Distribution Behaviour of Dimethoate in Tea Leaf

doi:10.4236/jep.2011.24056

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Journal of Environmental Protection, 2011, 2, 482-488

doi: 10.4236/jep.2011.24056 Published Online June 2011 (http://www.SciRP.org/journal/jep)

Distribution Behaviour of Dimethoate in Tea Leaf

Shivani Jaggi1*, Bikram Singh2, Adarsh Shanker2

1Maya Institute of Technology & Management Selaqui, Dehradun, Uttarakhand, India, 2Institute of Himalayan Bioresource Tech-

nology (IHBT), Council of Scientific & Industrial Research, Palampur (Himachal Pradesh), India

Email: [email protected]

Received February 9th, 2011; revised March 17th, 2011; accepted May 2nd, 2011.

ABSTRACT

A study was undertaken to assess the distribution behaviour of Dimethoate in tea leaf. Tea bushes were subjected to

Dimethoate spray at recommended dose and double the recommended doses. The extraction of pesticide was done using

chloroform and the analysis wa s done using a Hewlett-Packa rd 5890 series II gas chromatog raph with Nitrogen Phos-

phorus Detector (NPD). The penetration behaviour of Dimethoate was studied in dry and wet seasons. Variations in

penetration were observed in dry and wet seasons which was attributed to climatic factors like temperature, humidity,

rainfall, sunlight and physicoch emical properties of the residue like water solubility, partition coeffici ent and formula-

tion type. Residues observed in the cell wall and tissues confirm its good penetrating ability inspite of its hydrophilic

nature. Higher penetration in wet season as indicated can be attributed to the route through the stomatal pores.

Keywords: Tea, Cuticle, Dimethoate, Dissipation

1. Introduction

Tea, Camellia sinensis (L) O. Kurtze, is the most impor-

tant plantation crop in India occupying 420,000 ha of

land. All over the world it is a popular invigorating and

refreshing drink having excellent medicinal properties.

Considering that an estimated amount of 18 - 20 billion

cups are consumed daily in the world its economic and

social interest is clear [1]. Like any other crop tea planta-

tions are also subjected to ravages of insects, mites, plant

pathogens, nematodes etc. The perennial nature of the

crop and the more equitable weather pattern prevailing in

tea areas are favorable for insect pests resulting in

250-500 million kg of annual loss of crop. In terms of

monetary loss it could be approximately 500 million-1

billion US $ [2]. The insects target almost all plant

parts such as roots, stem, leaves and buds. Thus every part

of the tea plant is a potential target for a wide spectrum of

pests and disease causing organisms.

Tea is an unusual crop where leaves are sprayed di-

rectly with pesticides, harvested and processed even

without washing. The shoots of the tea plant are thin and

tender (233 - 291 m) with cuticle thickness 4 - 10 m

and surface area per unit weight of leaf (tender leaf 41.7

cm2/gm and mature leaf 15.9 cm2/gm respectively).Tea

leaf, with thin cuticle, relatively larger surface area and

short interval between pesticide application and harvest,

is expected to be permeable to various groups of chemi-

cals. Pesticide thus could have negative impact both on

ecology and quality of tea when applied [3]. Conse-

quently tea represents a significant potential of human

exposure to pesticide residues by virtue of high applica-

tion of pesticides to tea crop coupled with the average

intake of 6 gms of dried (made) tea per day per individ-

ual [4,5]. Some basic studies on plant cuticles as barriers

against the diffusion of chemicals have been reported

[6,7]. Pesticide penetration into the leaf surface plays

important practical implications as it allows the residues

to stick/persist thus maintaining its efficacy even if it

rains after the treatment [8]. Moreover once the pesticide

has penetrated the epicuticular wax solar radiations act-

ing on the pesticide molecule have to cross the cuticle

thus reducing the photodegradative activity [9]. Further-

more, cuticular wax can hold on the pesticide residues

resulting in low volatility from the leaf surface. The in-

formation available on the fate of pesticides on tea leaf

and role leaf cuticle, plays in the dissipation under dif-

ferent environmental conditions is scanty in literature.

