American Journal of Anal yt ical Chemistry, 2011, 2, 69-76
doi:10.4236/ajac.2011.228126 Published Online December 2011 (
Copyright © 2011 SciRes. AJAC
Persistence of Indoxacarb on Cauliflower (Brassica
oleracea var. botrytis. L.) and Its Risk Assessment
Reenu Takkar, S. K. Sahoo, Gurmail Singh, Kousik Mandal*, R. S. Battu, Balwinder Singh
Pesticide Residue Analysis Laboratory, Departme nt of Ent om ol o gy , Punjab Agricultural University,
Ludhiana, India
Received October 31, 2011; revised December 3, 2011; accepted December 11, 2011
A rapid, simple and an efficient method for the determination of indoxacarb in cauliflower and soil samples
was developed and validated using QuEChERS technique (Quick, Easy, Cheap, Effective, Rugged and Safe).
Recoveries at four different spiking concentrations of 0.01, 0.05, 0.1 and 0.2 mg·kg–1 ranged from 87% to 96%
were achieved with good repeatability and RSD of 1% - 6%. The average initial deposits of 0.23 and 0.45
mg·k g –1 were observed after last application of indoxacarb @ 52.2 and 104.4 g. a.i. ha–1 at recommended and
double the recommended dosages, respectively. The residues in cauliflower dissipated below its LOQ of 0.01
mg·k g –1 after 7 days and its half-life periods were observed to be 1.12 and 1.31 days, respectively, at single
and double the dosages. Keeping in view 80 g consumption of cauliflower curds per day for a 55 kg person,
theoretical maximum residue contribution (TMRC) of indoxacarb when calculated from maximum residues
observed on 0 day samples at recommended and double the recommended dosages, respectively, were found
to be 20.8 and 36.8 µg in comparison to its acceptable daily intake (ADI) of 550 µg, which is quite safe.
Keywords: Cauliflower, Indoxacarb, MRL, QuEChERS, Residue
1. Introduction
Indoxacarb, Methyl 7-Chloro-2,5-dihydro-2-[[(methoxy-
indeno[1,2-e][1,3,4]oxadiazine-4a(3H)-carboxylate, is a
non-systemic, synthetic organophosphate replacement
insecticide with broad spectrum activity [1,2]. This rela-
tively new pesticide was developed by E. I. du Pont de
Nemours and Company and was registered as Avaunt
and Steward in California, January 2001 [2]. Indoxacarb,
technical material is a white powered solid and it is for-
mulated as a wettable granule and soluble concentrate [3].
It is an oxadia zine insecticide that blocks the sodium
channels in insect nerve cells, causing lepidopteran lar-
vae to stop feeding within 4 hours, become paralyzed
and die within 2 to 5 days [4]. Indoxacarb is a highly
effective new insecticide has low side effect on non tar-
get insects [5,6] and allows most predators and immature
wasp parasites to survive [7,8] however, the wet resi
dues of indoxacarb are toxic to bees and adult wasp
parasites. It is considered as a reduced risk pesticide as
compared to that of conventional pesticide [9].
Cauliflower is one of the most important cruciferous
vegetable crops of India. It is widely cultivated through-
out the sub-tropical parts of north India. In Punjab, the
area under cauliflower was 5.59 thousand hactares with
the production of 131.40 thousand tonnes [10]. Cauli-
flower is low in fat, high in dietary fibers, contains water
and vitamin C, possessing a very high nutritional density
[11]. It contains other glucosinolates besides sulfuro-
phane, substances which may improve the livers ability
to detoxify carcinogenic substances. A high intake of
cauliflower has been found to reduce the risk of aggres-
sive prostate cancer [12]. One of the major constraints in
commercial growth of the crop is the severe damage
caused to its leaves and curds by insects pests such as
cabbage borer (Hellula undalis), leaf webber (Croci-
dolomia binotalis), diamond back moth (Plutella xylos-
tella), cut worm (Lepidoptera: Noctuidae), aphids (Li-
paphis psuedobrassicae), cabbage flea beetle (P. cru-
ciferae) and tobacco caterpillar (Spodoptera litura) re-
sulting in severe loss of quality and production [13,14].
