted at different retention times. 2 Mass-to-charge (m/z) of F parent compound; 3 FD; 4 FS and 5 FSO scanned alone in MRM
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Table 1 Programming parameters for MRM Compounds
Molecular mass
Precursor ion (m/z)
Collisions energies
Monitoring ions (m/z) and relative abundance (in brackets)
F
436
368
30, 20, 25, 30
213 (419.4) and 282 (748.6)
FD
388
388
35, 35, 10, 15
231 (629.4), 281 (1,485.6), 333 (3,117.4) and 368 (1,005)
FS
420
420
30, 35, 10, 15
351 (5,961)
FSO
452
383
35, 10, 15
213 (277.6), 255 (279.7) and 355 (16.5)
Table 2 Residuesa (mg kg-1) of F and its metabolites in cauliflower Days after treatment
F
FD
FS
FSO
Total F
Percent reduction of total F
0
0.959
0.422
0.067
0.166
1.61
-
1
0.768
0.195
0.045
0.075
1.08
32.9
3
0.597
0.155
0.035
0.039
0.83
48.4
7
0.307
0.083
0.030
0.021
0.44
72.7
10
0.165
0.045
0.023
0.010
0.24
85.0
15
0.060
0.004
0.006
0.003
0.08
95.0
30
BDL
BDL
BDL
BDL
BDL
100
Correlation coefficient r = - 0.996 Regression equation y = 3.18 - 0.082x tl/2 a
= 3.66 days
Average of thee replicates; BDL: 0.001 mg kg-1
and 1 lg mL-1 in triplicate. Implementing QuEChERS method with slight modification by using acetone as extraction solvent in place of acetonitrile in this study gave recoveries above 80 % in all samples. Pizzutti et al. (2007, 2009) in two studies on analysis of 169 pesticides by using acetone and acetonitrile as extraction solvent in QuEChERS observed both methods to be fast, efficient and robust, with good method performances showing good linearity of the calibration curves over the range from 0.1 to 10 ng. In cauliflower head, the average initial deposits on 0 day (2 h after application) of F, FD, FS and FSO have been shown in Table 2. Total F residues (sum of F and its three metabolites) were found to be 1.61 mg kg-1 which contained greater amount (0.959 mg kg-1) of F followed by other metabolites FD, FSO and FS as 0.422, 0.166 and 0.067 mg kg-1. It was observed that residues of F and its metabolites reduced with passage of time. Maximum reduction in total residues of F and other metabolites was observed to be 32.9 % after one day of application. Among various metabolites, FD was found higher followed by FSO and FS thereby showing faster degradation of FSO and FS. Degradation process of F occurs due to oxidation, reduction, hydrolysis and photolysis (Kumar and Singh 2013). Higher concentration of FD in comparison to other metabolites was mainly because of the photolytic degradation of F. FSO metabolite was supposed to be formed
due to oxidation process. Cauliflower head have rough surface and greater exposure to surface oxygen therefore involved greater oxidation by surface tissues due to greater availability of aerobic conditions. It may impart the higher concentration of FSO metabolite than FS. As cauliflower head do not have high moisture content; therefore the reduction process was less favored than oxidation and resulted in low conversion of F to FS. From the present study, it has been observed that the residues of all metabolites except F, reached below maximum residues limit of 0.02 mg kg-1 (EFSA 2012) on 15th day. Total reduction of insecticide up to day seven of treatment was to the extent of 72.7 % which reached to 100 % below detectable limit (BDL) of 0.001 mg kg-1 in 30 days. The half-life value of total F in unprocessed cauliflower head was found to be 3.66 days following first order kinetics. The results are fairly in agreement with the findings of Bhardwaj et al. (2012) who reported half-life of F as 3.43 and 3.21 days in cabbage at single and double dose, respectively. The authors also observed significantly higher presence of FD metabolite as compared to other metabolites in cabbage head. Dutta et al. (2008) observed that 76.4 % F residues dissipated in cabbage on 15th day with half-life of 7.5 days. Sunaina Saini (2012) observed that F and metabolites FSO, FS and FD reached BDL on 10th day in chilli with half-life period of 3.19 days. This may be due to the smooth surface of the chilli which prevented the inside
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movement of insecticide. On the other hand, cauliflower has its surface rough enough to facilitate the inside movement of the insecticides and resulting in longer persistence of residues. Mohaptra et al. (2010) reported residues of F in grapes leaves and berries degraded with halflife of 9.6 and 18.3 days and that of total F at 13.6 and 20 days at recommended and double the recommended dose, respectively. The authors also observed that the residues were having higher amount of F followed by FD, FSO and FS. Gupta et al. (2008) reported that in gram, F residues persisted beyond 7th day with half-life of 1.08 days at application rate of 50 g a.i. ha-1. They also reported 98 %–100 % dissipation of F in 7 days with halflife varying from 1.84 to 2.31 days in okra. Harvest time residues in paddy grain and straw were found to BDL by Kumari (2008). Kumar and Singh (2013) observed maximum concentration of F and its metabolites residues up to 7th day. Among metabolites, FSO was found as major one followed by FS, F amide and FD. Greater amount of FSO and FS was mainly due to greater oxidation reduction processes. On the other hand, low amount of FD was due to low photochemical degradation of F because of its immediate absorption by plants on application. Therefore, it can be concluded that the dissipation of F and its metabolites applied at different doses in different vegetables crops and fruits varied from each other. In soil samples, total F derived residues observed on 0 (2 h after application) day were found to be 0.098 mg kg-1 (Table 3). It constituted F (0.028 mg kg-1), FD (0.025 mg kg-1), FS (0.027 mg kg-1) and FSO (0.018 mg kg-1). The results showed that immediate oxidation–reduction and photolytic degradation resulted in greater conversion of F to FSO and FD. Residues of F and its metabolites reduced with passage of time. After 1, 3, 5, 7, 10, 15, 20 and 30 days of spray, the corresponding level of total F residues were found to be 0.062,
