Guidelines on Producing Pesticide Residues Data from Supervised Trials

FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS

Rome, 1990

CONTENTS GENERAL INTRODUCTION

PART I DEFINITION OF THE RESIDUE

1. 2. 3. INTRODUCTION METABOLITES AS COMPONENTS OF THE TOTAL RESIDUE NON-EXTRACTED OR "BOUND" RESIDUES; CONJUGATES 1 1 2

PART 2 METABOLISM/DEGRADATION STUDIES IN CROPS AND PLANT PRODUCTS

(To be completed)

PART 3 SUPERVISED RESIDUE TRIALS IN CROPS AND PLANT PRODUCTS

1. INTRODUCTION 2. DESIGN OF RESIDUE TRIALS 2.1 Trials Lay-out 2.1.1 Selection of Sites 2.1.2 Number of Sites 2.1.3 Replication 2.1.4 Plots 2.1.5 Type/Variety of Crop/Commodity/Cropping System 2.2 Application of the Pesticide 2.2.1 Formulation 2.2.2 Methods of Application 2.2.3 Dosage Rates 2.2.4 Number and Timing of Applications 2.2.5 Additional Pesticides 2.3 Degradation Studies 2.4 Residue Decline Studies 3. SAMPLING IN PESTICIDE RESIDUE TRIALS 3.1 General Recommendations 3.1.1 Methods of Sampling 3.1.2 Replication 3.1.3 Sample Handling 3.2 Contamination 3.3 Control Samples 3.4 Sampling in Decline Studies and at Normal Harvest Time 3.4.1 Sampling in Decline Studies 3.4.2 Sampling at Normal Harvest Ti me 3.5 Detailed Sampling Procedures 3.5.1 Fruits and Tree Nuts 3.5.2 Vegetables 3.5.3 Grasses 3.5.4 Seeds 3.5.5 Herbs and Spices; Tea Leaves; Hops; Beer 4. SAMPLING PROCESSED COMMODITIES 5. SAMPLING STORED COMMODITIES 5.1 Sampling from Bulk 5.2 Sampling Bagged Commodities 5.3 Sampling Fruit and Vegetables in Packing Houses Contents 1 1 3 3 3 3 3 4 4 4 4 4 5 5 6 6 6 6 6 7 7 7 8 8 8 9 9 9 10 13 15 15 16 16 16 16 16 iii

6. SAMPLE SIZE REDUCTION 7. SAMPLE PACKING AND STORAGE 7.1 Packing 7.1.1 Containers 7.1.2 Shipment of Samples 7.2 Labels and Records 7.3 Sample Reception and Handling 7.4 Storage 8. REPORTING ON FIELD ASPECTS OF RESIDUE TRIALS 8.1 General Information on the Supervised Trial 8.2 Application Data for Field Trials 8.3 Application Data for Post-harvest Trials 8.4 Sampling Data 9. ANALYTICAL METHODS FOR RESIDUES IN CROPS AND PLANT PRODUCTS 10.REPORTING RESULTS OF RESIDUE ANALYSES 11. FURTHER READING Figure 1. Methods of Systematic Sampling Appendix 1. Report on Pesticide Residue Trial. Part A. Field Report Appendix 2. Report on Pesticide Residue Trial. Part B. Analytical Report

17 17 17 17 18 18 19 19 20 20 20 21 21 22 22 23 24 25 28

PART 4 METABOLISM STUDIES AND SUPERVISED RESIDUE TRIALS IN ANIMALS

1. INTRODUCTION 2. DESIGN OF STUDIES 2.1 Studies with Radiolabelled Compounds (Metabolism Studies) 2.1.1 Oral Administration Studies 2.1.1.1 Sampling and Analysis 2.1.2 Dermal Treatment Studies 2.2 Animal Studies with Unlabelled Compounds ("Cold" Studies) 2.2.1 Oral Administration Studies 2.2.1.1 Sampling and Analysis 2.2.2 Dermal Treatment Studies 3. STORAGE OF SAMPLES 4. REPORTING OF RESIDUE TRIALS 4.1 In-life Part of the Study 4.2 Analysis of the Sample Appendix 1. Suggested Procedures for Dosing Animals with Radiolabelled Chemicals 1 1 2 2 4 4 5 6 7 7 7 7

