Harvest Residue Study of Fungicide Tebuconazole Ec Formulation in Groundnut and Paddy

doi:10.4236/jep.2011.24048

Paper Menu >>

Journal Menu >>

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.

Email: [email protected]

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.

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 (%)

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 (%)

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.

REFERENCES

[1] FAOSTAT, “FAO Statistical Database”, 2007.

http://www.fao.org

[2] Indian Council of Agricultural research, “Hand Book of

Agriculture, Pesticide residues,” 5th Edition, New Delhi,

2007, pp. 553-587.

[3] H. N. Swamy, S. Sannaulla and M. D. Kumar, “Evaluation

of New Fungicides against Rice Blast in Cauvery Delta,”

Karnataka Journal of Agricultural Sciences, Vol. 22, No. 2,

2009, pp. 450-451.

[4] R. Angelini, “Folicur (Tebuconazole): A New Triazole

Fungicide With A Wide Spectrum Of Activity,” Informa-

tore-Agrario-Supplemento, Vol. 52, No. 15, 1996, pp.

46-50.

[5] S. S. Adiver, K. H. Anahosur and K. Giriraj, “Triazoles

for Control of Foliar Diseases of Groundnut (Arachis

Hypogaea L.),” K arnatak a Journal o f Agricultu ral S c i e n c e s ,

Vol. 8, No. 1, 1995, pp. 65-68.

[6] S. S. Adiver and K. H. Anahosur, “Efficacy of Some

Triazole Fungicides Against Late Leaf Spot of Groundnut

and Their Subsequent Effects on Sclerotium Rolfsii,”

Indian Phytopathology, Vol. 48, No. 4, 1995, pp. 459-462.

[7] A. M. Tirmali, S. B. Latake and N. J. Bendra, “Evaluation

of New Fungicides for Control of Blast Disease of Rice,”

Journal of Maharashtra Agriculture University, Vol. 26,

2001, pp. 197-198.

[8] T. B. Moorman, “A Review of Pesticidal Effect in Soil

Under Vegetable Production,” Journal of Production and

Agriculture, Vol. 2, 1989, pp. 14-23.

[9] M. Sandra, Robert C. Menary and Noel W. Davies,

“Dissipation of Propiconazole and Tebuconazole in

Peppermint Crops,” Journal of Agricultural and Food

Chemistry, Vol. 47, No. 1, 1999, pp. 294-298.

doi:10.1021/jf980120e

Harvest Residue Study of Fungicide Tebuconazole EC Formulation in Groundnut and Paddy

428

[10] L. Chuan, “Determination of Tebuconazole Residue in

Soil and Apple,” Journal of Anhui Agricultural Sciences,

Vol. 37, No. 6, 2009, pp. 135-139.

[11] European Food Safety Authority, “Modification of The

Existing Mrls For Tebuconazole in Mandarins and Pass-

ion Fruit,” European Food Safety Authority Journal, Vol.

7, No. 10, 2009, pp. 1368.

[12] Food and Drug Administration of the United States,

“Pesticide tolerances”, 2003. http://www.cfsan.fda.gov

[13] M. A. Kastanias, S. Coward, A. Philippoussis and P.

Diamantopoulou, “Residue Evaluation of the Azole

Fungicides Prochloraz and Tebuconazole in the White

Mushroom Agaricus Bisporus,” Bulletin of Environmental

Contamination and Toxicology, Vol. 77, No. 1, 2006, pp.

149-154. doi:10.1007/s00128-006-1044-5

[14] K. D. Srivastava, D. V. Singh, R. Aggarwal, A. K. Dixit

and P. Bahadur, “Bioefficacy and Persistence of Tebu-

conazole against Loose Smut in Wheat,” Indian Phyto-

pathology, Vol. 50, No. 3, 1997, pp. 434-436.

[15] D. Shitienberg, “Integrated Management of Early and

Late Blights of Potatoes in Israel,” African Crop Science

Journal, Vol. 9, No. 1, 2001, pp. 203-207.

[16] S. Mohapatra, A. Ahuja, G. K. Jagadish, G.S. Prakash and

S. Kumar, “Behaviour of Trifloxystrobin and Tebucon-

azole on Grapes under Semi-Arid Tropical Climatic Con-

dition,” Pest Management Sciences, Vol. 66, No. 8, 2010,

pp. 910-915.