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IS 10630 (1983): Methods for Determination of MethylParathion Residues in Foods [FAD 1: Pesticides andPesticides Residue Analysis]

IS :10630 -1983

Indian Standard METHODS FOR

DETERMINATION OF METHYL PARATHION RESIDUES IN FOODS

Pesticides Residue Analysis Sectional Committee, AFDC 56

Chairman

DR H. L. BAMI Bungalow No. A, Malkaganj,

Delhi

Members Rqwcscnting

SHRI E. A. ALMEIDA SRHI F. QUADR~S ( Alternate )

Hindustan Ciba-Geigy Ltd. Bombay

SHRI K. D. AMKE National Organic Chemical Industries Ltd, Bombav

DR J. S. V~RMA ( Altcrnatc ) Dn K. C. GUHA Central Food Laboratory, Calcutta; and Central

Committee for Food Standards. New Delhi SHKI P. K. DEIINCRA ( Alternate )

DR S. S. GUPTA Bayer India Ltd, Thane DR R. L. KALRA Department of Entomology, Punjab Agricultural

University, Ludhiana DR R. P. CHAWLA ( Alternate )

DR KALYAN SINQ~ C. S. A. University OfAgriculture and Technology, Kanpur

Da K. KRISHNAMURTKY Department of Food ( Ministry of Agriculture ). New Delhi

SERI G. N. BZiARDWAJ ( Alternate ) DR V. LAKSHMINARAYANA Directorate of Plant Protection, Quarantine and

DH R. C. GUP~A ( Altcrnatc ) Storage, Faridabad

DR J. C. MAJUMDAR Pesticides Association of India, New Delhi DR V. SRINIVASAN ( Alternate )

DR M. S. MITHYANATHA Rallis Agro-Chemical Research Station, Bangalore DR A. L. MOOKEKJEE Cyanamid India Ltd, Bombay

( Continuad on page 2 )

Q Cqyrighr 1983

INDIAN STANDARDS INSTITUTION

This publication is protected under the Indian Co@ght Act ( XIV of 1957 ) and reproduction in whole or in part by any means except with written permission of the publisher shall be deemed to be an infringement of copyright under the said Act.

IS :10630- 1983

( Continuedfrom page 1 )

Members Represenfina

DR S.K. MUKERJEE Indian Agricultural Research Institute ( ICAR ), New Delhi _.

- __~_ DR S. K. HANDA ( Altcmatc )

DR NAQABHUSH~N Rao Regional Research Hyderabad

Laboratory (CSIR ),

PUBLIC ANALYST Public Analyst, Government of Chandigarh

Haryana,

DEPUTY PUBLIC ANALYST ( Alternate ) DR T. D. SETE Industrial Toxicolonv Research Centre ( CSIR \.

Lucknow -’ I,

DR R. K. SETBI DR K. SIVASANI~ARAN

Indofil Chemicals Ltd, Thane

DR S. Y. PANDEY I Alternate ) Union Carbide India Ltd, New Delhi

DR S. C. SRIVASTAVA ’ ’ Indian Veterinaiy Research Institute ( ICAR ),

DR R. C. NAITHANI ( Alternates Izatnagar

DR K. VISHESWARAIAE . Central Food Technological Research Institute

Dn J. R. RANGAS~AMY ( Al!ernatc ) ( CSIR ), Mysore

DR B. L. WATTAL National Institute of Communicable Diseases, Delhi

SIIRI G. C. JOSHI ( Alternate ) SHRI T. PURNANANDAM,

Director ( Agri & Food ) Director General, ISI (Ex-ojicie Member )

Secretary SHRI M. L. KUXAR

Senior Deputy Director ( Agri & Food ), IS1

Indian METHI

DETERMINATION 01 RESIDUE

0. FOE

0.1 This Indian Standard was Institution on 30 August 1983, afte Residue Analysis Sectional COI

Agricultural and Food Products 1

0.2 Methyl parathion formulations the control of many insect pests. 1 parathion formulations often result of their residues. Careful assessme step in safeguarding human heal regulatory policy.