Hence a study was initiated to understand the penetrating

and dissipation behavior of dimethoate on tea leaf sur-

face. Fate of dimethoate in tea and its brew have been

reported but still its exact behaviour on the tea leaf sur-

face is not known thoroughly [10,11].

Distribution Behaviour of Dimethoate in Tea Leaf483

2. Experimental Details

2.1. Field Trials

Field trials were carried out in dry and wet seasons at

Institute of Himalayan Bioresource Technology (IHBT)

tea experimental farm at Banuri, Palampur (1300 msl,

326′20″N × 7633′29″E), India. The maximum and

minimum temperature during dry and wet seasons as

recorded from the experimental farm was 32˚C, 23˚C and

30˚C, 18˚C respectively. Relative humidity was 64% and

82% respectively and the total rainfall was 97 mm during

wet season where as dry season was without any rainfall.

The commercial formulation of dimethoate was sprayed

(spray volume 400 l/ha) on tea bushes considering each

plot of 100 bushes per replicate at recommended dose

(200 gm a.i./ha) and double of the recommended dose

(400 gm a.i./ha). In control treatment (T0), water was

sprayed. Treatments were carried out in dry and wet sea-

sons. Spray was done with a calibrated Knapsack sprayer.

The weather parameters were continuously recorded in

the experimental period to monitor the effect of envi-

ronmental conditions.Green tea leaves (two leaves and a

bud) were plucked from each replicate of both the treat-

ment and control plots and brought to the laboratory each

time at 0 (immediately and 4 hours after spraying),

1,3,5,7,10,14 and 21days after the treatment. To see the

effect of leaky cuticle damaged leaves were also col-

lected from the same plots. Further to analyze the effect

of cuticle on the photodegradation of Dimethoate a labo-

ratory experiment was carried out in parallel by spraying

dimethoate on glass plate (i.e. without leaf) and other

with leaf under same environmental conditions to pro-

vide estimated behavioral information. The weather pa-

rameters during the experimental time are graphically

represented in figure below (see Figure 1).

2.2. Analytical Standards and Working Solutions

An analytical grade of dimethoate was obtained from

Dr.Ehrenstorfer Laboratories, Augsburg, Germany (re-

ported purity > 98%). For the field studies, formulation

grade of dimethoate (Rogor® 30 Emulcifiable Concen-

trate EC, Isagro Agrochemicals Pvt. Ltd., Mumbai) was

procured from the local market. Standard solution (1000

mg/l) was prepared in acetone and the spiking solution

(50mg/l) was diluted from the stock solution and the so-

lutions required for preparing a standard curve (0.2, 0.5,

1.0, 1.5, 5.0 and 10.0 mg/l) were prepared from the stock

solution by serial dilutions. All chemicals used for ex-

traction and analysis of the residues and activated carbon

were products of Merck India Limited, Mumbai India.

Anhydrous sodium sulphate AR (Analytical Reagent)

used was supplied by S.d. fine-chemicals, Mumbai.

Figure 1. Weather parameters.

2.3. Apparatus

Gas Chromatograph

A Hewlett-Packard 5890 series II gas chromatograph

(Avondale, PA-USA) supported by a nitrogen phospho-

rus detector (NPD), a HP-7673 autosampler and integra-

tor (Hewlett-packard) using split-splitless injector, con-

nected to HP 3365 Chemstation system software (Hew-

lett-packard) was used. The detection was done using

Nitrogen Phosphorus Detector (NPD). Peak resolution

was done on a HP-17 medium polar capillary column (25

m length × 0.2 mm id) containing 50% phenyl and 50%

methyl polysiloxane coated fused silica (0.25 µm film

thickness) (Hewlett-Packard, Co., Wilmington, DE). De-

tailed analytical conditions were as follows. The injec-

tion was made using a split mode (50:1), injector tem-

perature held at 260˚C. The temperature of the NPD was

held at 280˚C. The oven temperature was programmed at

150˚C for the initial 2 minutes and then ramped 10˚C

/min to 300˚C and finally maintained for 5 minutes. Car-

rier gas was nitrogen (purity 99.99%) at column flow rate

of 1 ml/min. The samples were filtered through millipore

Distribution Behaviour of Dimethoate in Tea Leaf

484

membrane teflon filters (0.45 µm particle size) before

injection into the chromatographic column.