PART 5 LOWER PRACTICAL ANALYTICAL LEVELS FOR THE DETERMINATION OF RESIDUES OF PESTICIDES
Lower Practical Analytical Levels for the Determination of Residues of Pesticides 1

iv

Contents

GENERAL INTRODUCTION The use of pesticides on food and forage crops, or on livestock for ectoparasite control, can lead to residues in food and the environment. The significance of pesticide residues depends upon 1. 2. The nature and level of residues present Their toxicology

Guidelines on the extent to which residues data are required and on the many facets of evaluating pesticide residues in the light of their toxicity have been developed separately. Guidelines for predicting dietary intakes of pesticide residues have been developed by the Joint UNEP/FAO/WHO Food Contamination Monitoring Programme in collaboration with the Codex Committee on Pesticide Residues (Document WHO/EHE/ FOS/88.2, 1988). One of the basic prerequisites of such predictions and assessments is the availability of reliable data on pesticide residues in food, feed and the environment, derived mainly from adequately designed supervised trials. Residues data on the whole commodity as it moves in trade should be supplemented, where appropriate, by data on the effects upon residue levels of processing and cooking. This document contains guidance on the various aspects of producing such data. These guidelines have been developed in the interests of improving and harmonising the procedures used in obtaining the data, to avoid undesirable variations in methods and techniques used in the selection, preparation and analysis of samples. They will be of particular value to all countries which are in the process of developing their procedures for the efficient control of pesticides and their use. The metabolism or degradation of the pesticides in or on plants or soil may have a major influence on the identity of the residues which need to be measured in samples taken in supervised field trials. It is often valuable to elucidate the behaviour of the different components of the residue with respect to translocation, leaching or volatilisation. Studies of binding to, or conjugation with, plant, animal or soil constituents provide data on the bioavailability of residues and their ease of extraction. Knowledge of the latter is essential in the design of residue analytical methods. Considerations which are basic to the decision as to whether or not specific metabolites should be included in the definition of the residue are reviewed in detail in Part 1 below. The main reason for carrying out supervised trials is to assist the evaluation, during both development and registration, of the efficacy and safety of the product. In some countries, another important reason for carrying out these studies is to obtain the data for establishing maximum limits for residues of the pesticide in food or animal feedstuffs. Arrangements need to be made in advance if the samples have to be stored for any length of time, or as frequently happens transported elsewhere, before analysis. To be of value, residue trials must be reported properly. Guidance on reporting is contained in individual parts of these guidelines as appropriate. Because of legal and commercial implications residue trials must be carefully planned, conscientiously executed, carefully evaluated and intelligently interpreted, to ensure that the decisions taken are meaningful and that they reflect the practical situation resulting General Introduction v

from approved uses of the chemical. Co-operation between scientists of several disciplines is usually necessary to achieve the desired result. Careful consideration must be given to all the factors and their variability. The analyst's residue data may be accurate but the results can be misleading because of inadequate sampling before material reaches the laboratory. An erroneous result is worse than none at all. Attention is drawn to the Guide to Codex Recommendations Concerning Pesticide Residues - Part 4, Codex Classification of Foods and Animal Feeds. - Part 6, Portion of Commodities to which Codex Maximum Residue Limits Apply and which is Analysed. Persons undertaking supervised residue trials which may be used to establish maximum residue limits for food and feed commodities are strongly urged to generate the information in a form which is consistent with these parts of the Codex Guide. These guidelines are being issued in loose-leaf form, to permit appropriate parts to be updated as necessary in the light of experience.