0.3 In the-preparation of this stan to the limits of methyl parathion under the provisions of Prevention specified test methods are sensitive

0.4 This standard will enable the in the field to follow uniform test parathion residues in various food

0.5 In reporting the result of a tee this standard, if the final value, ok off, it shall be done in accordance

1. SCOPE

1.1 This standard prescribes spectr methods for determination of II nitrophenyl ) phosphorothioate ] : foods.

*Rules for rounding off numerical va

Representing

Agricultural Research v Delhi

Institute ( ICAR ),

.l Research derabad

Laboratory (CSIR),

Analyst, Government mdigarh

of Haryana,

al Toxicology Research Centre (CSIR ), :know Chemicals Ltd, Thane Carbide India Ltd, New Delhi

Veterinary Research Institute ( ICAR ), tnagar

r*

Food Technological Research Institute SIR ), Mysore

1 Institute of Communicable Diseases, Ihi

r General, IS1 (Ex-o&is Member )

‘V . KUMAR~ ( Agri & Food), IS1

IS:10630-1983

Indian Standard METHODS FOR

DETERMINATION OF METHYL PARATHlON RESIDUES IN FOODS

0. FOREWORD

0.1 This Indian Standard was adopted by the Indian Standards Institution on 30 August 1983, after the draft finalized by the Pesticides Residue Analysis Sectional Committee had been approved by the Agricultural and Food Products Division Council.

0.2 Methyl parathion formulations are extensively used in agriculture for the control of many insect pests. Frequent and increased use of methyl parathion formulations often results in harmful effects due to toxic nature of their residues. Careful assessment of residues is, therefore, an important step in safeguarding human health and in the establishment of sound regulatory policy.

0.3 In the preparation of this standard, due consideration has been given to the limits of methyl parathion residues which have been laid down under the provisions of Prevention of Food Adulteration Rules and the specified test methods are sensitive to the prescribed levels of residues.

0.4 This standard will enable the health authorities and others engaged in the field to follow uniform test procedure for the estimation of methyl parathion residues in various foods.

0.5 In reporting the result of a test or analysis made in accordance with this standard, if the final value, observed or calculated, is to be rounded off, it shall be done in accordance with IS : ‘L-1960*.

1. SCOPE

1.1 This standard prescribes spectrophotometric and gas chromatographic methods for determination of methyl parathion [ O,O-dimethyl 0-( 4- nitrophenyl ) phosphorothioate ] and its oxygen analogue residues in foods.

*Rules for rounding off numerical values (revised).

3

IS : 10630 - 1983

1.1.1 Spectrophotometric method may be adopted as a limit test for routine purposes whereas gas chromatography method shall be the reference method with a minimum detection limit of 0.05 “g/g ( 0.05 ppm ) of methyl parathion residues.

1.1.2 Though no set procedure for thin layer chromatography ( TLC ) is being prescribed, any standardized TLC procedure may be followed if necessary for clean up, identification and confirmation of methyl parathion residues.

2. SAMPLING

2.1 The representative samples for the purpose of estimating methyl parathion residues in various agricultural and food products shall be in accordance with the sampling procedures as prescribed in the relevant Indian Standards, whenever available.

3. SPECTROPHOTOMETRIC METHOD

3.1 The residues of methyl parathion are extracted with benzene or isopropanol and the cleaned up solution is clarified. The nitro group of parathion is then reduced with zinc dust and hydrochloric acid solution to amino group. The resulting water soluble compound is diazotized and coupled with N-( I-naphthyl ) ethylene diamine to produce a magenta colour, the intensity of the colour is measured with a spectrophotometer at 555 to 560 nm.

3.2 Reagents

3.2.1 Ben.cene - Analytical reagent grade and glass redistilled.

3.2.2 Adsorbent Mixture -Ten part of anhydrous powdered sodium sulphate, 5 parts of Atta clay, 5 parts filter ccl, and 2 parts of activated carbon.

3.2.3 Parathion Standard Sol&m - Prepare a standard solution for 1

methyl parathion in such a manner that 1 ml of the final solution contains 20 pg of parathion.