0.044, 0.029, 0.027, 0.020, 0.015, 0.008 and 0.005 mg kg-1. On reaching 30th day of application, the total F residues were found to be dissipated by 94.9 %. All other metabolites were found below detectable level on 30th day. Half-life of F alone was observed to be 5.55 days whereas half-life of total F was observed to be 2.59 days. It was observed that the FS residues on 1st day of application was 0.008 mg kg-1 and reached below detectable level on 5th day. Contrary to above, the rates of degradation of FD and FSO were slow. Residues of both metabolites on 15th day degraded to 68.0 and 66.7 %. Persistence of FD and FSO for more than 15 days showed that degradation process of F in soil occurred mainly through photolysis and oxidation. The results in this study showed slight variation in half-life period from the study performed by Sunaina Saini (2012) who observed the half-life of total F as 8.14 and 13.05 days at single and double dose, respectively with FS as the major metabolite. Mulrooney et al. (1998) reported that degradation of F in loamy soil was slow with half-life period of 34 days. Kumari (2008) observed biphasic first order degradation kinetics of F in soil under paddy crop at two application doses of 15 and 30 g a.i. ha-1 with half-life periods of 9.50 and 10.3 days, respectively. Harwood (2007) reported low persistence of F with 96.0 % loss at Narrandera and 67.0 % loss in Sydney, Australia. Ying and Kookana (2002) reported half-life period of all F components in soil up to 178 and 198 days at application dose of 0.15 and 0.075 g m-2. Fenet et al. (2001) reported rapid degradation of F in soil under tropical field conditions, which corroborate the findings of present study.
Effect of Processing The results (Table 4) obtained after washing of cauliflower head revealed that the initial deposits of F, FD, FS and FSO reduced by 32.7 %, 61.4 %, 62.7 % and 34.9 %,
Table 3 Residuesa (mg kg-1) of F and its metabolites in soil Days after treatment 0
F 0.028
FD 0.025
FS
FSO
Total F
0.027
0.018
0.098
1
0.022 (21.4)
0.023 (8.0)
0.008 (70.4)
0.009 (50.0)
0.062 (36.7)
3 5
0.017 (39.3) 0.015 (46.4)
0.017 (32.0) 0.008 (68.0)
0.003 (88.9) BDL
0.008 (55.6) 0.006 (66.7)
0.044 (55.1) 0.029 (70.4)
7
0.013 (53.6)
0.008 (68.0)
–
0.006 (66.7)
0.027 (72.4)
10
0.011 (60.7)
0.005 (80.0)
–
0.004 (77.8)
0.020 (79.6)
15
0.010 (64.3)
0.003 (88.0)
–
0.003 (83.3)
0.015 (84.7)
20
0.008 (71.4)
–
BDL
0.008 (91.8)
30
0.005 (82.1)
–
–
0.005 (94.9)
a
BDL –
For F for total F correlation coefficient r = - 0.966
For F for total F correlation coefficient r = - 0.974
t1/2 = 5.55 days
t1/2 = 2.59 days
Average of thee replicates; figures in parenthesis represent percent reduction
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Bull Environ Contam Toxicol (2015) 94:260–266 Table 4 Effect of processing on reduction of residuesa (mg kg-1) of F and its metabolites in cauliflower
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Days after treatment
F
FD
FS
FSO
Total F
0
0.645 (32.7)
0.163 (61.4)
0.025 (62.7)
0.108 (34.9)
0.941 (41.6)
1
0.310 (59.6)
0.058 (70.3)
0.014 (68.9)
0.025 (66.7)
0.407 (62.3)
3
0.156 (73.9)
0.033 (78.7)
0.009 (74.3)
0.009 (76.9)
0.207 (75.1)
7
0.102 (66.8)
0.036 (56.6)
0.018 (40.0)
0.017 (19.0)
0.173 (60.7)
10
0.098 (40.6)
0.022 (51.1)
0.014 (39.1)
0.009 (10.0)
0.143 (40.4)
15
0.025 (58.3)
BDL
0.004 (33.3)
BDL
0.029 (63.8)
30
BDL
BDL
BDL
BDL
BDL
Washing
Washing followed by cooking or boiling a
Average of thee replicates; BDL: 0.001 mg kg-1; figures in parenthesis are percent reduction
0
0.355 (63.0)
0.180 (57.3)
0.027 (59.7)
0.084 (49.4)
0.646 (59.9)
1
0.210 (72.7)
0.049 (74.9)
0.015 (66.7)
0.026 (65.3)
0.300 (72.2)
3
0.126 (78.9)
0.025 (83.9)
0.006 (82.9)
0.005 (87.2)
0.162 (80.5)
7
0.095 (68.1)
0.028 (66.3)
0.015 (50.0)
0.009 (57.1)
0.147 (66.6)
Table 5 Residuesa (lg mL-1) of F and its metabolites in wash water Days after treatment
F
FD
FS
FSO
Tota F
0
0.440 (54.1)
0.291 (31.0)
0.032 (52.2)
0.098 (41.0)
0.861 (46.5)
a
Average of thee replicates; BDL: 0.001 lg mL-1; figures in parenthesis are percent reduction
respectively on 0 day. Total residues comprising of F and its metabolites reduced by 41.6 %. It has been observed that the washing first increases the percent removal of residues and reached maximum to 75.1 % on 3rd day and then decreased regularly on proceeding days. This suggests that insecticide may have penetrated in the inner tissues of cauliflower head. Residues of both major metabolites FSO and FD reached to BDL of 0.001 mg kg-1 after washing on 15th day. Reduction of total residues on 15th day was again increased to 63.8 % if compared with 10th day samples where total reduction was only 40.4 %. It may be due to natural degradation of total Bhardwaj et al. (2012) reported washing effect on cabbage and revealed that metabolites of F dislodged quickly in comparison to its parent compound on 1st day and total F derived residues reduced to the extent of about 45.0 %–55.0 %. The results pertaining to the practice of washing followed by cooking or boiling of cauliflower head (Table 4) revealed about 59.9 % reduction of F and its metabolites on 0 day where as in washing practice, only 41.6 % reduction was observed on the same day. After 1, 3 and 7 days, 72.2, 80.5 and 66.6 % reduction of total residues comprising of F and metabolites FD, FSO and FS were observed. From the data it can be inferred that effect of washing followed by cooking or boiling was comparatively more effective than washing only. Further washing and cooking or boiling treatments were found to be very effective in removing 72.2 % and 80.5 % of residues on 1st and 3rd day than on