3. Standard Calibration Curve

Standard curve was prepared by diluting the stock solu-

tion to five different concentrations in acetone. The col-

umn was conditioned by repeated injections (3 times) of

the standard under constant operating conditions until the

peaks obtained were reproducible. Dimethoate was injected

at 0.2, 0.5, 1.0, 5.0 and 10.0 mg/l to validate the method.

Recovery Assay

Before laying the experiments in the field, recovery

studies were performed at 50.0 mg/l fortification level of

active ingredient (three replicates) of each matrix (green

leaves, dried leaves and soil). These samples were pre-

pared by adding known amount of standard in matrix

before extraction. The extraction was carried out as de-

scribed below in section The duplicate injections of each

extract were made in Gas Chromatograph (GC-NPD).

4. Extraction of Pesticide from Leaf Surface

Extraction of dimethoate was done with Chloroform (re-

covery > 90%) The extract was agitated mechanically

with acetonitrile for 3 hours on a horizontal shaker. The

mixture was filtered through Whatman no.1 filter paper

and the cake was washed twice with 20 ml solvent each

time. The combined water extract was partitioned with

150ml of acetonitrile twice in a 500 ml separating funnel.

Discarding the aqueous layer, the organic layer was con-

centrated to near dryness on a water bath and reconsti-

tuted with 1 ml of acetone for final analysis. The effect

of washing was thus confirmed by laboratory washing

and in case of samples collected from the treated fields in

dry and wet seasons, the residue was extracted in dichloro-

methane. The final eluate was evaporated to near dryness and the

residue was reconstituted with 1ml acetone for quantification.

4.1. Extraction of Pesticide from Epicuticular

Wax

Epicuticular wax extraction was done using the method

described by Mc. Donald et al. [12]. After thorough

washing to remove the surface pesticides, the tea leaves

were soaked in 100 ml of chloroform and shaken on an

automatic horizontal shaker for 1 minute. Extract was

filtered through Whatman No.1 filter paper and concen-

trated to 5 ml followed by passing through an adsorbent

column containing florisil topped with 1 cm of anhy-

drous sodium sulphate prewashed with chloroform. The

extract was eluted with 200 ml of chloroform, concen-

trated on a vacuum rotatory evaporator using a water

bath at 35˚C - 40˚C. The residue was finally reconstituted

with 1ml of acetone and quantified by GC (NPD).

4.2. Extraction of Pesticide from Cell Wall

Extraction of dimethoate was done by soaking the tea

leaves in 100 ml of chloroform and shaken on an auto-

matic horizontal shaker for 1 minute. Extract was filtered

through Whatman No.1 filter paper and concentrated to 5

ml followed by passing through an adsorbent column

containing florisil topped with 1 cm of anhydrous sodium

sulphate prewashed with chloroform. The extract was

eluted with 200 ml of chloroform, concentrated on a

vacuum rotatory evaporator using a water bath at 35˚C -

40˚C. The extract was finally eluted with dichloro-

methane (200 ml) from the florisil column. The eluate

was evaporated to dryness and reconstituted to 1 ml in

acetone and 2 µl of it was analysed by GC (NPD).

5. Detection and Quantification

Detection limit test

To determine the limit of detection made tea samples

were spiked with different concentration levels of di-

methoate standard and analysed by GC (NPD). The de-

tection limit was evaluated by the peak signal/noise (S/N)

ratio. An S/N ratio greater than 3 was considered as a

detectable peak.

6. Results and Discussion

6.1. Quantification

The GC analytical conditions were optimized in terms of

temperature program that allowed an improvement of the

time and the chromatographic run resolution. Moreover

to avoid the cross contamination between high and low

spiked samples, the sequence of injections was in the

following order: solvent, blank sample, sprayed samples

and finally standard solution. No interfering peaks were

present during the analysis of any samples as before each

run the solvent was injected.

