Pesticide Residue Analysis Manual by Kisan Forum Pvt. Ltd.

PESTICIDE RESIDUE ANALYSIS MANUAL

Dr K.K. Sharma Network Co-ordinator All-India Network Project on Pesticide Residues

ICAR

DIRECTORATE OF KNOWLEDGE MANAGEMENT IN AGRICULTURE Indian Council of Agricultural Research Krishi Anusandhan Bhavan I, Pusa, New Delhi 110 012

First Edition Second Edition

June 2007 February 2013

Project Director (DKMA)

Dr Rameshwar Singh

Incharge, (English Editorial Unit) Editing

Dr R.P. Sharma Smt. Shashi A. Verma Dr Aruna T. Kumar Dr Sudhir Pradhan

Chief Production Officer Technical Officer (Production)

Dr V. K. Bharti Shri Ashok Shastri

New Delhi

ISBN : 978-81-7164-124-6

Price :

1500

Disclaimer The manual contains information from very authentic and highly reliable sources. Reasonable attempts have been made to publish reliable data and information. Extensive efforts have also been made to validate the methods described in this manual. However, neither the scientists involved nor ICAR can assume responsibility for the validity of all the materials or for the consequences arising as the result of their use.

Published by Dr Rameshwar Singh, Project Director, Directorate of Knowledge Management in Agriculture, Indian Council of Agricultural Research, Krishi Anusandhan Bhavan, Pusa, New Delhi. Lasertypeset at M/s Archana Printographics, 421-A, 1st Floor, Shahpur Jat, New Delhi 110 049 and printed at M/s Royal Offset Printers, A-89/1, Naraina Industrial Area, Phase-I, New Delhi.

Contents Page no.

Foreword

Foreword to the First Edition

vii

Preface Preface to the First Edition

Acknowledgements

1.0

Objectives of Pesticide Residue Analysis

2.0

Purification of Solvents and Reagents

3.0

Preparation of Pesticide Standard Solutions

4.0

Steps in Pesticide Residue Analysis Chromatography Instruments in Pesticide Residue Analysis

6 19

5.0

6.0

Mass Spectrometry

7.0

Multiresidue Methods (MRMs)

8.0

Multiresidue Methods for Specific Groups of Pesticides

150

9.0

Generalized Multiresidue Methods for Agricultural Chemicals

171

AppendixI

Fat, Water and Sugar Content in various Food Products

205

Appendix II

Insecticides/Pesticides Registered under section 9 of the Insecticides Act, 1968, for use in the Country

211

Appendix III Appendix IV

List of Pesticides/Pesticide Formulations Banned in India

216

MaximumResidueLimits (MRLs) for various Pesticides on different Crops

218

Appendix V Appendix VI Appendix VII

Acceptable Daily Intake (ADI)

230

Definitions of Terms used in Pesticide Residue Analysis

234

NABL Accreditation

240

References

247

Subject Index

248

xii

Pesticide Residue Analysis Pesticides are chemicals used to destroy or control weeds, insects, fungi, and other pests. Some of these pesticides and their metabolites may remain on food as residues. When pesticides are not employed as per the Good Agricultural Practices (GAPs), these pesticide residues can pose significant health risks to consumers as well as to non-target organisms and environment. Pesticide residue analysis may be defined as qualitative and quantitative analysis of samples drawn from agricultural fields, markets and environment for pesticides and their toxic metabolites.

1.0 OBJECTIVES OF PESTICIDE RESIDUE ANALYSIS ;

To study the persistence of the pesticide in or on the soil, plant and water. To establish ‘Maximum Residue Limits’ (MRLs) of pesticides by conducting field trials under supervision adopting Good Agricultural Practices (GAPs) in conjunction with the data obtained from the

toxicological studies. To establish safe waiting periods or pre-harvest intervals (PHIs) on the basis of multilocation trials. To screen agricultural produce drawn from farmers’ fields in order to judge the pesticide usage pattern, so as to educate and train farmers and extension workers in proper handling and safe use of pesticides. To conduct market survey of agricultural produce on thebasis of which dietary intake of pesticide can be predicted and thus assess the risk to general public on account of pesticide residues by comparing daily dietary intake of pesticides with Acceptable Daily Intake (ADI). To screen various methods for effective decontamination of pesticide residues. To monitor environmental samples for pesticide residues.