General Introduction

vi

Part 1 Definition of the Residue

INTRODUCTION The results of supervised residue trials depend upon analytical methods being available to determine the compounds which need to be included in the definition of the residue. There are two general considerations which are basic to the decision as to whether or not specific metabolites or degradation products should be included in the definition and expression of a residue: (1) (2) their basic toxicology; and their presence in significant amounts.

For these two points to be considered, the composition of the residue must be known. Furthermore it is often of some help to elucidate the behaviour of the components with respect to translocation, leaching or volatilisation. Studies of binding to, or conjugation with, plant, animal or soil constituents provide data on the bioavailability of residues and their ease of extraction, information on the latter being essential in the design of routine residue analytical methods. Recommendations concerning metabolism and degradation studies are contained in later parts of this guide. 2 METABOLITES AS COMPONENTS OF THE TOTAL RESIDUE A number of principles and subsequent specific options may be used in deciding which metabolites/degradation products to include in the definition of the residue, as listed below. They are based largely on the discussion in the report of the 1979 JMPR (FAO Plant Production and Protection Paper 20, Section 2.7). A. (1) (2) (3) Residues may be expressed as the parent compound if., there are no metabolites; metabolites are known to be insignificant and can be ignored; metabolites are known to be of toxicological concern but are not present in significant amounts; (4) metabolites of toxicological concern are known to be present in significant amounts and the analytical method measures the total residue as a single compound which may be numerically expressed as the parent compound. In this case the metabolites included in the residue are listed; and (5) metabolites of toxicological concern are known to be present in significant amounts and the analytical method measures the parent compound and metabolites separately. In such cases the compounds in the total residue may be expressed additively as parent compound, with recalculation for differences in molecular weight only when the differences are substantial (e.g. greater than 20%). B. Residues may be expressed as a single metabolite or alteration product if., (1) the parent is quantitatively converted to another chemical entity, e.g. aluminium phosphide to phosphine; (2) metabolites of toxicological concern are known to be present in significant amounts and the analytical method measures the total residue as a single

Definition of Residue

metabolite. The results may be expressed as that metabolite but the compounds included in the residue should be listed; and (3) metabolites of toxicological concern are known to be present in significant amounts and the analytical method measures the residue components, including parent compound if present, separately. The result may be expressed additively in terms of a metabolite with recalculation for differences in molecular weight only when differences are substantial (e.g. greater than 20%). C. Residues may be expressed as parent and metabolites separately if: metabolites are known to be present in significant amounts and the analytical method measures each component of the total residue separately. All metabolites and/or degradation products included in the definition of the residue should be listed regardless of the method of determination. Highly toxic impurities in pesticides must be treated separately. 3 NON-EXTRACTED OR "BOUND" RESIDUES; CONJUGATES A special problem in both metabolic studies and the description of residues is created by conjugates and bound residues. Any statement to the effect that residues are "bound" (to a substrate, or matrix) must necessarily be a function of the effort undertaken to "free" them, e.g. of the extraction procedure used. It is preferable, therefore, not to use the term "bound residue" in an analytical context but to replace it by "not extracted residue", each individual case being supported by a statement of the method of investigation. In instances where residues are found to be unextractable under usual chemical laboratory conditions, e.g. without running the risk of changing them by applying very rigorous extraction methods, they may be termed "bound" or "chemically unavailable". The question then remains whether such residues will be available to biological systems, e.g. to microorganisms, invertebrates, or following crops (when persisting in the soil), or to the digestive tract of ruminants or other warmblooded animals, including man (when persisting in certain constituents of feed or food). If chemical and biological unavailability of residues can be demonstrated, such residues should beor metabolites may be subject to hydrolysis in "Conjugated" residues considered negligible. biological systems different from those in which they were formed. If a conjugate is formed in edible plants but not in animals, it need not be regarded as a residue if it can be demonstrated, or it is considered highly likely, that the conjugate passes unchanged through animals. These considerations, for both unextractable and conjugated residues, will apply only in instances where the compound in question (e.g. the "bioavailable" substance, or the aglycone) satisfies the agreed definition of a "residue". If it does so, the compound should be included in the residue analytical procedure.