3.2.4 Standard Hydrochloric Acid Solution - 0.5 N.

3.2.5 Sodium .Nitrite Solution - Aqueous, 0.25 percent ( m/v ).

3.2.6 Ammonium SQhamate Solution - Aqueous, 2.5 percent (?rzlv ). Dissolve l-25 g in water and dilute to 50 ml. Prepare weekly.

.+

3.2.7 Jv-( I-Najhthyl ) Et hylenediamine D&Hydrochloride Solution - Aqueous, 1 percent ( m/v). Prepare fresh solution daily and store in a dark coloured bottle.

4

3.2.8 zinc Dust - analytical re:

3.2.9 IsopropVl Alcohol - analyti

3.3 Preparation of Sample C Parathion Residues

3.3.1 Firm a+zd Relatively Tough > Weigh 2 to 3 kg of fruit into a cle it can be turned with end-over-e motor. Add 500 ml of benzene tight-fitting cork, wooden bung, c sheet-cork or other suitable solver minutes at 75 to 100 rev/min ( se benzene as completely as possibl Note 4 ).

NOTE 1 - In all cases; treat the the adsorbent mixture, in proport Finally, filter through rapid, folded p;

NOTE 2 -As benzene vapours a inhaling them.

NOTE 3 - Since apples treated fc the time of stripping; on other firmer

NOTE 4 - It is assumed that con from apples is the same as that in the

3.3.1.1 Aliquots of this benze directly for parathion residue dett than 100 mg of foreign organic m

3.3.1.2 In order to remove p’ waxes, add, to each 100 ml of t mixture ( see 3.2.2 ). Stopper flasE filter on folded paper. Do not filtrate before determining parath

3.3.2 Soft Fruits, Such as Plums sample with 300 to 500 ml of the gently by hand for five minutes il

3.3.3 Fresh, Leafy Vegetables, Sue, 100 g of the sample into 100 ml o high speed blender. Add 200 m minutes, pour the mixture into 5 minutes. Transfer supernatants with 50 ml portions of benzene an up solids with stirring rod, stopper

Y

L

1

s

ay be adopted as a limit test for atography method shall be the n detection limit of 0.05 pg/g ies.

hin layer chromatography ( TLC ) ‘LC procedure may be followed if on and confirmation of methyl

:he purpose of estimating methyl ral and food products shall be in ures as prescribed in the relevant

rHOD

In are extracted with benzene or Nn is clarified. The nitro group of 1st and hydrochloric acid solution ;oluble compound is diazotized and

ne diamine to produce a magenta leasured with a spectrophotometer at

grade and glass redistilled.

,t of anhydrous powdered sodium :s filter ccl, and 2 parts of activated

Prepare a standard solution for Iat 1 ml of the final solution contains

ion - 0.5 N.

:ous, 0.25 percent ( m/v ).

:,;Llqueous, 2’5 percent ( m/v ). . Prepare weekly.

! Di-Hydrochloride Solution- Aqueous, olution daily and store in a dark

IS : 10630 - 1983

3.2.8 $nc Dust - analytical reagent grade.

3.2.9 Isopropyl Alcohol - analytical reagent grade and glass-redistilled.

3.3 Preparation of Sample Clean-up and Extraction of Methyl Parathion Residues

3.3.1 Firm aid Relatively Toqh Skinned Fruits, Such as .-l@les and Pears - Weigh 2 to 3 kg of fruit into a clean dry jar of 15 litres so mounted that it can be turned with end-over-end tumbling action by hand-crank or motor.

It

Add 500 ml of benzene (see Notes 1 and 2 ) and stopper with tight-fitting cork, wooden bung, or plastic screw-cap faced with gasket of sheet-cork or other suitable solvent-resistant material. minutes at 75 to 100 rev/min ( see Note 3 ).

Turn the jar for 5 Open the jar and drain off

i benzene as completely as possible into a 1-litre erlenmeyer flask ( see Note 4 ).

NOTE 1 - In all cases; treat the benzene extract by shaking for five minutes with- the adsorbent mixture, in proportion of approximately 10 g to 100 ml extract. Finally, filter through rapid, folded paper.