7th day (66.6 %). This may be due to penetration of insecticide into the interior parts of rough surfaced cauliflower head. Percent dissipation of F and its three metabolites were found with almost similar trend like that of washing process, which indicates that there was no special effect of cooking or boiling on dissipation of any particular metabolite. The amount of F and metabolites FD, FS and FSO on 0 day wash water was found to be 0.440, 0.291, 0.032 and 0.098 lg mL-1 thereby showing 54.1 %, 31.0 %, 52.2 %, 41.0 % reduction, respectively. Percent reduction of total residues was found to be 46.5 (Table 5). F degraded rapidly in water when exposed to light to form FD (Bobe et al. 1998). In this study, higher conversion of FS and FSO in comparison to FD may be attributed to greater oxidation– reduction of parent compound in water. F and its metabolite FD have less volatile nature which let them to concentrate under field conditions (Tomlin 2006) and may contaminate environment.
Conclusion The half-life periods of total F in cauliflower and soil were found to be 3.66 days and 2.59 days. About 95 % residues of total F got degraded after 30 days of application. Among the metabolites, residues of FSO and FD persisted for more than 15 days followed by FS. None of the F metabolites
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persisted after 20 days of application. Washing followed by cooking or boiling reduced residues of total F by 80.4 % whereas washing alone reduced 75.0 % residues in 3rd day cauliflower samples. From health safety point of view, application of F at studied dose seems to be safe before consumption of cauliflower as F and its metabolites reached below MRL before 30 days of application. In wash water, high amount of FS and FSO in comparison to FD was observed due to greater oxidation–reduction mediated conversion of parent compound.
References Aajoud A, Ravaneland P, Tissut M (2003) Fipronil metabolism and dissipation in a simplified aquatic ecosystem. J Agric Food Chem 51:1347–1352 Bhardwaj U, Kumar R, Kaur S, Sahoo SK, Battu RS, Singh B (2012) Persistence of fipronil and its risk assessment on cabbage Brassica oleracea var Capitata L. Ecotoxicol Environ Saf 79:301–308 Bobe A, Meallier P, Cooper JF, Coste CM (1998) Kinetics and mechanisms of a biotic degradation of fipronil (hydrolysis and photolysis). J Agric Food Chem 46:2834–2839 Cole LM, Nicholson RA, Casida JE (1993) Action of phenyl-pyrazole insecticides at the GABA-gated chloride channel. Pestic Biochem Physiol 46(1):47–54 Dutta D, Gopl M, Niwas R (2008) Persistence of fipronil and its metabolites in cabbage and soil. Pestic Res J 20(1):117–120 EFSA European Food Safety Authority (2012) Reasoned opinion on the review of the existing maximum residue levels (MRLs) for fipronil according to Article 12 of Regulation (EC). EFSA J 10(4):2688 FAO/WHO Food and Agriculture Organization of United Nation/ World Health Organization (2012) Pesticide residues in food 2012 report, joint FAO/WHO meeting on pesticide residues. http://www.fao.org/fileadmin/templates/agphome/documents/ Pests_Pesticides/JMPR/Report12/JMPR_2012_Report.pdf Fenet H, Beltran E, Gadji B, Cooper JE, Coste CM (2001) Fate of phenylpyrazole in vegetation and soil under tropical field conditions. J Agric Food Chem 49(3):1293–1297 Gaurav S (2011) Studies on lepidopterous insects associated with vegetables in Aravali range Rajasthan, India. Biol Forum Int J 3(1):21–26 Gupta S, Gajbhiye VT, Gupta RK (2008) Persistence of fipronil in/on okra, brinjal and gram. In: 1st international conference on agrochemicals protecting crop, health and natural environment held at New Delhi, 8–11 Jan 2008 Harwood MA (2007) Rapid degradation of termiticides under field conditions. Aust J Entomol 46(1):75–78
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Bull Environ Contam Toxicol (2015) 94:260–266 Kumar R, Singh B (2013) Persistence and metabolism of fipronil in rice (Oryza sativa L.) field. Bull Environ Contam Toxicol 90:482–486 Kumari B (2008) Degradation of fipronil in soil under paddy crop. In: IUPAC sponsored first international conference on agrochemicals protecting crop, health and natural environment, organized by Indian Agricultural Research Institute, Society of Pesticide Science India and Indian Council of Agricultural Research, held at New Delhi, 8–11 Jan, p 128 Kumari B, Madan VK, Kathpal TS (2008) Status of insecticide contamination of soil and water in Haryana, India. Environ Monit Assess 136:239–244 Mohaptra S, Deepa M, Jagdish GK, Rashmi N, Kumar S, Parkash GS (2010) Fate of fipronil and its metabolites in/on grape leaves berries and soil under semi arid tropical climatic conditions. Bull Environ Contam Toxicol 84:587–591 Mulrooney JE, Wollenbarger DA, Howard KD, Goli D (1998) Efficacy of ultra low volume and high volume application of fipronil against the boll weevil. J Cotton Sci 23:100–116 Pesticide Action Network- UK (PAN) (2000) Active ingredient fact sheet: