Identification of Metabolites of Methylparathion in ...

Analytical Letters

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Identification of Metabolites of Methylparathion in Plant, Water and Soil Ashesh K Tayal , Inderjeet Kaur & S N Tandon To cite this article: Ashesh K Tayal , Inderjeet Kaur & S N Tandon (1999) Identification of Metabolites of Methylparathion in Plant, Water and Soil, Analytical Letters, 32:12, 2521-2530, DOI: 10.1080/00032719908542985 To link to this article: http://dx.doi.org/10.1080/00032719908542985

Published online: 27 Feb 2008.

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Date: 15 September 2016, At: 04:16

ANALYTICAL LETTERS, 32(12), 2521-2530 (1999)

IDENTIFICATION OF METABOLITES OF METHYLPARATHION IN PLANT, WATER AND SOIL

Key Words: GC-MS, Methylparathion, Plants, Water, Soil, Metabolites, Radish Ashesh K Tayal1*, Inderjeet Kaur2, and S N Tandon2

'• Department of Biosciences & Biotechnology, University of Roorkee, Roorkee, India 2

Department of Chemistry, University of Roorkee, Roorkee, India

ABSTRACT

A GC-MS study was carried out to identify the decay products of methyl parathion in water, soil and rooted vegetable (radish) at different time intervals. The water (pH 5.5 and 8.0), soil (pH 7.5) and radish samples collected after the first half life shows the presence of

0 , 0 dimethyl O-p-nitro-2 or 3-

hydroxyphenyl phosphoorothioate which persists upto the 30th day in the alkaline water and soil samples. However, in acidic water methyl parathion is oxidized to give methylparaoxon.

* Address for Correspondence. Dr. Ashesh Tayal, Environmental Biotechnology Division, Wockhardt Limited, 167, Readymoney Terrace, Dr. AB Road, Worli, Mumbai-400 018, India. 2521 Copyright © 1999 by Marcel Dekker. Inc.

www.dekker.com

2522

TAYAL, KAUR, AND TANDON

Radish samples show • the presence of two other products. These are identified as O,O-dimethyl o-p-aminophenyl -phosphoorothioate and 0 , 0 dimethylo-p-hydroxyphenyl phosphoorothioate. The study indicates that the half life of methyl parathion in different media is nearly the same but its metabolites are not identical. INTRODUCTION

The toxicity of a pesticide depends upon its chemical nature, persistence in the environment and the toxicity of its decay products. It is known that the metabolites are sometimes more toxic than the parent compound. Therefore, before accepting a pesticide for agriculture use, the metabolites should be properly identified and their toxicity should be evaluated. Widespread agricultural use of organophosphates and their potential mammalian toxicity have dictated the development of several methods for the isolation and detection of parent compound12. Methyl parathion is the second most widely used organophosphate after malathion being used extensively in forestry and crop protection. A number of studies have been reported on the degradation of methyl parathion. Baker and Applegate3 studied the effect of temperature and UV radiation on methyl parathion in soil. They found that after application of methyl parathion to soil, it undergoes oxidation to oxon. The metabolism of methyl parathion has been studied extensively in mice4'5. In these studies, urinary dialkyl phosphates were recovered as the major metabolites of methyl parathion metabolism. The data on human subjects suggest that dimethyl phosphate is the major metabolite after oral exposure to methyl parathion. Dowling and Lomley5 have proposed the possible metabolic pathways of methyl-parathion in fenton reagent. The data on human subjects suggests that dimethyl phosphate is a major metabolite after oral exposure to methyl parathion7.

METABOLITES OF METHYLPARATHION

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A closer look at the above literature on the metabolites of methyl parathion indicates that different groups of workers have conducted studies confined to a particular aspect. A sizable information deals with the identification of possible metabolites in biological samples and those formed as a result of its chemical degradation in the laboratory-. None of these studies reflect on the formation of different metabolites in various matrices with the passage of time. In order to arrive at any useful conclusion it becomes necessary to look into decay products in water, soil and plant at different intervals of time and suggest possible pathways. The content of this paper includes identification of metabolites of methyl parathion in water (pH 5.5. 8.0), soil (pH-7.5), and radish.

MATERIAL AND METHODS

Methyl parathion was obtained from Bayer (India) Limited, Mumbai, India. All the solvents used were of chromatography grade from E Merck (India). Potassium dihydrogen phosphate and disodium hydrogen phosphate of analytical grade (E Merck) were used for preparing buffer solution.

Sodium sulfate.

magnesium sulfate, charcoal and silica gel used for column chromatography were of analytical purity from E.Merck, India.

Gas Chromatography-Mass Spectrometry System-Hewlett Packard (HP) G1800 A, Mass spectrometer coupled with GCD series Gas Chromatography with Electron lonization Detector Column: HP 5 (30m x 0.25 mm id.) Temperature: 120°C to 250° C @ 10°C/ minute Run Time: 22 minutes

Metabolism of Methyl parathion in Radish Methyl parathion (100 ppm) was sprayed on the two week old radish plants. The samples were collected at different time intervals for 30 days. The

2524


minimum and maximum temperature, average humidity and wind velocity ranged from 25-35°C, 53.1%, and 1.3 to 5.3 km/h, respectively.