Definition of Residue

Part 2 Metabolism/Degradation Studies in Crops and Plant Products

This section of the guidelines will be issued at a later date.

Metabolism Studies in Crops

Part 3 Supervised Residue Trials in Crops and Plant Products

1 INTRODUCTION Residues remaining on or in crops depend on many interacting influences of varying importance. These include the manner and timing of application, the formulation used, subsequent growth dilution, ratio of crop surface to mass, volatility of deposit and degree of adsorption on to and absorption into surface layers. The residue resulting from a given method, timing and dosage of pesticide application will also vary with site and climate; the limits of such variation are important to the assessment of safety and particularly to the establishment of maximum residue limits. In order to obtain the data necessary to estimate a maximum level, samples should be analysed from crops treated with pesticide according to good agricultural practice and grown under a representative range of agricultural and climatic conditions. Factors which may influence the disappearance of residues should be recorded. The procedures outlined in these guidelines refer to "supervised trials" which have served for many years as the primary source of residue information for the registration of pesticides and for setting maximum residue limits. These studies must be carefully planned, conscientiously executed, carefully evaluated and intelligently interpreted to ensure that the decisions taken are meaningful and that they reflect the practical situations which result from approved uses of the chemical. Co-operation between scientists of several disciplines is usually necessary to achieve the desired result. Where appropriate, studies should be carried out according to Good Laboratory Practice (GLP) standards. It may be helpful to consult registration authorities regarding specific details of protocols.

DESIGN OF RESIDUE TRIALS In designing a residue trial, early consideration must be given to the intended use of the residue data to be obtained. If data are to support applications for registrations, or the establishment of maximum residue limits through registration procedures, results from a number of experiments in several geographical areas, e.g. of different soil or climatic conditions, are usually required. These should encompass typical farming practices and periods of the year when the product will be used. When a product is applied to a crop at or near maturity, studies on residue decline with time are usually needed. Major trials should be done with proposed commercial formulations. Work with laboratory formulations is frequently of limited value since the fate of the residue may be influenced by the nature of the formulation. It is preferable to make the application with commercial equipment in a manner analogous to that used by farmers. Trials should be designed to cover a range of representative field conditions, typical periods of the year, and those cropping and farming practices which are commonly encountered. Since climatic conditions have an important influence on the persistence and performance of a chemical, trials should be carried out in those areas where the product is to be finally used. Whenever possible, and certainly whenever it is considered likely to influence residue levels, trials should be repeated on different varieties, different stages of

ResidueTrials in Crops

growth at typical periods of the year and under different agricultural regimes, to determine residue levels under various conditions. Since one of the objectives of residue studies is to provide the basis for the estimation of maximum residue levels, the design of the experiments should be directed to the determination and evaluation of the conditions and factors which lead to the highest residue levels following recommended use patterns. If it is anticipated that the interactions of various factors could produce widely varying residue levels, experiments should be designed to demonstrate the effect of such interactions on residues. Residue trials have to be especially designed in most cases and the presence of a target organism is not necessary. Trials intended for biological evaluation may be suitable for obtaining residue samples if the full range of the recommendations is reflected and if the plot size is large enough to obtain adequately representative samples. Where the product is applied to the growing plant, the prime objective should be to obtain data on the residue remaining in or on the crop at the time of harvest. If significant residues are expected at the time of harvest, it will be necessary to obtain information on the effects of storage and processing on the residue after harvesting, as this will provide a basis for assessing the likely intake by consumers. After postharvest treatments, commodities should also be sampled when they leave the store. When the product is applied to the harvested crop, information should be obtained on the alteration of the amounts and nature of the residues during the normal course of storage and handling of the crop after treatment. It is desirable to know in the case of a fumigant, for example, how much is taken up by a foodstuff during treatment and whether and how quickly it is lost by aeration. Residue data will normally not be required for a crop which is not used for human or animal consumption, such as flower bulbs and ornamental shrubs, but the possible persistence of pesticides in the soil and their subsequent uptake by edible crops should not be overlooked. Where the use of a pesticide is likely to result in soil residues after harvest of the treated crop or residues in water used for irrigation purposes, data on residues in edible parts of subsequent crops should be obtained. Because of the large variety of crops and commodities on which a pesticide may be used, it may not always be necessary to carry out trials on all crops, species or commodities. The Codex Committee on Pesticide Residues has recently adopted a Classification of Foods and Animal Feeds (Part 4 of the Guide to Codex Recommendations Concerning Pesticide Residues) in which assignment of a commodity to a group involved considerations such as botanical family, part(s) of the commodity to be used as food or feed, potential for acquiring and retaining residues, and agricultural practices. Although residue data will normally be required for most major commodities in a group, a study of this classification will suggest circumstances in which the results of trials on one or more major commodities may be regarded as applicable to others in the group, provided the rates and methods of application of the pesticide and cultural conditions are similar. However, care must be exercised in the extrapolation of the results from one commodity to another.