fipronil. Pestic News Lond 48:20–22 Pizzutti IR, Kok AD, Zanella R, Adaime MB, Hiemstra M, Wickert C, Prestes OD (2007) Method validation for the analysis of 169 pesticides in soya grain, without clean up, by liquid chromatography-tandem mass spectrometry using positive and negative electrospray ionization. J Chromatogr A 1142(2):123–136 Pizzutti IR, Kok AD, Heimstra M, Wickert C, Prestes OD (2009) Method validation and comparison of acetonitrile and acetone extraction for the analysis of 169 pesticides in soya grain by liquid chromatography-tandem mass spectrometry. J Chromatogr A 1216:4539–4552 Raina A, Raina M (2008) Dissipation of chlorpyriphos on cauliflower (Brassica oleracea var. Botrytis L.). Pestic Res J 20:263–265 Sharma D, Rao DV (2012) A field study of pest of cauliflower cabbage and okra in some areas of Jaipur. Int J LifeSc Bt & Pharm Res 1(2):122–127 Sunaina Saini (2012) Persistence of fipronil and its metabolites in chilli and soil. PhD thesis. Department of Chemistry and Physics, CCS Haryana Agricultural University Hisar India Tomlin CDS (2000) The Pesticide Manual. The British Crop Protection Council, Surrey, pp 413–415 Tomlin CDS (2006) The Pesticide Manual, A World Compendium, 14th edn. British Crop Protection Council, Hampshire, pp 462–464 US Environmental Protection Agency (1996) New pesticide fact sheet: Washington, DC, US PB96-181516. EPA737-F-96-005. U.S. EPA Office of Prevention, Pesticides and Toxic Substances, pp 10 World Trade Organization (2007) Maximum residues levels for pesticides–Impact on exports from developing countries. http:// www.wto.org/english/newsm e/news07 e/sps 29june07 e.htm Ying G, Kookana R (2002) Laboratory and field studies on the degradation of fipronil in a soil. Aust J Soil Res 40:1095–1102
Determination of Residues of Fipronil and Its Metabolites in Cauliflower by Using Gas Chromatography-Tandem Mass Spectrometry Anil Duhan • Beena Kumari • Saroj Duhan
Received: 24 January 2014 / Accepted: 19 December 2014 / Published online: 1 January 2015 Ó Springer Science+Business Media New York 2015
Abstract Fipronil is a widely used insecticide with a welldescribed toxicological pathway. Recently it has been widely used in India to control vegetable pests. The present study has been carried out to observe the persistence pattern of fipronil and its metabolites—fipronil sulfone, fipronil sulfide, fipronil desulfinyl in cauliflower and soil so as to know the potential risk if any to consumers and environment. Fipronil was applied @ 56 g a.i. ha-1. Samples of cauliflower and soil were collected periodically; processed using QuEChERS method and analyzed by GCMS/MS. In cauliflower, residues of fipronil and its metabolites reached below detectable level before 30 days of application whereas in soil about 95 % of total fipronil residues got degraded within same time period. Washing and washing followed by cooking or boiling was found effective in reducing residues. A safe waiting period of 15 days is therefore suggested before consuming cauliflower. Keywords Cauliflower Fipronil Metabolites Residues GCMS/MS
A. Duhan (&) Agrochemicals Residues Testing Laboratory, Department of Agronomy, CCS Haryana Agricultural University, Hisar 125004, India e-mail: [email protected] B. Kumari Department of Entomology, CCS Haryana Agricultural University, Hisar 125004, India S. Duhan Department of Chemistry, Guru Jambheshwar University of Science and Technology, Hisar 125001, India
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India is the second largest producer of vegetables after China, but Indian vegetable’s export is very low because of high domestic requirement and other limitations like extensive crop devastation due to increased pest menace. The insect pests inflict crop losses to the tune of 40 % in vegetable production (Gaurav 2011; Sharma and Rao 2012). Cauliflower (Brassica oleracea var. Botrytis L.) is an important vegetable crop grown extensively in Asia. One of the major constraints in commercial growth of this crop is the heavy damage caused to its leaves and heads by large number of insect pests. The intensive insecticide application required to protect this crop against pests, are likely to leave residues on cauliflower heads and soil under crop cover, which may be hazardous to consumers (Raina and Raina 2008). Fipronil [±5 amino-1-(2,6-dichloro–a,a,a-trichlorop-tolyl)-4-trifluoromethylsulfinyl pyrazole-3-carbonitrile] is a phenyl pyrazole insecticide (Tomlin 2000). Due to its broad efficacy, it has been widely used for controlling major lepidopterous and orthopterous pests in vegetables and coleopterous larvae in soil. Its mode of action do not follow the common biochemical pathways of some classical insecticides like pyrethroids (sodium channel blockers), organophosphates and carbamates (cholinesterase inhibitors). Some insects have developed resistance against them (Aajoud et al. 2003). Fipronil (F) has a different mode of action as it prevent the inhibition of principal nerve transmitter amino butyric acid (GABA) receptors in insects (Cole et al. 1993), that is why it is also known as ‘new generation insecticide’. The chemical characteristics indicate that fipronil (F) and its three metabolites fipronil sulfone (FSO), fipronil sulfide (FS) and fipronil desulfinyl (FD) have different mobility and environmental fate. The degradation products of F are also very toxic. FD is 10 times, FSO 6.6 times and FS is 1.9 times more toxic than the parent compound (US