NOTE 2 -As benzene vapours are toxic, take precautions at all times against inhaling them.

NOTE 3 - Since apples treated for longer periods become swollen and soft, limit the time of stripping; on other firmer products time factor is not so important.

NOTE 4 -It is assumed that concentration of parathion in benzene poured off from apples is the same as that in the solvent retained by the apples after wetting.

3.3.1.1 Aliquots of this benzene extract obtained in 3.3.1 may be used directly for parathion residue determination if-they do not contain more than 100 mg of foreign organic matter.

3.3.1.2 In order to remove partially any colouring matter and plant waxes, add, to each 100 ml of the benzene solution, 10 g of adsorbent mixture ( see 3.2~.2 ). Stopper flask, shake vigorously for five minutes, and filter on folded paper. Do not allow appreciable evaporation of the filtrate before determining parathion in aliquot.

3.3.2 Soft Fruits, Such as Plums, Tomatoes, Berries, etc - Use 1 to 2 kg sample~with 300 to 500 ml of the redistilled benzene, and strip by shaking gently by hand for five minutes in a jar of suitabIe size.

3.3.3 Fresh, Leafy Vegetables, Such as Cabbage, Lettuce and Greens - Blend 100 g of the sample into 100 ml of isopropyl alcohol for two minutes in high speed blender. Add 200 ml of benzene and blend again for two minutes, pour the mixture into a centrifuge bottle and centrifuge for 5 minutes. Transfer supernatants to a 1-litre separator. Wash blender with 50 ml portions of benzene and transfer to centrifuge bottles. Break up solids with stirring rod, stopper and shake for 2 minutes. Centrifuge

5

as before and add solvent layer to separator. Wash and extract with equal volume of distilled water to remove isopropyl ~alcohol. Dry benzene layer with 30 g-of anhydrous sodium sulphate and dilute to suitable volume with benzene and finally make the volume to 200 ml with benzene.

3.3.4 Other Products -Grind dried products, in a suitable mill and extract with benzene in a large Soxhlet-type extractor for a period of one to two hours. Satisfactory extraction may be achieved in many cases by letting ground material steep overnight in stoppered jars with measured volume bf redistilled benzene. in 3.3.3.

Follow the ~same procedure as detailed

3.4 Preparation of Standard Curve for O-200 tig Range - Add 2.0, 4.0, 6.0, 8.0 and 10.0 ml parathion standard solution (see 3.2.3) to a series of 250-ml conical flasks. Make up the volume to 25 ml with benzene in all cases and provide a blank of 25 ml benzene. Add 20 ml of dilute hydrochloric acid ( see 3.2.4) and 200 mg of finely powdered zinc dust and connect to a condenser by all-glass adapter fitted with thermometer. Place on a hot-plate at medium heat and rapidly distil off the benzene. When the vapour temperature is about 7O”C, ebullition stops, benzene is eliminated and vapour temperature falls. Do not let aqueous sollltion boil at this point. Disconnect the flask, add 10 ml of alcohol and reconnect to reflux condenser. Reflux for 5 minutes and cool the flask in ice water. Treat the other flasks likewise in succession.

3.4.1 Add about 100 mg of filter ccl to each flask and filter into 50-ml volumetric flask through whatman No. 44 or equivalent filter paper. Use long thin stirring rods during transfer of solutions and washings to filter. Take care not to exceed the volume by 45 ml in each of the flasks. Volume of wash water that may be used is limited to about 15 ml.

3.4.2 Add 1 ml of sodium nitrite solution to each flask, mix and let stand for 10 minutes. Add I ml of ammonium sulphamate solution to each flask, mix, and let stand for 10 minutes. Then add 2’0 ml of N-( 1-napthyl ) ethylene diamine dihydrochloride solution, dilute to 50 ml volume and let stand for 10 minutes. Take the absorbance of the solution at 555 nm in spectrophotometer ( see Note ). Colours should be stable for at least one hour.

NOTE- With l-cm cell absorbance for 100 pg of parathion is about 0.33. If available, 5-cm cell may be preferred.

-3.4.3 Read the absorbance in succession using blank standard as reference and plot optical density against concentration in eg of parathion to obtain standard curve.

