Journal of Environmental Protection, 2011, 2, 424-428
doi: 10.4236/jep.2011.24048 Published Online June 2011 (http://www.SciRP.org/journal/jep)
Harvest Residue Study of Fungicide Tebuconazole
EC Formulation in Groundnut and Paddy
Chiranjit Kundu, Arnab Goon, Anjan Bhattacharyya
Pesticide Residue Laboratory, Department of Agricultural Chemicals, West Bengal, India.
Received November 10th, 2010; revised February 7th, 2011; accepted March 12th, 2011.
ABSTRACT
A field trial was conducted under West Bengal condition during July 2009 to October 2009 to evaluate the harvest
residue of Tebuconazole (25.9% EC) in paddy at two application rates (750 and 1500 mLha–1). Another field tria l was
conducted during August 2009 to December 2009 to evaluate the harvest residue of the same molecule in groundnut.
The quantitative analysis of the fungicide residue was performed using Liquid Chromatography-Mass Spectrometry
(LC-MS/M S). The average recovery was found in between 86.33% to 91.87% for different substrates o f groundnut. In
case of paddy the average recovery was ranges in between 86.40% to 90.86% for different substrates. In a ll the cases, it
was found that the fung icide residue s were below the detection lim it of the instrum ent (<0.01 ppm) irrespective of doses
in different substrates of paddy and groundnut. Based on these findings, the use of Tebuconazole in paddy and
groundnut may be advocated for the control of diseases in paddy and groundnu t without any residual toxicity problem.
Keywords: Control, Fungicides, Pyricularia Oryzae, Rice Blast, Oryza Sat i va L
1. Introduction
Rice (Oryza sativa), one of the three most important food
crops in the world, forms the staple diet for 2.7 billion
people [1]. It is grown in all the continents except
Antarctica, occupying 150 million ha, producing 573
million tonnes paddy with an average productivity of
3.83 tonnesha–1 [2]. More than 70% grain loss may occur
in India as a result of rice blast disease caused by fungus
Pyricularia or yzae [3].
Similarly groundnut is one of the most important
oilseed crop grown in wide range of soil and climate.
Leaf spot is one of the most important diseases of
groundnut caused by fungus Sclerotium rolfsii, causes
significant yield losses under Indian climatic condition
[4].
Tebuconazole [IUPAC Name: (RS)-1-(4-chloro-
phenyl)-4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)-pe
ntan-3-ol. Ratio (1:1)], a triazole fungicide used as a seed
dressing chemical is a systemic fungicide with protective,
curative and eradicant action and acts by inhibiting the
demethylation of steroids. Used as a spray, it controls
numerous pathogens in various crops out of which leaf
spot of groundnut, blast disease of paddy are major one
[4]. Tebuconazole when applied in the form of fungicide
accumulated in surface layers of soil and became toxic to
susceptible plants. Also it is rapidly absorbed by
vegetative parts of the plants with translocation
principally acropetally. The objective of the present work
was to study the harvest residue of Tebuconazole EC
formulation in groundnut and paddy.
N
N
N
OH
Cl
Chemical structure of Tebuconazole.
2. Materials and Methods
A field study was conducted at University Experimental
Field, Mohanpur, BCKV, West Bengal, India, during
July 2009 to October 2009 on paddy [variety-Khitish].
Another field study was conducted at University
Experimental field, Gayeshpur, BCKV, West Bengal,
India, during August 2009 to December 2009 on
groundnut [variety-JL-24]. The formulation Tebucona-
zole 25.9% EC was applied with the help of knapsack

Harvest Residue Study of Fungicide Tebuconazole EC Formulation in Groundnut and Paddy425
sprayer equipped with WFN 40 nozzle @ 750 mLha–1
(T1) and @ 1500 mLha–1 (T2) in Randomized Block
Designed (RBD) plots and maintained untreated control
(T3) plots. Three replications were used for each
treatment. The area of every plot was 20 m2. Spraying of
fungicide was done twice at 15 days interval both for
paddy and groundnut.
2.1. Collection of Samples and Processing
Different substrates of groundnut (groundnut cropped
soil, groundnut plant and groundnut) were collected at
the time of harvest following standard sampling
procedures. Also different substrates of paddy (paddy
plant and paddy cropped soil) were collected at the time
of harvest following the same procedure. Groundnut
plant, paddy plant, groundnut (0.10 kg) and field soil
(0.25 kg) samples were collected from 5 - 7 places
randomly from each treatment plots. Samples from
untreated control plots were also collected in the same
way. Soil samples were collected from a depth of 6″ with
the help of soil auger.