Moreover, the adopted oven programming allowed a

good chromatographic separation of dimethoate. The total

run time was 14 minutes and the retention time of the

dimethoate in the given chromatographic conditions was

8.68 minutes and constant for each series of samples.

Chromatographic separation by HP-17 column provided

good results for the quantification of the samples.

6.2. Linearity

The calibration curve of the analysed dimethoate gave a

good regression line (R2 = 0.8827) in the range of ex-

plored concentrations, 0.1 - 10.0 mg/l. The detection

limit of dimethoate was taken to be 0.01 mg/kg, which

were much lower than the maximum residue limits fixed

by European Commision for dimethoate in tea (0.2

mg/kg). Residues below 0.01 mg/kg were detected but

not quantified. This low detection limit was achieved

Distribution Behaviour of Dimethoate in Tea Leaf485

because of the efficient cleanup step that allowed the

elimination of all the possible interfering peaks, giving a

low noise value.

6.3. Method Validation

The recovery of the fortified (50 mg/kg) samples of di-

methoate in made tea (in triplicate) ranged from 92.9% -

94.6%. The results showed good recovery and repro-

ducibility. The compounds of interest were well resolved

from other co-extractives. These results indicated that the

method used in this study provided a good cleanup.

6.4. Field Studies

The shoots of the tea plant are thin and tender (233 - 291

m) with cuticle thickness 4 - 10 m and relatively larger

surface area per unit weight of leaf (tender leaf 41.7 and

mature leaf 15.9 cm2/gm respectively). It is an unusual

crop where leaves are sprayed directly with pesticides,

harvested and processed even without washing. In the

preliminary experiments with tea leaf cuticle penetration

of dimethoate was measured immediately after applica-

tion as evaporation of water also affected the rate of

penetration [13].As indicated by data presented in Tables

2-5, the residues of dimethoate on leaf surface at 0 day

(immediately after spray) was found to be 9.07  0.14

mg/kg and 18.31  0.34 mg/kg whereas its concentration

was 9.01  0.10 mg/kg and 18.19  0.34 mg/kg respec-

tively at two different treatments when the samples were

collected 4 hours after spray. No penetration effect ob-

served in the epicuticular wax and cell wall when the

leaves were collected immediately after spray. This indi-

cated that during the evaporation of water the penetration

rate was negligible. In dry season the residue concentra-

tion in the epicuticular wax was 0.14  0.05 and 0.11 

0.10 mg/kg and in the cell wall was 2.97  0.02 and 4.41

 0.08 mg/kg in normal leaf when the samples were col-

lected 4 hours after spray at 200 and 400 gm a.i/ha re-

spectively. While the concentration in injured leaf was

0.17  0.02 and 0.68  0.06 mg/kg in epicuticular wax

and 3.04  0.06 and 6.12  0.12 mg/kg in cell wall.

Further, the concentration in leaf surface was found to

Figure 2. Standard curve.

be 0.02  0.00 mg/kg and 0.04  0.00 mg/kg at two dif-

ferent treatments on 7th day after spray. No residues

were detected on leaf surface after 10th day of treatment.

In wet season, the residue concentration in the cell

wall was 3.24  0.06 mg/kg and 6.25  0.19 mg/kg re-

spectively at two different treatments in normal leaf and

3.32  0.11 mg/kg and 7.20  0.17 mg/kg in injured

leaves whereas in wax the residue concentration was 0.03

 0.00 mg/kg, 0.08  0.01 mg/kg and 0.08  0.00 mg/kg,

0.08  0.00 mg/kg in normal and injured leaves respec-

tively. Further, this concentration in wax was negligible

in normal leaf and injured leaf on 3rd day after spray,

which declined to no detectable limit in 7th day after

spray. As observed from the data, the residues found on

the epicuticular wax and cell wall was comparatively

more in the damaged cuticle.