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EPA 1996). The metabolites FSO and FS are also more toxic to fresh water invertebrates (Pesticide Action Network-UK, PAN 2000). During application of pesticides on crop, less than 0.1 % of the pesticides applied for pest control reach their target pests. Thus, more than 99.9 % of pesticides used move into the environment where they adversely affect public health and beneficial biota, and contaminate soil, water and atmosphere of the ecosystem. Improved pesticide application technologies can improve pesticide use efficiency and protect public health and the environment. Now, the time has come when everyone is talking about residue free food as improved and strict guidelines from World Trade Organization and World Health Organization have demanded residue free food commodities in international market (WTO 2007; FAO/ WHO 2012). Most of the countries have taken serious concern and have implemented these guidelines in their foreign import and export policies. Therefore, the persistence behavior must be studied for different pesticides in crop and soil. In order to estimate the trace level residues, the role of modern analytical techniques and instruments like GCMS/MS with high sensitivity and accuracy cannot be neglected. Keeping this point in view, present study was carried out in order to assess the residues of F and its metabolites FSO, FS and FD in cauliflower and soil. Additionally, effect of processing like washing and washing followed by cooking or boiling have also been studied to know the role of these practices in reducing residues from the cauliflower head. Residues of F and its metabolites were also studied in wash water.
Materials and Methods The analytical standards of F (purity 97.5 %), FSO, FS and FD and formulation (Regent 80 WG, used for field application) were supplied by M/s Bayer Crop Science Limited, Mumbai, India. Solvents like acetone, dichloromethane, hexane and other chemicals were procured from Merck, Darmstadt, Germany. All the solvents were glass distilled before use so as to remove traces of non-volatile impurities, water and acids like acetic acid. Cauliflower crop was raised during October 2012 in University Research Farm, Department of Entomology, CCS Haryana Agricultural University Hisar, Haryana, India under recommended agronomical practices. There were three replications for each treatment, recommended dose of 56 g a.i. ha-1 and a control. In control plots, no insecticide was applied. The experiment was laid out in randomized block design having size of each plot as 23.5 m 9 6.8 m. Periodic sampling of cauliflower was done on 0 (2 h), 1, 3, 7, 10, 15 and 30 days after spray. In order to assess the effect of household processing, one part
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of the sample was subjected to washing for one min under tap water. Second part of the sample was first washed and then cooked. For this, 20 g of sample was taken in a 250 mL conical flask and cooked till softness (10–15 min) after adding 15 mL water to it. Soil samples were drawn in triplicate from top 15 cm of soil profile on 0 (2 h), 1, 3, 5, 7, 10, 15, 20 and 30 days of treatments at 6–8 different places from each plot in polythene bags and brought to the laboratory for further analysis. The samples of cauliflower heads were extracted and cleaned up by QuEChERS method with slight modification using acetone as extraction solvent (Pizzutti et al. 2007, 2009) in place of acetonitrile. The speed and user friendliness of the acetone extraction with comparable recoveries to acetonitrile motivated us for performing modification in above method. The samples were chopped into small pieces and after quartering, a representative 20 g sample was macerated with 5–10 g anhydrous sodium sulphate in mixer blender (Remi make, Mumbai, India). The macerated samples were taken in centrifuge tubes and added 20 mL acetone and rotary shaked at 50 r.p.m on a roto-spin for 1 h. The extracts were filtered though a Buchner funnel and concentrated to 10 mL on rotary evaporator. The acetone extract obtained in previous step was taken in centrifuge tube along with 4 g of anhydrous MgSO4 and 1 g of NaCl. The extract was centrifuged at 10,000 r.p.m. for 10 min. After centrifugation, 5 mL of supernatant was taken out and concentrated to 1 mL under a stream of nitrogen and subjected to clean-up by dispersive solidphase extraction using 75 mg primary secondary amine sorbent and 150 mg anhydrous MgSO4. The extract was again centrifuged for 5 min at 5,000 r.p.m. The supernatant was taken, concentrated to dryness and final volume of 2 mL was prepared in n-hexane for analysis by GCMS/MS. Similarly, samples of cauliflower after washing and washing followed by cooking or boiling were also processed. For extraction of F and its metabolites FSO, FS and FD in wash water, 250 mL of water was taken in a separatory funnel and 15 g of NaCl was added to it. The aqueous layer was partitioned with n-hexane thrice 50, 30 and 20 mL. The organic phases were collected, mixed together and concentrated up to 10 mL. Clean-up was done in similar manner as performed in cauliflower samples. Soil samples were extracted and cleaned-up as per method of Kumari et al. (2008). The samples were air dried, ground and sieved (2 mm) before use. About 20 g of soil was mixed with activated charcoal and florisil (0.3 g each) along with 10 g of anhydrous sodium sulphate before its compact packing in a glass column (60 cm 9 22 mm i.d.) in between two layers of anhydrous sodium sulphate. Residues were eluted with 125 mL of hexane: acetone (9:1 v/v) at flow rate of 2–3 mL min-1.