Methyl

parathion from the radish samples was extracted and metabolites were identified by GC-MS. The radish samples used for the GC-MS study correspond to 3rd (tl/2), 15th, 21st, and 30th day samples.

Metabolism of Methyl parathion in water and Soil Distilled water (10 ml) at pH 5.5 and pH 8.0 and cotton black soil (pH 7.5, 2.0 gin) were taken in glass stopper test tubes. Analytical samples of methyl parathion (2 ml of 100 ppm) in acetonitrile were added to each tube. These treated samples of water and soil were kept in a thermostatic water bath at 20°C. Samples were withdrawn at different time intervals for a period of 30 days. GCMS was used for the identification of metabolites in the 7th and 30th day old samples of water at pH 5.5, 6th and 30th day old samples of water at pH 8.0 and 5th and 30th day old samples of soil as it nearly corresponds to the half life in that particular medium.

Extraction from Radish The whole radish plant sprayed with methyl parathion was crushed and taken in a flask and shaken with equal amounts of methanol and water (5:1 v/v) The mixture was centrifuged and partitioned with the same volume of dichloromethane. The organic layer was separated and passed through anhydrous Na2SO.i and cleaned up by a silica, charcoal, magnesium sulfate (2:1:1) column (Figure 1).

Extraction from Water Water samples were withdrawn at different time intervals and extracted with 10 ml of dichloromethane. The lower layer was separated and dried over anhydrous Na2SO4 (Figure 2).


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Homogenize plant sample (ca lOOgni) spiked with pesticide Add 100 ml methanol- water (5:1 v/v) Shake well for 30 minutes Filter the sample Repeat the process with 2x100 ml methanol-water (5:1 v/v) Combine the extracts Extract with CH,C1, (2x100 ml)

I Dry on anhydrous Na2SO4 (100 gm) Pass throudi column of silica gel. charcoal, maanesium sulfate (2:1 by \vt.) Evaporate at ambient temperature Dissolve in organic solvent

Figure 1.

Extraction Procedure for Plant Sample

Water Sample (10 ml)

1 Extract with CH.C1, (10x2 ml)

1 Pass through anhydrous Na:SOj (5 gm) Rinse with 20 ml CH,Cli

1 Evaporate at ambient temperature

1 Dissolve in Appropriate organic solvent

Figure 2. Extraction Procedure for Water Sample

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Extraction from Soil Soil samples withdrawn at different time intervals were taken in a flask and shaken well with known amounts of methanol and water. The mixture was centrifuged and partitioned with dichloromethane. The organic layer was passed through anhydrous Na2SOj and cleaned up by column chromatography (Figure 3). RESULTS AND DISCUSSION

Identification of Metabolites of Methyl parathion in Radish, Water and Soil Metabolites of methyl parathion were identified at different stages in radish plants. Figure 4 shows the various metabolites of methyl parathion identified in radish, water and soil. The water (pH 5.5 and 8.0), soil and radish samples collected after the first half life show only the presence of product XV which is identified as 0 , 0 dimethyl O-p-nitro-2 or 3- hydroxyphenyl phosphoorothioate. Its mass spectrum shows a molecular peak at m/z 279 along with fragment ion peak at m/z 265, 156, 142, 125, 109, 79, and 65. In the 30th day sample of water (pH 8.0) and in soil only 0 , 0 - dimethyl- O- p-nitro 2 or 3 hydroxyphenyl phosphoorothioate is identified. OH _ +

m/z 279

i 1 OH

P-O-NO 2 m/z 265

+

+

3

m/z

142

m/z |

109

CH3OPOH


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Soil sample (ca 2.0 urn) spiked with pesticide 20 ml methanol- water (5:1)

I Blend

I Centrifime

4 Stand for 10 minutes

I Residue

i

20 ml methanol

•

Supernatant

I * Discard Residue

I Combine Extract * Partition with CH^CN (20x2)

I Dry on anhydrous Na^SO4 (5 gin)

I Pass throuuh column silica uel. charcoal, maunesium sulfate (2:1:1 by wt.) Evaporate at the ambient temperature

I Dissolve in organic solvent

Figure 3.

Extraction Procedure for Soil Sample

In the 30th day water sample (pH- 5.5) a different product is identified (XVIII). The mass spectrum of the product XVIII shows a molecular ion peaks at m/z 247, (M+) along with fragment ion peaks at m/z 186, 138, 135, 109, 79 and 65. The product is identified as methylparaoxon. O

tCH :H 3O n

O ii p

CH3OPOH

CH3O m/z

247

m/z

109

m/z

79


2528

OH P-0 '•0-O-NO,

(XVI)

(XV) ; p-o-