2.0 PURIFICATION OF SOLVENTS AND REAGENTS Before any analysis is attempted, it is of utmost importance that the purity of the solvents and the reagents should be ensured. If reagent blank analysis does not indicate any contamination, purification of reagents and solvents can be avoided.

Pesticide Residue Analysis Manual

. 2.1 Florisil Florisil, a synthetic magnesium silicate, used as an adsorbent, is subjected to variations in adsorptivity common to most analytical grade adsorbents. Pesticide Residue (PR) grade Florisil is specified because others may exhibit drastically different adsorptivity from what is required. Handling directions are designed to prevent contamination that may interfere with subsequent analysis and to ensure consistent adsorptivity throughout the use of a particular lot of Florisil. Florisil, 60-100 mesh, is calcined (heated) for 3 hr at 500°C and stored in vacuum desiccator or air tight bottle. Before use, reactivate Florisil at 130°C for 2 hr. 2.2

Sodium sulphate/sodium chloride/ magnesium sulphate All methods specify use of anhydrous, granular, reagent grade sodium sulphate/sodium chloride/magnesium sulphate. To remove phthalates that interfere in determination using electron capture detector (ECD), heat sodium sulphate/ sodium chloride/magnesium sulfhate for 4 hr in muffle furnace at 500°C. Store in an air tight glass container or vacuum desiccator. If reagent blank tests indicate that sodium sulphate contributes to interferences, rinse it several times with acetone and ethanol, and then dry.

2.3

Glass wool Pyrex glass wool can have contaminants that interfere with determination. If reagent blank tests indicate glass wool is contaminated, rinse it with hexane and air-dry or heat for lhr at 400°C. Utmost safety precautions should be taken while handling glass wool.

2.4

Celite 545 Purification of Celite shouldbe done by making slurry with 6M hydrochloric acid (HC1) while heating on a steam bath. Then the slurry is washed with distilled water several times until neutral. Again wash with several solvents ranging from

high to low polarity and dry. Impurities interfering with phosphorus selective detectors are removed by heating Celite at 500°C in a muffle furnace for a period of 4 hr. 2.5

Charcoal

Make 200 g slurry with 500 mL concentrated hydrochloric acid, stir magnetically while boiling for one hour. Add 500 mL of water; stir and boil for another 30 minutes. Recover the charcoal by filtering through a Buchner funnel, wash with distilled water until washings are neutral and dry at 130°C. Store in an air tight bottle.

2.6

Magnesium oxide Make 500 g slurry with enough distilled water in one litre Erlenmeyer flask. Heat with occasional shaking for 30 minutes on a steam bath and filter with suction.

Pesticide Residue Analysis Manual Dry for 2-4 hr at 105-130°C and pulverize to pass through No. 60 sieve. About 10% water is absorbed in this process. Store this in an air tight bottle.

2.7

Silica gel Heat silica gel at 130°C for 12 hr. Cool down to room temperature and store in an air tight bottle.

2.8

Solvents The solvents viz., hexane, chloroform, toluene, isopropanol, methanol, dichloromethane, ethyl acetate, isooctane etc., should be of high purity. Before use, purity of the solvents must be checked with gas chromatograph (GC)/high performance liquid chromatograph (HPLC). However, solvents like acetone, diethyl ether and acetonitrile need special treatment for purification.

2.8.1 Acetone Reflux acetone (2.5 L) with KMn04 (0.5 g) until the violet colour persists and collect fractions at 55-60°C. 2.8.2 Diethyl ether Use all-glass distilled diethyl ether and add 2% ethanol as a stabilizer to prevent formationof peroxides. Its practical shelf life is limited, even when alcohol is added.Peroxides form readily and so ‘Peroxide Test’ using appropriate peroxide test paper needs to be done.