Studies revealed from the field persistence test con-
firmed that indoxacarb were effective against P. xylos-
tella [15-18]. It is highly toxic and very strong ovicidal
action with LC50 value of 0.0064% against S. litura [19].
Indoxacarb has been used to control of leaf folder, fruit
borer in chillies [20] tomato fruit borer [21] and cabbage
looper [22] also. So, indoxacarb may play an important
role in integrated pest management programmes with its
novel mode of action, quick cessation of feeding, persis-
tence under field conditions and compatibility with natu-
ral enemies [17]. Keeping in view, the present studies
were undertaken to determine the residue dynamics and
final residues of indoxacarb on cauliflower and to find
out the safe period for harvest of the produce for con-
sumer safety under Punjab climate conditions at different
time intervals.
2. Experimental Sections
2.1. Materials
The organic solvents including acetonitrile and acetone
used were of HPLC grade and GR grade respectively,
purchased from Merck (Darmstadt, Germany). Sodium
chloride (ACS reagent grade 99.9%) and sodium sulfate
anhydrous (AR grade) were obtained from SD fine
chemicals, Mumbai, India. Analytical-grade MgSO4
(was purchased from Merck, India) were activated by
heating at 400˚C in muffle furnace for 3 hrs, cooled and
kept in a desiccators before use. Graphatised carbon and
primary secondary amine (PSA) were obtained from
Sigma Adrich and Varian, Mumbai, India, respectively.
2.2. Stock Solutions
Technical grade pesticide indoxacarb (99.9% purity)
was purchased from Dr. Ehrestorfer Augusburg, Ger-
many. The stock solution of indoxacarb containing 1000
μg·mL–1 of analyte was prepared using acetone as sol-
vent and kept at –20˚C. Working solutions of 0.01, 0.05,
0.1, 0.5, 1.0 and 5.0 µg·mL–1 concentrations were prepared
by serial dilutions and stored at 4˚C. These standard so-
lutions were used for fortification of the matrices and
instrument calibration purposes.
2.3. Field Trial
Field experiment was conducted during November 2009
- March 2010 following good agronomic practices at
Vegetable Research Farm, Punjab Agricultural Univer-
sity, Ludhiana, using randomized block design (RBD)
[10]. There were three replications for each treatment
and the plot size of each treatment was 100 m2 Figure
1 .Three applications of insecticide were made starting at
fruit initiation followed by second and third spraying at
10 days intervals with the help of Aspee Knapsack
sprayer equipped with hollow cone nozzle. The treat-
ments were untreated control (T1), single dose @ 52.2
(T2) and double dose @ 104.4 g a.i. ha–1 (T3). Untreated
control was sprayed with water only.
2.4. Sampling
About 5 - 6 cauliflower curds were collected at 0, 1, 3, 5,
7, 10 and 15 days after the last application of the insecti-
cide. The samples were collected at random from each
plot separately, ensuring the samples were reliable and
representative. Cauliflower curd samples were packed in
polyethylene bags and transported to the laboratory
where they were chopped and mixed thoroughly. The
soil samples of about 1 kg were collected from each plot
separately after 15 days following the last application.
Pebbles and other unwanted materials were removed
manually from field soil, air-dried, powered and pass
through a 3 mm sieve to achieve uniform mixing. Soil
samples were collected separately from 15 sites of each
treated plot with the help of tube auger at a depth of
about 10 - 15 cm; the soil from the 15 sites were pooled
and sieved, and extraneous matter, including stones/
pebbles, were removed. The texture and characteristics
@ 104.4 g a.i. ha–1)
@ 52.2 g a.i. ha–1)
@ 52.2 g a.i. ha–1)
@ 104.4 g a.i. ha–1)
@ 104.4 g a.i. ha–1)
@ 52.2 g a.i. ha–1)
Water channel
Figure 1. Layout of the field experiment.
Copyright © 2011 SciRes. AJAC
of field soil were sand 78.0%, slit 10.2%, clay 11.8%,
organic carbon 0.30%, EC 0.30 dsm–1 and pH 8.0.