6

3.5 Determination - The total parathion residues obtained as investigation ia~shaken with chilled a separating funnel. The solvent this clarified solution is placed in z aliquot taken depending upon the is less than 25 ml, dilute to 25 ml 1 0.5 N hydrochloric acid and 200 n standard solution of parathion for

3.6 Reporting of Parathion absorbance against blank standarc the aliquot taken from the stal residues in the material in terms o

4. GAS CHROMATOGRAPHR

4.1 The residues of methyl parat extracted methyl parathion is mes with flame photometric detector. ( ppm ) is determined by compari: known standard of similar concen

4.2 Reagents

4.2.1 Acetone - analytical grade

4.2.2 Precipitating Solution - Pre_ chloride and 25 g of orthophosphc

4.2.3

4.2.4

4.2.5

4.2.6

4.2.7

Hyjlo Super- Gel

Chloroform - analytical gx

Sodium Sulfate - anhydrol

Benzene - analytical grad

Silica Gel Neutral Column -

4.3 Preparation of Sample, C Parathion Residues

4.3.1 Initial Extraction - Crush blender ror 2-3 minutes. To t further for 2-3 minutes. Filter tl to the blender for re-extraction and rinse the blender with furthe

c

.

. .

larator. Wash and extract with equal isopropyl alcohol. Dry benzene layer )hate and dilute to suitable volume >lume to 200 ml with benzene.

I products, in a suitable mill and %-type extractor for a period of one may be achieved in many cases by

It in stoppered jars with measured w the same procedure as detailed

rve for O-200 fig Range - Add n standard solution (see 3.2.3 ) to a e up the volume to 25 ml with .nk of 25 ml benzene. Add 20 ml and 200 mg of finely powdered zinc

by all-glass adapter fitted with medium heat and rapidly distil off

perature is about 70°C; ebullition 3ur temperature fa-11s. Do not let Xsconnect the flask, add 10 ml of Iser. Reflux for 5 minutes and cool . flasks likelvise in succession.

I to each flask and filter into 50-ml . 44 or equivalent filter paper. Use of solutions and washings to filter. : by 45 ml in each of the flasks. ed is limited to about 15 ml.

llution to each flask, mix and let ammonium sulphamate solution to ) minutes. Then add 2.0 ml of rochloride solution, dilute to 50 ml Fake the absorbance of the solution ote ). Colours should be stable for

r 100 pg of parathion is about 0’33. IF

cession using blank standard as 1st concentration in rcg of parathion

IS : 10630 - 1983

3.5 Determination - The total benzene extract containing methyl parathion residues obtained as under 3.3 from the material under investigation is shaken with chilled dilute sodium bicarbonate solution in a separating funnel. The solvent extract is separated and an aliquot of this clarified solution is placed in a 250-ml conical flask, the volume of aliquot taken depending upon the expected residue content. If the aliquot is less than 25 ml dilute to 25 ml with redistilled benzene, add 20 ml of 0.5 N hydrochloric acid and 200 mg of zinc dust and proceed as for the standard solution of parathion for the preparation of standard-curve.

3.6 Reporting of Parathion Residues - Develop colour, read absorbance against blank standard and determine content of parathion in the aliquot taken from the standard curve and report of parathion residues in the material in terms of clg/g ( ppm ).

4. GAS CHROMATOGRAPHIC METHOD

4.1 The residues of methyl parathion are extracted with acetone. The extracted methyl parathion is measured by- gas chromatograph equipped with flame photometric detector. The content of methyl parathion pg/g ( ppm ) is determined by comparing the response with the response for a known standard of similar concentration.

4.2 Reagents

4.2.1 Acetone - analytical grade and glass redistilled.

4.2.2 Precipitating Solution - Prepared by dissolving 2 g of ammonium chloride and 25 g of orthophosphoric acid in 1 000 ml water.