2.8.3 Acetonitrile All methods specify use of acetonitrile distilled from all-glass apparatus. To make use of reagent grade acetonitrile, test it for impurities by holding moistened litmus paper over mouth of storage container. If litmus paper turns blue, purify 4 L acetonitrile by adding 1mL 85% phosphoric acid, 30 g phosphorus pentoxide and boiling chips, then allowing standing overnight. Distil using all¬ glass apparatus at 81-82°C, discarding first and last 10% of distillates.

2.9 Cotton Purify cotton using hexane: acetone (1:1 v/v) for 6 hr in Soxhlet apparatus. Dry cotton in an oven at 60°C for 1 hr and store in air tight container. 2.10 Filter paper Pre-wash filter paper (WhatmanNo.l) with acetone to remove contaminants.

3.0 PREPARATION OF PESTICIDE STANDARD SOLUTIONS Organic solvents of high purity are required for the preparation of standard solutions because any interference due to impurities in solvents will lead to error. Besides the purity, the selection of correct solvent as per the individual pesticide solubility and its stability is very important. In general, solvents like acetone,

Pesticide Residue Analysis Manual hexane, isopropanol, toluene, isooctane are used for standard solution preparations. While stock solution and intermediate concentrations are prepared in toluene, acetone or hexane, the working standard is preferably prepared in nhexane. Pesticides analysed on HPLC should be dissolved in acetonitrile/methanol/ water. Ethyl acetate is not desirable for preparation of standards while working with electron capture detector (ECD) since the solvent gives a broad peak. Pesticide-residue grade solvents are essential for preparation of reference standard solutions. Solvents should be checked before use for the presence of interfering substances, if any, by injecting solvent into the determinative system(s) to be used.

3.1 Certified reference materials Reference standard must have a known purity, certified by an authorized agency having National or International traceability. The purity of certified reference materials (CRMs) and the use of appropriate preparation and storage techniques for standard solutions significantly affect analytical results. Reliable and accurate data can be obtained only if correct analytical standard solutions are used for identification and quantification. The laboratory must have a Quality Assurance (QA) programme plan that includes an element of certifiedreference materials and standard solutions. Standard Operating Procedures (SOPs) should include protocols for obtaining, labeling, storing, and handling standards. Reference materials procured from authentic sources should be stored in frost free refrigerator below -10°C in order to prevent undesirable reactions, such as oxidation, rearrangement, or hydrolysis. Improper storage can lead to loss of integrity of previously acceptable standards. Storage conditions also must prevent the possibility of external contamination. Storage requirements are dependent on the chemical and physical properties of the chemical of interest and are much more stringent for volatile, reactive or unstable compounds. It is essential to have a reference materials file for maintaining records. Certified reference material must be brought to room temperature in order to avoid absorption of moisture. Primary stock solutions Preparation of stock solutions is of utmost importance in pesticide residue analysis. Any mistake in the preparation of stock solutions would give erroneous results and the entire exercise will be futile. Check the certificate of analysis and the date of expiry of the certified reference material (CRM). • Take out the CRM bottle from the refrigerator and wait for 1hr until the CRM attains room temperature. • Weigh the calibrated volumetric flask (25 mL or 50 mL) using a calibrated analytical balance and tare the weight. Use spatula for transferring solid CRMs, and capillary tube for viscous/ semisolid CRMs.

3.2

•

Pesticide Residue Analysis Manual

•

Transfer carefully about 10-20 mg of CRM into the volumetric flask and note the weight. the CRM using approximately 2-3 mL of analytical grade Dissolve • acetone for GC analysis. For LC analysis, use acetonitrile or methanol. • Make up the volume with analytical grade hexane: toluene (1:1 v/v) for GC and use acetonitrile or methanol for LC. However, in some cases, the solvent may be very specific depending upon the solubility of the pesticide. Label the flask with name of the standard, concentration of the stock • solution, solvent used, date of preparation and date of expiry. Calculate the final concentration of the primary stock solution using the formula: Weight of CRM (mg)

Purity of CRM (%)

Concentration =

x 1,000 pg/mL (ppm)

Final volume (mL)

100

For example: If purity of CRM is 98.4% and the weight is 12.43 mg, the final concentration of the primary stock solution in 25 mL will be: 12.43 98.4 Concentration = x 1,000 = 489.25 pg/mL (ppm) 100 25