2.5. Extraction and Clean Up Method
QuEChERS method with slight modifications was used
for the extraction and cleans up of cauliflower samples
for estimation of indoxacarb residues [23,24]. Cauli-
flower curd samples were macerated in a blender (Blixer
6 V.V. by robot coupe, France) and a representative 15 g
(±0.1 g) of the sample was transferred to 50 mL centri-
fuge tube. Added 30 mL of acetonitrile to each tube with
the help of dispenser and homogenized @ 15,000 rpm
for 2 - 3 min using a high speed homogenizer (High
Speed Silent Crusher-Heidolph) to ensure that the sol-
vent interacted well with entire sample. Mixed 5 - 10 g
NaCl to each tube and shook the tubes on rotaspin for 5
minutes followed by centrifugation for 3 min @ 2500
rpm. The acetonitrile layer was collected in another cen-
trifuge tube containing 5 g activated anhydrous sodium
sulfate and again shaken for 5 minute at 50 rpm on the
rotaspin to remove the moisture completely. The samples
were cleaned up by dispersive solid phase extraction
method. An aliquot of 6mL was dispensed into 15 mL
centrifuge tube containing 0.15 ± 0.01 g (PSA) primary
secondary amine sorbent ( to remove fatty acid among
other components) and 0.9 ± 0.01 g anhydrous MgSO4
(to reduce the remaining water in the extract) and 0.05 ±
0.01 g graphatised carbon. The tubes were vortexed for 3
minutes followed by centrifugation for 1 min @ 2500
rpm. From the upper layer of the prepared samples, 4mL
of the extract were transferred into another 15 mL tube
and were rotary evaporated at <35˚ to remove the ace-
tonitrile completely. Finally, the volume was reconsti-
tuted to 2 - 5 mL with distilled acetone. Soil samples
were processed by adding 10 mL of distilled water in 15
g of soil sample and rest of the procedure was same as
per the method described above.
3. Instrumental Analysis
The cleaned up extracts were analysed by Gas liquid
chromatograh (Perkin Elmer Clarus 500) equipped with
an electron capture detector (ECD) 63Ni operated at
310˚C. Chromatographic separation was carried out us-
ing a capillary column Elite 608 (50 m × 0.53 mm i.d,
1.5 μm film thicknesses) held at 290˚C with spilt ratio
1:10 was used for the estimation of indoxacarb residues.
The carrier gas flow was 2 mL·min–1 of high purity ni-
trogen with makeup flow of 30 mL·min–1. The injector
port and detector was held at 300˚C and 310˚C respec-
tively. Under these operating conditions the retention
time of indoxacarb was 6.20 minutes. Various concentra-
tions of standard solutions varying from 0.01 to 0.20
µl·mL–1 were prepared and 1 µL of each was injected to
prepare the standard curve.
The residues of indoxacarb on cauliflower curds were
confirmed by gas chromatograph-mass spectrometer
(GC-MS) in selective ion monitoring (SIM) mode. The
gas chromatograph (Shimadzu-QP 2010) with auto in-
jector, equipped with mass spectrometer and capillary
column Rtx-5 Sil MS (30 m × 0.25 mm i.d. × 0.25 μm
film thickness) was used to verify the results. The GC-
MS operating conditions were: injector temperature
285˚C, oven initial temperature was 200˚C and held for
4.0 min, raised to 280˚C at a rate of 10˚C·min1 and held
for 10 min, ion source temperature 200˚C, interface
temperature was 290˚C. Helium was used as a carrier gas
with a flow rate of 1.0 mL·min1.
4. Results and Discussions
4.1. Efficiency of Analytical Method
Validation is an essential requirement to ensure quality
and reliability of the results for all the analytical applica-
tions [25].The factors considered in the validation in-
cluded recovery, precision (relative standard deviation),
determination coefficient (R2), linearity, limit of detec-
tion (LOD) and limit of quantification (LOQ) [26-29]. In
order to evaluate the efficiency of extraction, cleanup
and determinative steps the analytical method was stan-
dardized by processing spiked samples, before taking up
the analysis of the test samples. The experiment was
performed by spiking the cauliflower and soil samples
with the pesticide being studied. The recoveries were
found to be consistent and more than 80% at different
concentration (0.01, 0.05, 0.1, 0.2 mg·kg1) with relative
standard deviation RSD below 15% confirmed a good
repeatability of the method shown in Table 1.