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Analysis of the F and its three metabolites were carried out at Agrochemicals Residues Testing Laboratory, Department of Agronomy, CCS Haryana Agricultural Universirty, Hisar, Haryana, India using GCMS/MS (Agilent 7890A series). The instrument was tuned properly before injection of standard of F and its metabolites. The operating parameters were: injection port temperature: 280°C. Column: HP-5 (30 m 9 0.32 mm i.d. 9 0.25 lm film thickness) containing 5 % diphenyl and 95 % dimethyl polysiloxane. Oven temperature ramping was: 70°C (2 min) ? @ 25°C min-1 ? 150°C (0 min) ? @ 15°C min-1 ? 200°C (0 min) ? @ 8°C min-1 ? 280°C (2 min). Detector: Mass 7000 GCMS/MS; detector parameters were: source temperature, 230°C; emission current, 35 lA; energy, -70 eV; repeller voltage, 11 V; ion body, 12 V; extractor, -7.2 V; ion focus, -7.4 V; quadrupole one (MS1) temperature, 150°C; quadrupole two (MS2) temperature, 150°C. Gas flow rates: helium (carrier gas), 1 mL min-1 though column and 2.25 mL min-1 as collision flow/quench flow, nitrogen (collision cell), 1.15 mL min-1. Other parameters: split ratio, 1:10; vacuum (high pressure), 2.23 9 10-05 torr; rough vacuum, 1.51 9 10?02 torr; injection volume, 2 lL. Under these
Bull Environ Contam Toxicol (2015) 94:260–266
conditions retention time of F was 26.3 min; FD, 23.1 min; FS, 26.1 min and FSO, 28.3 min (Fig. 1). The confirmation and quantification of F and its three metabolites was achieved by developing a programming in SCAN, product ion and finally multiple reaction monitoring (MRM). Characteristic ions with relatively high intensity and strong anti-turbulence were selected as monitoring and quantitative ions [Table 1; Fig. 1(2–5)].
Results and Discussion Different known concentrations of F, FSO, FS and FD were injected and measured the peak area resulting from the elution of the compound and its metabolites. Calibration curves plotted for concentration of the standard injected versus area for the standards of F, FSO, FS and FD were found linear within dynamic range of 0.001–1 lg mL-1. LOD and LOQ for F and its three metabolites were observed to be 0.001 and 0.003 lg mL-1 respectively. Recovery experiments were carried out to check the validity of the method in cauliflower, soil and water by fortifying the control samples of each matrix @ 0.25, 0.5
Fig. 1 1 F and its metabolites eluted at different retention times. 2 Mass-to-charge (m/z) of F parent compound; 3 FD; 4 FS and 5 FSO scanned alone in MRM
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Table 1 Programming parameters for MRM Compounds
Molecular mass
Precursor ion (m/z)
Collisions energies
Monitoring ions (m/z) and relative abundance (in brackets)
F
436
368
30, 20, 25, 30
213 (419.4) and 282 (748.6)
FD
388
388
35, 35, 10, 15
231 (629.4), 281 (1,485.6), 333 (3,117.4) and 368 (1,005)
FS
420
420
30, 35, 10, 15
351 (5,961)
FSO
452
383
35, 10, 15
213 (277.6), 255 (279.7) and 355 (16.5)
Table 2 Residuesa (mg kg-1) of F and its metabolites in cauliflower Days after treatment
F
FD
FS
FSO
Total F
Percent reduction of total F
0
0.959
0.422
0.067
0.166
1.61
-
1
0.768
0.195
0.045
0.075
1.08
32.9
3
0.597
0.155
0.035
0.039
0.83
48.4
7
0.307
0.083
0.030
0.021
0.44
72.7
10
0.165
0.045
0.023
0.010
0.24
85.0
15
0.060
0.004
0.006
0.003
0.08
95.0
30
BDL
BDL
BDL
BDL
BDL
100
Correlation coefficient r = - 0.996 Regression equation y = 3.18 - 0.082x tl/2 a
= 3.66 days
Average of thee replicates; BDL: 0.001 mg kg-1
and 1 lg mL-1 in triplicate. Implementing QuEChERS method with slight modification by using acetone as extraction solvent in place of acetonitrile in this study gave recoveries above 80 % in all samples. Pizzutti et al. (2007, 2009) in two studies on analysis of 169 pesticides by using acetone and acetonitrile as extraction solvent in QuEChERS observed both methods to be fast, efficient and robust, with good method performances showing good linearity of the calibration curves over the range from 0.1 to 10 ng. In cauliflower head, the average initial deposits on 0 day (2 h after application) of F, FD, FS and FSO have been shown in Table 2. Total F residues (sum of F and its three metabolites) were found to be 1.61 mg kg-1 which contained greater amount (0.959 mg kg-1) of F followed by other metabolites FD, FSO and FS as 0.422, 0.166 and 0.067 mg kg-1. It was observed that residues of F and its metabolites reduced with passage of time. Maximum reduction in total residues of F and other metabolites was observed to be 32.9 % after one day of application. Among various metabolites, FD was found higher followed by FSO and FS thereby showing faster degradation of FSO and FS. Degradation process of F occurs due to oxidation, reduction, hydrolysis and photolysis (Kumar and Singh 2013). Higher concentration of FD in comparison to other metabolites was mainly because of the photolytic degradation of F. FSO metabolite was supposed to be formed