3.3 Working standard solutions Stock solutions are normally diluted to prepare 100 ppm intermediate working standards. For example, from the above primary standard of 489.25 ppm, to prepare 100 ppm intermediate standard in 25 mL volumetric flask, the formula is as follows. N.V, = N2V2 100 ppm x 25 mL V, (mL) = = 5.10 mL 489.25 ppm

Thus, 5.10 mL of primary stock solution is required to be diluted in 25 mL for preparing 100 ppm intermediate stock solution. Generally, acetone, toluene, hexane are used for preparation of primary and intermediate standards. For LC, methanol or acetonitrile should be used for serial dilution from stock solution. Label the flask with name of the standard, concentration of the stock solution, solvent used, date of preparation and date of expiry. Note: Care should be taken to see that the working standard solutions do not get concentrated due to repeated opening of the volumetric flask. The stability of the working solutions must be monitored at regular intervals. After taking required volume from primary stock solution for preparation of intermediate stock, note the weight of the primary stock solution and note the same in records before keeping it back in refrigerator. This helps making sure that the weight is made-up with the same solvent before further use, if any loss or evaporation during storage.

3.4. Standard checking New stock solutions that are prepared from neat pesticides currently used

Pesticide Residue Analysis Manual in the laboratory shall be compared to the old stock solution. The two standards must match within 10% relative per cent difference (RPD). Stock solutions of neat pesticide standards not previously prepared or not currently in use in the laboratory shallbe prepared in duplicate and the two standards shouldbe compared to each other. Responses for standards of comparable concentrations must match within 10% RPD. RPD--

(RF,-RF2) RF,+RF2

x 100

where RFj is the response factor (peak area or peak height) of the first analytical standard and RF2 is the response factor of the second standard. If standards do not match, a third standard shall be made and compared. This process shallbe continueduntil two matching standards are prepared. New stock solutions that are prepared from neat pesticides currently used in the laboratory shall be compared to the old stock solution. The two standards must match within 10% RPD.If the two standards do not match, the problem must be identified and solved before the standard is used for quantitation. A suggested approach is to make new dilutions of both the old and new standards to check for dilution errors. If no dilution errors are found, a second stock dilution should be made to determine whether an error was made in the original preparation from neat material. If these two stocks match, then the standard may be used. If they do not match, a third stock solution should be made. Whenever possible, duplicate injections shall be used. Documentation of the standard checking process shall be kept through appropriate records (i.e. logs). Chromatograms of all standards shall be kept indicating the standard comparisons of old and new standards and the calculated difference.

4.0 STEPS IN PESTICIDE RESIDUE ANALYSIS Pesticides may occur in food at very low concentrations, usually at ppm levels. Measuring such small amounts of pesticides is the function of pesticide residue analysis. A variety of analytical methods are currently used to detect pesticide residues, and all contain certain basic steps, that include: • Sampling (collection, transport and storage of sample) • Sample preparation (preparation of laboratory sample): Sample is prepared by chopping, grinding, blending • Extraction: removal of pesticide residue from components that are present naturally • cleanup: cleanup and purification is the removal of constituents that interfere with the analysis of the pesticide residue of interest—this step includes partitioning and purification (detection), quantification (determination) and Identification • confirmation

Pesticide Residue Analysis Manual 4.1 Sampling Sampling may be defined as the procedure or step adopted to obtain a representative quantity from a large consignment so that the selected representative quantity can be handled conveniently. The analytical results will be meaningful and would truly reflect the quality of the entire consignment only if sampling is done properly and the sample drawn is true representative of the entire lot. The samples are usually drawn in a randomized manner and mixed well. The quantity of samples may differ from one commodity to another.