Table 1. Recovery studies and RSD values obtained for
indoxacarb in cauliflower and soil at different spiking lev-
Substrates Level of spiking
0.05 96 1
0.50 94 2
0.05 91 5
0.01 87 1
aEach value is mean of four replicate determinations; *Relative standard
Copyright © 2011 SciRes. AJAC
Further, linearity of the calibration curves was studied
using pesticide standard solution at concentration rang-
ing between 0.01 - 2 µg·mL1 in GC-ECD detector. The
response function was established to be linear. The cali-
bration curve that constructed followed linear relation-
ships with good correlation coefficients (R2 > 0.99) with
the equation of analytical graph was y = 3E + 06x – 7417
shown in Figure 2.
The LOQ of indoxacarb was found to be 0.01 mg·kg–1,
and no substrate interferences were observed at this de-
tection limit as evidenced by control samples analysis
shown in Figure 3. The limit of detection was deter-
mined as the concentration having a peak area three
times higher in relation to the noise of the base line at the
retention time of the peak of interest. The LOD was cal-
culated to be 0.003 mg·kg–1. Residues were estimated by
comparison of peak height/peak area of the standards
with that of the unknown or spiked samples run under
identical conditions. The average data on indoxacarb
residues were subjected to statistical analysis [30].
4.2. Residues of Indoxacarb in Cauliflower and
The average initial deposits of indoxacarb on cauliflower
were found to be 0.23 and 0.45 mg·kg–1 following the
last application of indoxacarb 14.5 SC @ 52.2 and 104.4
g a.i. ha–1 shown in Table 2. Residues of indoxacarb
Figure 2. Linearity calibration curve for indoxacarb at
concentration of 0.01 to 2 ng.
(a) (b)
(c) (d)
Figure 3. GC Chromatograms (a) Indoxacarb 0.02 ng standard; (b) Untreated cauliflower sample; (c) Spiked cauliflower
sample; (d) Field treated cauliflower sample.
Copyright © 2011 SciRes. AJAC
Table 2. Mean and range of indoxacarb residues (mg·kg–1) in cauliflower and soil at different time interval after the applica-
tion indoxacarb @ 52.2 and 104.4 g a.i. ha–1.
Time after application (Days) Indoxacarb @ 52.2 g a.i. ha–1 Indoxacarb @ 104.4 g a.i. ha–1
1 R
2 R
3 Mean ± SD R1 R
2 R3 Mean ± SD
0 day (1hr after application) 0.26 0.20 0.210.23 ± 0.03 0.430.460.46 0.45 ± 0.017
1 0.15 0.13 0.150.14 ± 0.01 (39.14)* 0.23 ± 0.01 (44.44)*
3 0.05 0.06 0.050.05 ± 0.002 (78.26)* 0.14 ± 0.04 (68.88)*
5 0.01 0.01 0.020.01 ± 0.001 (95.65)* 0.03 ± 0.002 (93.33)*
Soil sample after 15 days BDL BDL BDLBDL BDLBDLBDL BDL
#T1/2 days 1.12 1.31
()* Per cent dissipation after spraying; BDL < 0.01 mg·kg–1; #T1/2= Half-life time.
dissipated more than 78 and 68 percent after 3 days of
the last application at single and double the dosages re-
spectively. Sinha et al. [31] reported initial deposits of
indoxacarb residues of 0.11 and 0.21 mg·kg–1 on brinjal
following the application @ 70 and 140 g a.i. ha–1,
whereas, Gupta et al. [32] reported initial deposits of
0.26 and 0.67 mg·kg–1 on okra at the same dosages, re-
spectively. Though the dose used in case of brinjal is
higher than the dose used in cauliflower but the initial
deposits of indoxacarb residues in brinjal were low be-
cause of the nature of substrate.
The results of dissipation of indoxacarb in cauliflower
are shown in Figure 4. The residues of indoxacarb in
cauliflower dissipated below its LOQ of 0.01 mg·kg–1
after 7 days with half-life periods observed to be 1.12
and 1.31 days, respectively, at single and double the
dosages. The results were in agreement with Gupta et al.