Residue Trials in Crops

2.1 2.1.1

Trials Lay-out Selection of Sites Trials should be carried out in major areas of cultivation or production and should be sited to cover the range of relevant representative conditions (such as climate, season, soil, cropping system and farming practices) likely to be met during the intended use of the pesticide. Areas or sites where atypical conditions occur and which are not representative should be avoided unless it is expected that use under these conditions could result in higher residues. Number of Sites The number of sites needed depends upon the range of conditions to be covered, the uniformity of crops and agricultural practice, and the data already available. Whilst it may not be necessary to require that trials be repeated for all regions with different ecological and climatic conditions in which the use of the product is intended or all seasons with widely varying climatic conditions, sufficient data must be available to confirm that the patterns determined hold for all regions and the total range of conditions including those which are likely to give rise to the highest residues. Trials in at least two growing seasons are normally needed.

2.1.2

2.1.3 Replication Since the variations in residue levels between replicates at individual sites are small compared with those between sites, it is usually not necessary to replicate treatments at individual sites. However, it is useful to have three or four replicates at one site to study experimental uniformity and determine the within-site variation. In glasshouses or stores, the use of products with a high vapour pressure (fumigants, aerosols, smokes or fogs) will generally not allow true replication at one site. If an efficacy trial with replicated plots has to be sampled, then samples taken from plots receiving "identical" treatments may be analysed separately to provide an indication of the within-site variations. 2.1.4 Plots Residue data should not be generated from plots which are too small to be representative. The size of the individual plots will vary from crop to crop but should be large enough (1)to apply the pesticide in an accurate and realistic manner, preferably under the same conditions as in normal local commercial practice; and (2) to provide representative crop samples (see 3. SAMPLING IN PESTICIDE RESIDUE TRIALS). A control plot for the supply of untreated samples is necessary for the reasons indicated in 2.2.3. The control plot should be large enough to satisfy these requirements and should be located close enough to treated plots to secure identical growing and climatic conditions. However, it has to be sufficiently separated to exclude any contamination from the treated plots (e.g. by drift, volatilisation or leaching). For products with a high vapour pressure (e.g. fumigants, aerosols, smokes or fogs) used in glasshouses or in stores, provision

Residue Trials in Crops

should be made for control samples from untreated crops or stored products, e.g. in separate glasshouses, stores or compartments, grown or kept under almost the same conditions. A sufficient buffer zone (lanes, guard rows) should be left between plots to prevent cross-contamination. In general, close proximity of a high-dose treatment and control plot should be avoided and untreated plots should be upwind of treated plots. 2.1.5 TypeNariety of Crop/Commodity/Cropping System The type or variety of a crop and the way in which it is grown may influence the residue pattern. In these circumstances, data should be available on the most commonly used type or variety or cropping system and on the factor or combination of factors most likely to result in the highest residue levels. 2.2 Application of the Pesticide