4.1.1 Sampling procedures for raw andpartially processedfoods â&#x20AC;&#x2DC;Sample Collectionâ&#x20AC;&#x2122; is the process of taking portion(s) of a food or other substance for laboratory analyses, so that the resulting analytical data and conclusions can be applied to the original food or substance sampled. Remember that quality results can be obtained only from a true representative sample. The sample should always be a representative and sufficiently large one. Nearly any type of food can contain pesticide residues which can occur either from direct application by way of spraying/fumigation, or indirectly from migration of theresidue (contaminated milk, meat etc.) into the food. Therefore, a variety of sampling implements and tools are used. Minimum sample sizes for various commodities are given in Table 1. 4.1.1.1 Vegetables and fruits: As far as possible, a sample of a fruit or vegetable must be taken directly from the field or from grower at the time of, or shortly after harvest. In such cases, the sampler must decide how to divide the lot if a random sample is desired. The history of pesticides applied to a given field is extremely important and such information is vital for the analysis. Superimpose an imaginary grid on the field dividing into approximately 100 areas and randomly select 10 of these areas to form a representative sample of the field. Collect 0.51 kg portions from each of these 10 areas and combine them to form the sample. In case of leafy vegetables (cabbage, lettuce etc.), the portions collected from a growing field should be representative of the local commercial practices. For example, if the local practice is to strip the outer leaves at the time of harvest, then this should be done while collecting the sample. If the fruit and vegetable samples are to be taken at transport (or) storage (wholesale/retail shops), collect 10 samples, 1kg each, randomly from the lot. It is recommended that a minimum total sample consisting of the recommended number of portions, depending upon the lot size, should be collected. 4.1.1.2 Grains: Grains are almost never sampled at the grower or primary producer. If such samples are to be taken, the sampler can use the procedure recommended for fruits and vegetables. Nonetheless sampling of grains during transport/shipment and storage is important, because residues on grains are typically due to post harvest use of fumigants. Sampling of bulk lots can be done using the large sector trier to probe the surface of the bulk lot in several places. A sample size of 5 kg should be taken from five locations. 4.1.1.3 Milk: Fluid milk, like grains, should never be sampled at primary

Pesticide Residue Analysis Manual producer. Since fluid milk is relatively homogeneous, a total sample size of two litres is usually sufficient from the producer or each tank. Sample portions need not be packed separately and can be combined in one sample container. Glass jars are most commonly recommended. Where pouched milk is to be sampled, a minimum of three 500 mL pouches of similar nature are to be taken. 4.1.1.4 Eggs and egg products: Shell eggs can be sampled at the primary producer or at the packer, prior to shipment. The sample size for unpackaged shell eggs sampled at the producer is six dozen eggs. Shell eggs are quite fragile and if suitable packaging is not available for transport to the laboratory, then the eggs can be broken, magma placed in glass jars and the shells can be discarded. For the sampling of shell eggs at packer, take one dozen eggs for 15 cases. For 16 cases or more, take a total of 10 dozen eggs, each from 10 cases selected at random. If a sample of liquid eggs prior to freezing is to be taken at a processor, then collect 0.5 -1 L portion size. 4.1.1.5 Fish and shellfish (shellfish includes shrimp, lobsters, crabs and oysters): When samples of fish are to be taken at the dock or at a processor who is cleaning and cutting for the fresh market, the lots should be divided into three sizes, the number of portions to be taken to represent each size can be determined using following formula. Ns = 12 (NS/N) where Ns, is no. of fish of a given size to be sampled; NS, total no. of fish of that size in a lot; N, total no. of fish of all sizes in a lot; 12; total no. of portions to be taken

Draw the required number of fishes randomly from each lot. 4.1.1.6 Animalfeeds: Animal feeds including hay, silage, grains, byproducts and commercial feed rations, are normally sampled, only when there is reason to believe that a pesticide problem exists in foods. Therefore, sampling procedure is typically selective, not random. If the feed is the bulk, collect 10 x 1 kg portions to comprise one sample. 4.1.2 Sampling procedures for processedfoods The key consideration is the packaging size, because the degree of processing can vary considerably. The foods to be sampled need not be divided into groups as is done for raw and partially processed foods. When the samples of processed food are taken at the processor, the sampler has a unique opportunity to obtain information about the sample that usually is not available. Codex recommended sampling schedule for all processed packaged foods is given in Table 2. 4.1.3 Sampling procedures for soil and water The sampling of soil and water is typically done for either to investigate the pesticide residues or to routinely monitor environmental samples. Soil samples should be taken from growing fields in the grid pattern uniformly distributed, so that each area of the field is sampled. A 3 x 3 grid with 9 total sample portions is suggested for smaller fields, with 4x4 (16 sample portions) for the medium sized fields, 5x5 and even larger grids are used for very large fields. Each sample