[32] who reported the half-life of 1.6 and 2.3 days on
okra following application of indoxacarb @ 70 and 140 g
a.i. ha–1, respectively. However, indoxacarb residues
were below the determination limit of 0.01 mg·kg–1 in
the soil samples collected at harvest.
The residues of indoxacarb on cauliflower curds were
confirmed by gas chromatograph-mass spectrometer
(GC-MS) in selective ion monitoring (SIM) mode with
low concentration. The fragmentation of indoxacarb
produced selective m/z ions of 249, 293, 496, 529 and
base peak at 529 shown in Figure 5. The treated samples
also showed the presence of these ions which confirmed
the presence of indoxacarb.
4.3. Risk Assessment
The maximum residue limit (MRL) of indoxacarb on
cauliflower has been prescribed as 0.2 mg·kg–1 by Codex
[33]. The statistical analysis revealed that the residues of
indoxacarb on cauliflower dissipated below the MRL in
2.2 days. MRL for Indoxacarb under Indian condition is
not available to make an assessment of the results ob-
tained. Therefore, a risk assessment of indoxacarb resi-
dues on cauliflower was made on the basis of its total
intake through consumption of cauliflower and compar-
ing it to its acceptable daily intake (ADI). ADI of indox-
acarb is 0.01 mg·kg–1 body weight·day–1. Maximum Per
missible Intake (MPI) was obtained by multiplying the
ADI with the average weight (55 kg) of an Indian person
(Mukherjee and Gopal, 2000). MPI was calculated to be
550 μg·person–1·day–1. Theoretical Maximum Residues
Contribution (TMRC) has been calculated at considering
recommended consumption of cauliflower as 80 g in
Indian context [34]. The TMRC values were derived
through maximum residue level observed from recom-
mended the double the recommended dosages, respec-
tively, and were observed to be 20.8 and 36.8 µg, respec-
tively shown in Table 3. Both these values are signifi
Figure 4. Semi logarithm graph of indoxacarb showing
dissipation kinetics in cauliflower with Regression equation
y = –0.269x + 1.408 (single dose) and y = –0.232x + 1.673
double dose). (
Copyright © 2011 SciRes. AJAC
(a) (b)
(c) (d)
Figure 5. GC-MS Chromatograms (a) Standard indoxacarb; (b) Field treated caulifower sample; (c) Mass spectra of stan-
dard indoxacarb; (d) Mass spectra of indoxacarb field treated cauliflower sample.
Table 3. Theoretical maximum residue contribution (TMRC) in cauliflower curds.
Days Maximum residues in
cauliflower curds (mg·kg–1) in T1 TMRC μg·person–1·day–1 Maximum residues in
cauliflower curds (mg·kg–1) in T2 TMRC μg·person–1·day–1
0 0.26 20.8 0.46 36.8
1 0.15 12.0 0.25 20
3 0.06 4.8 0.19 15.2
5 0.02 1.6 0.03 2.4
10 - - - -
15 - - - -
BDL Below determination limit (<0.01mg·kg–1); T1 Indoxacarb@ 52.2 g a.i. ha–1; T2 Indoxacarb@ 104.4g a.i. ha–1.
cantly lower as compared to MPI. So, the dietary expo-
sure to indoxacarb is within health standard with in
safety zone and no health hazard is expected.
5. Conclusions
An analytical method for estimation of indoxacarb resi-
dues was standardised and validated. The residues of
indoxacarb dissipated below its LOQ of 0.01 mg·kg-1
after 7 days with half-life periods of 1.12 and 1.31 day,
respectively, at single and double the dosages. The statis-
tical analysis revealed that the residues of indoxacarb
dissipated below its prescribed MRL of 0.2 mg·kg–1 in
2.2 days. The TMRC values of indoxacarb were below
its acceptable daily intake (ADI) at both the application
rates when calculated from maximum residues levels
observed on 0-day. Hence the use of indoxacarb is safe
from crop protection point of view and residues in food
are unlikely to pose any undue hazard to the consumers.
6. Acknowledgements
Authors would like to thank Head, Depatment of ento-
mology, PAU Ludhiana for providing necessary research
Copyright © 2011 SciRes. AJAC
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