2.2.1 Formulation The formulation to be marketed (or one of similar type and composition) should be used in the residue trials. Before the introduction of a major change of formulation, it may be necessary to obtain a limited amount of information from comparative trials to check that the residue levels will not be significantly affected by the change. The need for such information will depend upon the extent of the formulation change involved: it would probably not be necessary for minor changes. 2.2.2 Methods of Application The method of application should reflect the intended recommendation. As far as possible, applications should be made with equipment similar to that used in local commercial practice. Experimental plot applicators are convenient and readily calibrated and can be used in residue trials as an alternative method of application provided they are compatible with normal practice. Care should be taken to ensure uniformity of application and to avoid contamination of neighbouring plots, either during or after application. In glasshouses, when using products with a high vapour pressure (fumigants, aerosols, smokes or fogs), the whole glasshouse, store or compartment has to be treated. It will not be possible, in general, to have replicated plots, other dosage rates, and untreated controls in the same glasshouse, store or compartment. With fumigants, aerosols, smokes and fogs, special attention should be paid to equal and uniform distribution and a preliminary check on this particular aspect may be required. Furthermore, recommended procedures in the glasshouse or store during and after the application (e.g. doors and windows shut or open) should be carefully followed. 2.2.3 Dosage Rates The thrust of the work should be directed towards studies in which the product is applied at the rate or rates at which it is likely to be used commercially. However in some of the trials inclusion of a higher rate, preferably double the commercial
4 Residue Trials in Crops

rate, can be useful if considerations of phytotoxicity allow. This will give guidance on likely residue levels should dosage rates exceed recommendations and allow some assessment of the relationship between dosage and residue level. When sprays are used, the volume per unit area should reflect practical conditions and should be the same for all sites in the region. The volume applied should be recorded, if relevant. The concentration of pesticides should be expressed as units of active ingredient per unit area, recorded in international (SI) units. In glasshouses and stores, dosage rates of products with a high vapour pressure (fumigants, aerosols, smokes or fogs) should be expressed both per unit area and per unit volume. In addition to the treatment rates mentioned already, a control plot should always be included in any residue experiments, to provide the analyst with a sample known to be free from residues of the pesticide under investigation. Control samples are needed: (a) to ascertain that no artefact in the crop derived from local conditions could give rise to interference in the analysis; (b) to establish the recovery level of the pesticide from the crop or soil by the analytical method; (c) in the case of a new crop or pesticide, to investigate the storage stability of any residue. When two or more dosage rates are included, particular care should be taken to avoid crosscontamination. In glasshouses or stores, the use of products with a high vapour pressure (fumigants, aerosols, smokes or fogs) will not allow in general for more than one dosage rate per glasshouse, store or compartment, nor for an untreated control. Provision has to be made to obtain samples from untreated crops or commodities and from treatments at another dosage rate, e.g. from separate glasshouses, stores or compartments, in which the crops are grown or stored under as nearly the same conditions as possible. 2.2.4 Number and Timing of Applications The presence of a target organism is not essential and, regardless of the occurrence or level of infestation in the residue trial, the number of treatments and the intervals between applications should reflect the latest application and highest dosage rate of the product to be recommended. 2.2.5 Additional Pesticides Unless unavoidable, no pesticide in addition to that to be analysed should be applied to control or test plots before or during the period of the trial. However, since it is of primary importance that both the untreated and treated plants should be healthy, the use of other pesticides may be necessary. In this case only those pesticides that will not interfere with the analysis for the residues of the test compound may be used. The pesticides used should be noted; where possible the advice of the analyst should be obtained beforehand. It is important that control and test plots receive the same treatment, with the exception that the product under study is not applied to the control plot.

Residue Trials in Crops

2.2

Degradation Studies Residue trials are sometimes used to obtain information which, although supplementary to the main purpose of the trial, is extremely valuable in studying the properties of the compound under test and in enabling a fuller safety assessment to be made. The trial may be used, for example, to supplement studies of the metabolism and degradation of a pesticide under field conditions. Such uses should be given early consideration in the planning of the trial.