Residues observed in the cell wall and tissues confirm

its good penetrating ability inspite of its hydrophilic na-

ture. Higher penetration in wet season as observed from

the tables indicated the route could be through the

stomatal pores also. Little variation in the results as ob-

served from Tables 2-5 for normal and damaged cuticle

confirmed that cuticle was also acting as a barrier during

penetration of dimethoate residue. The tea leaf having

thin cuticle is expected to be permeable to various groups

of pesticides and dimethoate residue in the wax and cell

wall confirmed it but cuticle thickness alone may not be

responsible for the dimethoate penetration in the leaf.

Data in Table 1 proved that physical properties of the

Table 1. Physico chemical properties of Dimethoate.

COMMON

NAME DIMETHOATE

CHEMICAL

NAME

O,O-DIMETHYLS-METHYL-CARBAMOYL-

METHYL PHOSPHORODITHIOATE

EMPIRICAL

FORMULA C5H12NO3PS2

MOLECULAR

WEIGHT 229.3

VAPOUR PRES-

SURE 2.5 × 10–4 Pa at 25˚C

PHYSICAL

STATE COLOURLESS CRYSTALLINE SOLID.

MELTING

POINT 45 - 52.5˚C

BOILING

POINT 107˚C at 0.05 mmHg

VOLATILITY 1.107 mg/m3

SOLUBILITY

IN WATER 39 g/l

SOLUBILITY

IN ORGANIC

SOLVENTS

HIGHLY SOLUBLE IN CHLOROFORM,

METHYLENE CHLORIDE, BENZENE,

TOULENE, ALCOHOLS, ESTERS AND KE-

TONES.

SPECIFIC

GRAVITY AT

25˚C

1.281

Distribution Behaviour of Dimethoate in Tea Leaf

486

Table 2. Dimethoate distribution in tea leaf in dry season (200 gm a.i/ha).

Residue in mg/kg  standard deviation

Distribution in normal leaf surface Distribution in injured leaf

S Leaf surface Wax Cell wall Leaf surface Wax Cell wall

0(1) 9.07 ± 0.14 ND ND 8.96 ± 0.17 0.02 ± 0.00 ND

0(2) 9.01 ± 0.10 0.14 ± 0.05 2.97 ± 0.02 8.65 ± 0.13 0.17 ± 0.02 3.04 ± 0.06

1 6.17 ± 0.15 0.11 ± 0.09 2.03 ± 0.00 5.92 ± 0.12 0.21 ± 0.02 2.79 ± 0.06

3 1.46 ± 0.07 0.02 ± 0.00 0.03 ± 0.00 1.30 ± 0.01 0.02 ± 0.00 0.02 ± 0.00

5 0.05 ± 0.00 ND ND 0.04 ± 0.00 ND ND

7 0.02 ± 0.00 ND ND ND ND ND

10 ND ND ND ND ND ND

14 ND ND ND ND ND ND

21 ND ND ND ND ND ND

Table 3. Dimethoate distribution in tea leaf in dry season (400 gm a.i/ha).

Residue in mg/ kg  standard deviation

Distribution in normal leaf Distribution in injured leaf

S Leaf surface Wax Cell wall Leaf surface Wax Cell wall

0(1) 18.31 ± 0.34 ND ND 17.60 ± 0.30 0.03 ± 0.00 0.02 ± 0.00

0(2) 18.19 ± 0.31 0.11 ± 0.10 4.41 ± 0.08 17.36 ± 0.25 0.68 ± 0.06 6.12 ± 0.12

1 11.39 ± 0.23 0.90 ± 0.07 4.10 ± 0.10 11.18 ± 0.16 0.33 ± 0.07 5.93 ± 0.06

3 3.13 ± 0.13 0.06 ± 0.00 0.04 ± 0.00 2.95 ± 0.07 0.03 ± 0.00 0.03 ± 0.00

5 0.10 ± 0.01 ND ND 0.06 ± 0.00 ND ND

7 0.04 ± 0.00 ND ND ND ND ND

10 ND ND ND ND ND ND

14 ND ND ND ND ND ND

21 ND ND ND ND ND ND

Table 4. Dimethoate distribution in tea leaf in wet season (200 gm a.i/ha).