due to oxidation process. Cauliflower head have rough surface and greater exposure to surface oxygen therefore involved greater oxidation by surface tissues due to greater availability of aerobic conditions. It may impart the higher concentration of FSO metabolite than FS. As cauliflower head do not have high moisture content; therefore the reduction process was less favored than oxidation and resulted in low conversion of F to FS. From the present study, it has been observed that the residues of all metabolites except F, reached below maximum residues limit of 0.02 mg kg-1 (EFSA 2012) on 15th day. Total reduction of insecticide up to day seven of treatment was to the extent of 72.7 % which reached to 100 % below detectable limit (BDL) of 0.001 mg kg-1 in 30 days. The half-life value of total F in unprocessed cauliflower head was found to be 3.66 days following first order kinetics. The results are fairly in agreement with the findings of Bhardwaj et al. (2012) who reported half-life of F as 3.43 and 3.21 days in cabbage at single and double dose, respectively. The authors also observed significantly higher presence of FD metabolite as compared to other metabolites in cabbage head. Dutta et al. (2008) observed that 76.4 % F residues dissipated in cabbage on 15th day with half-life of 7.5 days. Sunaina Saini (2012) observed that F and metabolites FSO, FS and FD reached BDL on 10th day in chilli with half-life period of 3.19 days. This may be due to the smooth surface of the chilli which prevented the inside
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movement of insecticide. On the other hand, cauliflower has its surface rough enough to facilitate the inside movement of the insecticides and resulting in longer persistence of residues. Mohaptra et al. (2010) reported residues of F in grapes leaves and berries degraded with halflife of 9.6 and 18.3 days and that of total F at 13.6 and 20 days at recommended and double the recommended dose, respectively. The authors also observed that the residues were having higher amount of F followed by FD, FSO and FS. Gupta et al. (2008) reported that in gram, F residues persisted beyond 7th day with half-life of 1.08 days at application rate of 50 g a.i. ha-1. They also reported 98 %–100 % dissipation of F in 7 days with halflife varying from 1.84 to 2.31 days in okra. Harvest time residues in paddy grain and straw were found to BDL by Kumari (2008). Kumar and Singh (2013) observed maximum concentration of F and its metabolites residues up to 7th day. Among metabolites, FSO was found as major one followed by FS, F amide and FD. Greater amount of FSO and FS was mainly due to greater oxidation reduction processes. On the other hand, low amount of FD was due to low photochemical degradation of F because of its immediate absorption by plants on application. Therefore, it can be concluded that the dissipation of F and its metabolites applied at different doses in different vegetables crops and fruits varied from each other. In soil samples, total F derived residues observed on 0 (2 h after application) day were found to be 0.098 mg kg-1 (Table 3). It constituted F (0.028 mg kg-1), FD (0.025 mg kg-1), FS (0.027 mg kg-1) and FSO (0.018 mg kg-1). The results showed that immediate oxidation–reduction and photolytic degradation resulted in greater conversion of F to FSO and FD. Residues of F and its metabolites reduced with passage of time. After 1, 3, 5, 7, 10, 15, 20 and 30 days of spray, the corresponding level of total F residues were found to be 0.062,
0.044, 0.029, 0.027, 0.020, 0.015, 0.008 and 0.005 mg kg-1. On reaching 30th day of application, the total F residues were found to be dissipated by 94.9 %. All other metabolites were found below detectable level on 30th day. Half-life of F alone was observed to be 5.55 days whereas half-life of total F was observed to be 2.59 days. It was observed that the FS residues on 1st day of application was 0.008 mg kg-1 and reached below detectable level on 5th day. Contrary to above, the rates of degradation of FD and FSO were slow. Residues of both metabolites on 15th day degraded to 68.0 and 66.7 %. Persistence of FD and FSO for more than 15 days showed that degradation process of F in soil occurred mainly through photolysis and oxidation. The results in this study showed slight variation in half-life period from the study performed by Sunaina Saini (2012) who observed the half-life of total F as 8.14 and 13.05 days at single and double dose, respectively with FS as the major metabolite. Mulrooney et al. (1998) reported that degradation of F in loamy soil was slow with half-life period of 34 days. Kumari (2008) observed biphasic first order degradation kinetics of F in soil under paddy crop at two application doses of 15 and 30 g a.i. ha-1 with half-life periods of 9.50 and 10.3 days, respectively. Harwood (2007) reported low persistence of F with 96.0 % loss at Narrandera and 67.0 % loss in Sydney, Australia. Ying and Kookana (2002) reported half-life period of all F components in soil up to 178 and 198 days at application dose of 0.15 and 0.075 g m-2. Fenet et al. (2001) reported rapid degradation of F in soil under tropical field conditions, which corroborate the findings of present study.