3. Residue Analysis
3.1. Extraction and Clean Up of Samples
Plant samples ( Paddy stra w/Paddy g rain/Hu sk/Ground-
nut plant):
The samples were blended using Polytron homogen-
izer. In each case five gram (5 g) of the homogenized
sample was taken in a 50 mL centrifuge tube and 10 mL
(Ethyl Acetate: Cyclohexane = 9:1) mixture was added
and subjected to vortex for 2 min. After that added 5 gm
of activated Na2SO4, the sample was again vortex for
3 min. Then the sample was centrifuged for 15 min at
10,000 rpm and then 5 ml supernatant liquid was taken in
10 ml centrifuge tube. Afterwards 25 mg florisil & 25 mg
PSA was added to it and vortex for 2 min and the sample
was again centrifuged for 10 min at 5000 rpm. Then 3 ml
supernatant liquid was collected from it and evaporated
to dryness in a N2–Evaporator at 25˚C. The residue was
then reconstituted in 3 ml of [MeOH: H2O (9:1, v/v) + 5 mM
CH3COONH4] and subsequently filtered through 0.2 µ
membrane filter. Now the sample is ready for the final
analysis with LC-MS/MS.
Groundnut Oi l:
Deshelled groundnut sample (50 g) was grinded in a
grinder and was subjected to Soxhlet extraction with 150
mL of hexane for 6 hours. The extracted oil was
collected and the rest portion (deoil cake) was kept
separately. The hexane extract was concentrated in rotary
vacuum evaporator below 40˚C. The oil thus obtained
was collected and from it 1 g of oil was weighed and was
subjected to extraction. The oil taken was redissolved in
100 mL of hexane and was partitioned thrice (100 + 50 +
50) with acetonitrile. The acetonitrile fraction was
collected over anhydrous Na2SO4 and the organic phase
was evaporated in a rotary vacuum evaporator below
40˚C and was subjected to column clean up. The oil
sample thus obtained was cleaned up using silica gel
column conditioned with hexane. The sample was
applied in the column and kept for 15 min. It was then
eluted with 50 mL hexane and discarded. Then 100 mL
toluene was passed through the column and the fraction
was collected and concentrated in a rotary vacuum
evaporator below 40˚C. Finally, it was reconstituted with
[MeOH: H2O (9:1, v/v) + 5mM CH3COONH4] which is
ready for analysis in LC-MS/MS.
Groundnut Deoil cake:
The deoil cake (10 g) obtained from the oil extraction
step was analysed by following same procedure as
described for groundnut plant samples.
Soil (Both paddy and groundnut cropped soil):
Five gram (5 g) soil was taken in a 50 mL centrifuge
tube & similar method as mentioned in plant samples
was followed.
Instrumentatal Parameters:
Chromatographic condition.
Column Waters Symmetry C-18, 5 µm, 2.1 × 100 mm
Eluent
A: 5% {CH3OH: H2O (1:9) + 5 mM
CH3COONH4}
B: 95% {CH3OH: H2O (9:1) + 5 mM
CH3COONH4}
Elution Isocratic (Binary Solvent)
Flow rate 0.2 ml/min
Stop time 5 min
Post time 5 min
Injection volume5 µl
Column temp. 25˚C ± 0.8˚C
Mass Spectrometric condition.
Instrument Micromass Quattro micro API
Ionization mode ESCi multi-mode
Scan type MRM
Capillary voltage (kV) 1.00 kV
Cone voltage (V) 35 V
Extractor (V) 2 V
Source temperature 120˚C
Desolvation temperature 350˚C
Desolvation gas flow 650.0 (L/hr)
Cone (L/hr) 25.0 (L/hr)
Molecular ion 308.14 amu (used for quantation)
Mass transition
308.14 → 69.6 amu (for qualitative
confirmation)
308.14 → 125.1 amu (for qualitative
confirmation)
308.14 → 150.9 amu (for qualitative
confirmation)
LOD 0.005 ppm. or 0.005 μg/mL
LOQ 0.01 ppm. or 0.01 μg/mL (For all
substrate)

Harvest Residue Study of Fungicide Tebuconazole EC Formulation in Groundnut and Paddy
426
3.2. Linearity Check
A calibration curve (Figure 1) was made by plotting
seven concentrations (0.01 - 1.00 μg/g) of standard Tebu-
conazole versus absorption. Also, to know the interfe-
rence of each substrate, matrix match calibration standard
for each substrate was prepared. In this study calibration
curve was prepared by taking the areas corresponding to
different concentrations of matrix match calibration stan-
dard, against which final quantification was done.
3.3. Recovery Test
Recovery studies were carried out in order to establish
the reliability of the analytical method and to know the
efficiency of extraction and clean up steps employed for
the present study, by fortifying the samples with different
levels of analytical standard solution of Tebuconazole. It
was carried out by fortifying different substrates of paddy and
groundnut samples at the level of 0.01, 0.10 and 0.50 ppm
with the analytical standard solution of Tebuconazole and
was analyzed following the procedure. Results of
recovery study are shown in Table 1 and Table 2.