4.2.3 Hyfto Super- Cel

4.2.4 Chloroform - analytical grade, glass redistilled.

4.2.5 Sodium Sulfate - anhydrous.

4.2.6 Benzene - analytical grade, glass redistilled.

4.2.7 Silica Gel Neutral Column - chromatography grade.

4.3 Preparation of Sample, Clean-up and Extraction of Methyl Parathion Residues

4.3.1 Initial Extraction-Crush 100 g of sample at high speed in a blender for 2-3 minutes. To this add 150 ml acetone and macerate further for 2-3 minutes. Filter the mixture and transfer the residue back to the blender for re-extraction with further 100 ml of acetone. Filter and rinse the blender with further- 100 mI of acetone and wash the residue

7

IS:lM3~- 1983

with the acetone rinsings. Transfer the filtrate to a 500.ml round-bottom flask and distil off the solvent on a rotary vacuum evaporator below 45°C.

4.3.2 Clean-@ Plant Co-Extracts by Precipitation - i)issolve the residue in 45 ml of acetone and to it add 50 ml of precipitating solution. Cool in deep freezer for 45 minutes. Add 5 g of filter aid and filter under vacuum. Rinse the flask and wash the residue 2 x 30 ml with 45 : 50 acetone : precipitating solution. Transfer the filtrate and washings to 250-ml separating funnel and extract it with chloroform 3 x 50 ml. Pass the organic solvent phase through anhydrous sodium sulfate and collect it in a 500-ml round bottom flask. Distil off the solvent on a rotary vacuum evaporator below 45’C.

4.3.3 Clean-up of Extract by Column Chromatography - Prepare a chromatography column using 10 g of activated neutral silica gel and glass column of 500 mm height and 18 mm internal diameter, using 90 : 10 benzene acetone mixture. Dissolve the residue in the benzene acetone solvent mixture and transfer it to the column. Elute the column with 30 ml of solvent mixture at the rate of 5-6 ml/min and collect the eluent in 50-ml round-bottom flask. Evaporate the eluent on a rotary evaporator below 45°C to dryness. Re-dissolve the residue in 2 ml acetone added by a volumetric pipette. Swirl well to get a uniform solution. Also prepare a standard solution of methyl parathion in acetone containing 0’05 rcglml.

4.4 Apparatus

4.4.1 Gas Chromatograph - A gas chromatograph equipped with flame photometer detector is operated under the following suggested parameters. These parameters may be varied according to the available facilities provided standardization is done:

Column Glass, 90 cm long, 6.25 mm diameter

Packing 10 percent QV-1 on Gas Chrom Q ( 80-100 mesh )

Carrier gas Nitrogen

Carrier gas flowrate 60 ml/min

Combustion gases Hydrogen 200 ml/min

Oxygen 30 ml/min

Air 100 ml/min

Column temperature 210°C

Injection port temperature 25O*C

8

IS : 10630 - 1983

Detector Flame photometric

Detector temperature 250°C

Retention time for methyl 2 min 45 s. parathion

4.5 Procedure -Inject suitable aliquot ( 3 ~1) into the column using a microlitre syringe. Identify the peak by its retention time and measure the peak area.

4.6 Calculations

Methyl parathion jig/g ( ppm) = A1 x V, x Vs x c A

2

x v 1

ic M xf

where

A, = peak area of sample;

A2 = peak area of standard;

vi = volume of sample injected, in ~1;

V, = volume of standard injected, in ~1;

V, = total volume of sample Solution, in ml;

C = concentration of standard solution, in ppm;

A4 = mass of sample taken for analysis, in g; and

f = recovery factor = 100

Percent mean recovery

Percent mean recovery is determined by taking untreated control sample to which known amount of methyl parathion are added and analysed as described above.

9

i&bIk& SThNDAHUS

ON

PESTICIDES RESIDUE ANALYSIS

IS:

5863 (Part I )-I970 Method for determination of malathion residues: Part I Cereals and oilseeds

5864-1983 Methods for determination of DDT residues in food commodities ( first reuision)

5952-1970 Method for determination of parathion residues in foodgrains and vegetables 6169-1983 Methods for determination of BHC ( HCH ) residues in food commodities

(Jut rtwision ) 10168-1982 Methods for determination of fenitrothion residues in foods 10169-1982 Methods for determination of carbaryl residues in fruits and vegetables