2.3

Residue Decline Studies The decline of a pesticide deposit may be due to one or more of several factors, principally: 1. 2. Physical removal, e.g. by washing or volatilisation Chemical degradation or metabolism in or on the plant Apparent decline due to crop growth dilution

3.

Decline studies are of particular value in understanding the significance of these factors, especially when at the moment of application a considerable amount of the future consumable part of the crop is already developed or when soil-applied, volatile or systemic pesticides are used.

3.
3.1

SAMPLING IN PESTICIDE RESIDUE TRIALS General Recommendations The best information about the residue behaviour of the pesticide under study would be obtained by the analysis of the entire yield of a plot. Since this is not practicable, representative samples have to be taken. Careful attention to the details of sampling is essential if worthwhile samples are to be obtained. Valid analytical results can only be obtained if the samples have been properly taken, despatched and stored before analysis. In selecting sampling points and/or the sampling method, all factors that control the residue distribution over the entire experimental plot must be considered. The best approach for any given plot can only be determined by a sufficiently trained person who is capable of recognising the importance and usefulness of the residue data sought, and who can interpret the results. The samples must be representative to enable the analytical result to be applied to the entire experimental unit. The greater the number of plants sampled in a field plot, the more representative will the sample be. However, economics and the practical problems involved in handling large samples affect the magnitude of the sampling programme. The size of sample suggested is the minimum that experience has shown is needed to give a representative, valid sample. The sizes are not usually dictated by the analytical method, which can often determine minute amounts of pesticides in small amounts of sample.

3 . 1 . 1 Method of Sampling Generally, the selection of the portions that make up the field sample should be

made depending on the circumstances:

Residue Trials in Crops

randomly, e.g. by the use of random numbers. Systematically, .e.g, in the case of field crops on a diagonal ("X"- or an "S"course (see Figure 1 a and b). Selectively from predetermined sampling-points, e.g. in the case of tree fruits take both exposed samples and those covered by foliage so that each fruit has an equal chance of being taken. Points to be borne in mind are: Avoid taking samples at the beginning or at the extreme end of plots (starting and stopping of spraying). Take and bag the required weight or number of samples in the field and do not subsample until the samples are in a clean field laboratory or in the analytical laboratory. Sample all parts of the crop that can be consumed by humans or livestock. Samples the parts of the crop that normally constitute the commercial commodity as described in Part 6 of the Guide to Codex Recommendations Concerning Pesticide Residues. Where appropriate, consider commercial harvesting practice, which reflects normal "Good Agricultural Practice" (see also 3.2).

3.1.2 Replication
In certain cases where there is likely to be considerable within-plot variation, such as orchard and glasshouse trials, there should be at least three sample replicates per plot at or near harvest. Sample integrity should be maintained throughout to leave it to the discretion of the analyst to analyse individual or composite samples. 3.1.3 Sample Handling Take care not to remove surface residues during handling, packing or preparation. Avoid any damage to or deterioration of the sample which might affect residue levels. To provide a representative sample of the raw commodity, adhering soil may have to be removed from some crops, such as root crops. This may be done by brushing and if necessary gentle rinsing with cold running water (see also 3.5.2). Sample control plots before treated plots (see also 3.2 and 3.3).

3.2 Contamination It is vital to avoid any contamination with the pesticide under study or with other chemicals during sampling, transportation or subsequent operations. Special attention should, therefore, be paid to the following:

Residue Trials in Crops

Make sure that sampling tools and bags are clean. To avoid contamination use new bags and containers of suitable size and adequate strength. The bags or containers should be made of materials which will not interfere with the analysis. Avoid contamination of the sample by hands and clothes which may have been in contact with pesticides. Do not allow the samples to come into contact with containers or equipment (including vehicles) that have been used for transporting or storing pesticides. Avoid sampling at the plot borders because of the possibility of spray drift or overlap, especially where the plot is small and particularly when various pesticides and dosages are applied to adjacent areas. Take special care to avoid contamination when commercial mechanical harvesting practices are used (see also 3.5.3, 3.5.4 and 3.5.5). Avoid cross-contamination of crop and soil samples. Sampling should proceed from the control to the lowest treatment and so on to the highest treatment (see also 3.3). 3.3

Control Samples
Control samples are in every way as important as samples from test plots. The quality of control samples should be similar to that of the test samples. The following procedure is recommended: Always take control samples. In decline studies of up to 14 days duration, control samples from the start and from the end of the study may suffice (see also 3.4.1). Sampling should proceed from the control to the lowest treatment and so on up to the highest treatment.