Figure 1. Calibration curve of analytical standard of
Tebuconazole.
Table 1. Recovery study of Tebuconazole in different
substrates of groundnut.
Substrate Amount
fortified
(ppm)
Amount
recovered*
(ppm) % Recovery Average %
recovery
0.01 0.009 90.00
0.10 0.089 89.00
Field Soil
0.50 0.483 96.60
91.87
0.01 0.008 80.00
0.10 0.086 86.00
Groundnut
Plant
(Haulm) 0.50 0.465 93.00
86.33
0.01 0.009 90.00
0.10 0.093 93.00 Oil
0.50 0.449 89.80
90.93
0.01 0.009 90.00
0.10 0.087 87.00 Deoil Cake
0.50 0.457 91.40
89.23
*Average of three replicates.
Table 2. Recovery study of Tebuconazole in different
substrates of paddy.
Substrate Amount fortified
(ppm)
Amount
recovered*
(ppm) % Recovery Average %
recovery
0.01 0.008 80.00
0.10 0.086 86.00
Paddy
Straw 0.50 0.466 93.20
86.40
0.01 0.009 90.00
0.10 0.093 93.00
Paddy
Grain 0.50 0.448 89.60
90.86
0.01 0.009 90.00
0.10 0.087 87.00 Husk
0.50 0.457 91.40
89.46
0.01 0.008 80.00
0.10 0.091 91.00 Field Soil
0.50 0.473 94.60
88.53
* Average of three replicates.
4. Results and Discussion
4.1. Recovery Study
The recovery percentage of Tebuconazole from different
substrates of groundnut and paddy were presented in
Table 1 & Table 2 respectively. As the recovery
percentage is quite high for all the substrates, hence the
method can be adopted for harvest residue study of
Tebuconazole in different substrate of paddy and
groundnut.
4.2. Residues of Tebuconazole in Harvest
Samples
All the data regarding residues of Tebuconazole in
harvest substrate of paddy and groundnut have been
presented in Tabl e 3 and Table 4 respectively. In all the
cases, it was found that the fungicide residues were
below the detection limit of the instrument (<0.01 ppm)
irrespective of doses in different substrates of paddy and
groundnut. Adiver et al. [5,6], stated that tebuconazole
effectively control groundnut diseases with no residual
toxicity problem. Tirmali et al. [7] in 2001 stated same
trend in their evaluation study of some new fungicides
against rice blast. Moorman et al. [8] also reported that,
application of Tebuconazole does not possess any
residual toxicity problem in soil under vegetable
production. Sandra et al. [9] and Chuan et al. [10] also
reported the effectiveness of Tebuconazole against fungal
diseases of peppermint crops and apple respectively
without possessing any residual toxicity problem. So, it
might be stated that Tebuconazole may not pose any
residual toxicity problem in paddy and groundnut. Similar
observations were also reported earlier about the safety
issue of Tebuconazole [11-16]. Based on these findings,
he use of Tebuconazole 25.9% EC in paddy and ground- t

Harvest Residue Study of Fungicide Tebuconazole EC Formulation in Groundnut and Paddy
427
Table 3. Harvest residue of Tebuconazole in different substrates of paddy.
Chemical applied Substrate (Harvest) Treatment Residues in ppm.
R1 R2 R
3 Mean ± S.D Dissipation (%)
T1
BDL BDL BDL - - Cropped Soil
T2 BDL BDL BDL - -
T1 BDL BDL BDL - -
Straw
T2 BDL BDL BDL - -
T1 BDL BDL BDL - -
Husk
T2 BDL BDL BDL - -
T1 BDL BDL BDL - -
Tebuconazole
Grain
T2 BDL BDL BDL - -
BDL: Below detection limit (<0.01 ppm).
Table 4. Harvest residue of Tebuconazole in different substrates of groundnut.
Chemical applied Substrate (Harvest) Treatment Residues in ppm.
R1 R2 R
3 Mean ± S.D Dissipation (%)
T1
BDL BDL BDL - - Cropped Soil
T2 BDL BDL BDL - -
T1 BDL BDL BDL - -
Groundnut
Plant T2 BDL BDL BDL - -
T1 BDL BDL BDL - -
Oil
T2 BDL BDL BDL - -
T1 BDL BDL BDL - -
Tebuconazole
Deoil Cake
T2 BDL BDL BDL - -
BDL: Below detection limit (<0.01 ppm).
nut in West Bengal may be advocated for the control of
fungal diseases in paddy and groundnut.
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