Effect of Processing The results (Table 4) obtained after washing of cauliflower head revealed that the initial deposits of F, FD, FS and FSO reduced by 32.7 %, 61.4 %, 62.7 % and 34.9 %,
Table 3 Residuesa (mg kg-1) of F and its metabolites in soil Days after treatment 0
F 0.028
FD 0.025
FS
FSO
Total F
0.027
0.018
0.098
1
0.022 (21.4)
0.023 (8.0)
0.008 (70.4)
0.009 (50.0)
0.062 (36.7)
3 5
0.017 (39.3) 0.015 (46.4)
0.017 (32.0) 0.008 (68.0)
0.003 (88.9) BDL
0.008 (55.6) 0.006 (66.7)
0.044 (55.1) 0.029 (70.4)
7
0.013 (53.6)
0.008 (68.0)
–
0.006 (66.7)
0.027 (72.4)
10
0.011 (60.7)
0.005 (80.0)
–
0.004 (77.8)
0.020 (79.6)
15
0.010 (64.3)
0.003 (88.0)
–
0.003 (83.3)
0.015 (84.7)
20
0.008 (71.4)
–
BDL
0.008 (91.8)
30
0.005 (82.1)
–
–
0.005 (94.9)
a
BDL –
For F for total F correlation coefficient r = - 0.966
For F for total F correlation coefficient r = - 0.974
t1/2 = 5.55 days
t1/2 = 2.59 days
Average of thee replicates; figures in parenthesis represent percent reduction
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Bull Environ Contam Toxicol (2015) 94:260–266 Table 4 Effect of processing on reduction of residuesa (mg kg-1) of F and its metabolites in cauliflower
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Days after treatment
F
FD
FS
FSO
Total F
0
0.645 (32.7)
0.163 (61.4)
0.025 (62.7)
0.108 (34.9)
0.941 (41.6)
1
0.310 (59.6)
0.058 (70.3)
0.014 (68.9)
0.025 (66.7)
0.407 (62.3)
3
0.156 (73.9)
0.033 (78.7)
0.009 (74.3)
0.009 (76.9)
0.207 (75.1)
7
0.102 (66.8)
0.036 (56.6)
0.018 (40.0)
0.017 (19.0)
0.173 (60.7)
10
0.098 (40.6)
0.022 (51.1)
0.014 (39.1)
0.009 (10.0)
0.143 (40.4)
15
0.025 (58.3)
BDL
0.004 (33.3)
BDL
0.029 (63.8)
30
BDL
BDL
BDL
BDL
BDL
Washing
Washing followed by cooking or boiling a
Average of thee replicates; BDL: 0.001 mg kg-1; figures in parenthesis are percent reduction
0
0.355 (63.0)
0.180 (57.3)
0.027 (59.7)
0.084 (49.4)
0.646 (59.9)
1
0.210 (72.7)
0.049 (74.9)
0.015 (66.7)
0.026 (65.3)
0.300 (72.2)
3
0.126 (78.9)
0.025 (83.9)
0.006 (82.9)
0.005 (87.2)
0.162 (80.5)
7
0.095 (68.1)
0.028 (66.3)
0.015 (50.0)
0.009 (57.1)
0.147 (66.6)
Table 5 Residuesa (lg mL-1) of F and its metabolites in wash water Days after treatment
F
FD
FS
FSO
Tota F
0
0.440 (54.1)
0.291 (31.0)
0.032 (52.2)
0.098 (41.0)
0.861 (46.5)
a
Average of thee replicates; BDL: 0.001 lg mL-1; figures in parenthesis are percent reduction
respectively on 0 day. Total residues comprising of F and its metabolites reduced by 41.6 %. It has been observed that the washing first increases the percent removal of residues and reached maximum to 75.1 % on 3rd day and then decreased regularly on proceeding days. This suggests that insecticide may have penetrated in the inner tissues of cauliflower head. Residues of both major metabolites FSO and FD reached to BDL of 0.001 mg kg-1 after washing on 15th day. Reduction of total residues on 15th day was again increased to 63.8 % if compared with 10th day samples where total reduction was only 40.4 %. It may be due to natural degradation of total Bhardwaj et al. (2012) reported washing effect on cabbage and revealed that metabolites of F dislodged quickly in comparison to its parent compound on 1st day and total F derived residues reduced to the extent of about 45.0 %–55.0 %. The results pertaining to the practice of washing followed by cooking or boiling of cauliflower head (Table 4) revealed about 59.9 % reduction of F and its metabolites on 0 day where as in washing practice, only 41.6 % reduction was observed on the same day. After 1, 3 and 7 days, 72.2, 80.5 and 66.6 % reduction of total residues comprising of F and metabolites FD, FSO and FS were observed. From the data it can be inferred that effect of washing followed by cooking or boiling was comparatively more effective than washing only. Further washing and cooking or boiling treatments were found to be very effective in removing 72.2 % and 80.5 % of residues on 1st and 3rd day than on
7th day (66.6 %). This may be due to penetration of insecticide into the interior parts of rough surfaced cauliflower head. Percent dissipation of F and its three metabolites were found with almost similar trend like that of washing process, which indicates that there was no special effect of cooking or boiling on dissipation of any particular metabolite. The amount of F and metabolites FD, FS and FSO on 0 day wash water was found to be 0.440, 0.291, 0.032 and 0.098 lg mL-1 thereby showing 54.1 %, 31.0 %, 52.2 %, 41.0 % reduction, respectively. Percent reduction of total residues was found to be 46.5 (Table 5). F degraded rapidly in water when exposed to light to form FD (Bobe et al. 1998). In this study, higher conversion of FS and FSO in comparison to FD may be attributed to greater oxidation– reduction of parent compound in water. F and its metabolite FD have less volatile nature which let them to concentrate under field conditions (Tomlin 2006) and may contaminate environment.
Conclusion The half-life periods of total F in cauliflower and soil were found to be 3.66 days and 2.59 days. About 95 % residues of total F got degraded after 30 days of application. Among the metabolites, residues of FSO and FD persisted for more than 15 days followed by FS. None of the F metabolites
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persisted after 20 days of application. Washing followed by cooking or boiling reduced residues of total F by 80.4 % whereas washing alone reduced 75.0 % residues in 3rd day cauliflower samples. From health safety point of view, application of F at studied dose seems to be safe before consumption of cauliflower as F and its metabolites reached below MRL before 30 days of application. In wash water, high amount of FS and FSO in comparison to FD was observed due to greater oxidation–reduction mediated conversion of parent compound.
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