Pesticide Residue Analysis Manual Table 1. Minimum sample size for various commodities required for sampling Type of food

Minimum amount of sample to be collected

Fruits and vegetables Small or light foods with unit weight of 25 g or less (e.g. berries, peas) Medium-sized foods with unit weight usually between 25 and 250 g (e.g. apples, oranges, carrots, potatoes) Large-sized foods with unit weight more than 250 g (e.g. cabbage, cauliflower, melons, cucumbers etc.) Dairy products Whole milk, cheese, cream, butter Eggs (whole) Meat Poultry, fat, fish, and other fish and animal products Oils and fats Cereals and cereal products Spices (chillies, cumin, coriander etc.) Tea (CTC/ fermented/ black) Honey Animal feed

1 kg 1 kg (at least 10 fruits) 2 kg (at least 5 fruits)

0.5 kg 10 nos or 0.5 kg 0.5 kg 0.5 kg 1 kg 0.25 kg 0.25 kg 0.25 kg 1.0 kg

Table 2. Codex recommendations for sampling of processed packaged foods

Number of retails units in the lot

Minimum number of retail units to be sampled

1-25 26 -100 101-250 Over 250

1 5 10 15

site represents one portion of the total sample, and at each site 2 soil plugs about 15 cm deep and 3 to 5 cm in diameter are to be taken.And 2 plugs, when combined, become the sample portion of that sample site. Another common soil sampling method for a field or other area is to take ‘5’ portions in a ‘Z’ pattern. An example of 3x3 sampling grid is designed by X pattern sampling (Fig. 1). Sampling tools include soil augers or conical triers. Place each portion of soil sample into separate glass jars covered with aluminium foil. It is recommended to chill soil samples to 4°Cfor transport to the laboratory. The glass jars for collecting soil and water samples should be rinsed thoroughly with methanol and dried. For water samples, collect 3 L of water to represent one sample from one water source, and transport the sample to the laboratory at 4°C or lower temperature.

Pesticide Residue Analysis Manual X - pattern soil sampling

Z - pattern soil sampling

X X

Fig.1. Sampling patterns

Storage of the sample Polythene bags, glass bottles or vials may be used as containers for holding the sample. It shouldbe borne in mind that if the samples are not stored properly, changes may take place during storage leading to unreliable results. Usually the samples are stored in deep freezer maintained at -10°C or below. Alternatively, instead of storing the sample, the extract of the sample may be stored in a refrigerator. The later method is better, more convenient and hence should be preferred. Control sample extracts fortified with pesticides can be stored along with samples to know total degradation of pesticides during storage.

4.2

Sample preparation Once the sample has arrived at the laboratory, the analyst must prepare the sample for an analyzable sample portion that represents the whole sample. The first step is to reduce the total sample (when necessary) to a manageable portion i.e. ‘sub sampling’. A ‘reserve portion’’ of the original unprepared sample is necessary or desirable, and hence should be stored for future use, such as for check analysis or for additional analysis required for confirmation test. This is sometimes done so that there is ‘unanalyzed reserve’ and the composite of the remaining sample (or) sub-samples becomes the ‘analyzed reserve’. Then determine what portion of the sample can be used for analysis i.e. for raw and partially processed foods (this is called as the ‘edible portion’) and for processed foods (this is the ‘required portion’). Both represent the part of the food that is normally consumed. The final step is to physically homogenize the sub-sample aggregate by chopping, grinding, blending etc. Different types of choppers, knives, grinders, and commercial warring blenders are used for this purpose. Soil samples are always non-uniform in both particle size and composition. Hence to ensure proper extraction, the soil sample must be ground to a uniformly small particle size. Portions of raw agricultural commodities to be analyzed for pesticide residue analysis are given in Table 3. The portions of processed food to be analyzed for pesticide residues are given in Table 4. 4.3

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