Residue in mg/ kg  standard deviation

Distribution in normal leaf surface Distribution in injured leaf

S Leaf surface Wax Cell wall Leaf surface Wax Cell wall

0 (1) 4.15 ± 0.13 ND ND 3.86 ± 0.10 ND ND

0 (2) 4.00 ± 0.01 0.03 ± 0.00 3.24 ± 0.06 3.65 ± 0.08 0.02 ± 0.10 3.32 ± 0.11

1 0.08 ± 0.00 0.05 ± 0.05 4.14 ± 0.09 0.06 ± 0.00 0.08 ± 0.13 4.25 ± 0.03

3 0.02 ± 0.00 0.02 ± 0.00 0.82 ± 0.03 0.03 ± 0.00 0.01 ± 0.00 0.94 ± 0.02

5 ND 0.03 ± 0.00 0.02 ± 0.00 ND 0.02 ± 0.00 0.02 ± 0.00

7 ND ND ND ND ND ND

10 ND ND ND ND ND ND

14 ND ND ND ND ND ND

21 ND ND ND ND ND ND

Distribution Behaviour of Dimethoate in Tea Leaf487

Table 5. Dimethoate distribution in tea leaf in wet season (400 gm a.i/ha).

Residue in mg/ kg standard deviation

Distribution in normal leaf Distribution in injured leaf

Leaf surface Wax Cell wall Leaf surface Wax Cell wall

0(1) 8.03 ± 0.07 ND ND 7.90 ± 0.05 ND ND

0 (2) 7.83 ± 0.05 0.08 ± 0.01 6.25 ± 0.14 7.44 ± 0.08 0.05 ± 0.00 7.20 ± 0.17

1 0.14 ± 0.02 0.08 ± 0.01 5.24 ± 0.91 0.10 ± 0.01 0.05 ± 0.00 6.64 ± 0.08

3 0.04 ± 0.00 0.05 ± 0.01 1.34 ± 0.09 0.04 ± 0.00 0.04 ± 0.00 1.42 ± 0.07

5 ND 0.05 ± 0.00 0.05 ± 0.00 ND 0.04 ± 0.00 0.03 ± 0.00

7 ND ND ND ND ND ND

10 ND ND ND ND ND ND

14 ND ND ND ND ND ND

21 ND ND ND ND ND ND

residue and environmental conditions might be equally

involved. Low partition coefficient Kow (0.7) and high

water solubility of 39 g/l might have enabled it to pene-

trate inside the inner region of the cuticle. They are rap-

idly absorbed under high humid conditions, supporting

the view that there was an aqueous route traversing the

cuticle and maximum penetration might be due to stomatal

pores. Thus the permeation of active ingredients was

influenced by their solubility characteristics as evident by

their partition coefficients. The Kow reflected the lipo-

philicity of the compound and was related to the degree

and rate at which it would be absorbed by leaf [14]. Fur-

thermore, penetration as observed in the leaf surface could

be attributed to the emulsifiable concentrate formulation

used as they allow better deposits and different adjuvants

keep the deposit in binded form and minimize the early

loss of pesticide by increasing the rate of penetration.

The adhension, retention and distribution of agrochemi-

cals sprayed on plant surfaces also depends on target

wettability [15]. However it could be envisaged that tem-

perature and humidity could have roles to play.

8. Conclusions

Above studies confirmed that the dimethoate distribution

on the tea leaf surface and decrease in surface residue is

due to the collective behaviour of cuticle, its solubility,

penetration, partition coefficient, vapour pressure and the

formulation along with the environmental conditions at

the time of experiments.

9. Acknowledgements

We are especially grateful to Director, IHBT for provid-

ing necessary facilities and CSIR, India for financial as-

sistance. The authors wish to thanks Guru Nanak Dev

University, Amritsar, India for the guidance.

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