3.4

Sampling in Decline Studies and at Normal Harvest Time

Representative and valid sampling protocols might be different for decline studies and residue trials at normal harvest time.

3.4.1 Sampling in Decline Studies

The first sampling may take place on the day of application. These samples have to be taken immediately after application or, in the case of spray application, immediately after the spray has dried (approx. 2 hours). Take great care to avoid contamination especially at the first sampling (see also 3.2). Take samples so as to be representative of the average size or weight of crop on the plot.

Residue Trials in Crops

3.4.2 Sampling at Normal Harvest Time Take samples so as to be representative of typical harvesting practice. Avoid taking diseased or undersized crop parts or commodities at a stage when they would not normally be harvested. Detailed Sampling Procedures The following recommendations refer to the sampling of mature crops at normal harvest time, unless otherwise stated. The classification of the crops is contained in Part 4 of the Guide to Codex Recommendations Concerning Pesticide Residues. 3.5.1 Fruits and Tree Nuts Circle each tree or bush and select fruit from all segments of the tree or plant, high and low, exposed and protected by foliage. For small fruits grown in a row, select fruit from both sides, but not within 1 metre of the end of the row. Select the quantity of the fruit according to its density on the tree or plant, i.e. take more from the heavily-laden parts. Take both large and small fruits where appropriate, but not so small or damaged that they could not be sold (except when taking immature samples for a residue decline study). Take samples of fruit juices, cider and wine in a manner reflecting common practice. Quantities: Codex Code No Group 001

3.5

e.g. orange, lemon, mandarin, pomelo, grapefruit, clementine, tangelo, tangerine Group 002

Citrus fruits

Pome fruits
e.g. apples, pears, quinces, medlars

12 fruits from several places on 4 individual trees. (If this produces a sample weight of less than 2 kg, more fruit should be taken to yield a 2kg sample)

Large stone fruit

e.g. apricots, nectarines, peaches, plums

Group 003

Miscellaneous fruit
e.g. avocados, quavas, mangoes, papayas, pomegranates, persimmons, kiwifruit, litchi

Group 006

1 kg from several places on 4 trees Group 003

Small stone fruit

e.g. cherries
Residue Trials in Crops

Grapes

FB 0269

12 bunches, or parts of 12 bunches, from separate vines to give at least 1 kg

Currants, raspberries and other small berries Strawberries, Gooseberries Miscellaneous small fruits e.g. olives, dates, figs Pineapples Bananas

Group 004 FB 0275, 0276 FB 0268 Group 005 Fl 0353 F10327

0.5 kg from 12 separate areas or bushes 1 kg from 12 separate areas or bushes 1 kg from several places on 4 trees 12 fruits 24 fruits. Take two fingers each from top, middle and lowest hand of four harvest able bunches 1 kg (with or without shells)

Tree nuts e.g. walnuts, chestnuts, almonds Coconut Fruit juices, wine, cider 3.5.2 Vegetables

Group 022

TN 0655 Group 070

12 nuts 1 litre

Bulb vegetables, root vegetables, tuber vegetables: Take samples from all over the plot, excluding 1 metre at the edges of the plot and the ends of the rows. The number of sampling points depends on the sample size of the crop (see below). To provide a representative sample of the raw commodity, adhering soil may have to be removed. This may be done by brushing and if necessary gentle rinsing with cold running water. Trim off tops according to local agricultural practice. Details of any trimming should be recorded. Where the tops are not used as animal feed (carrots, potatoes) they should be discarded; otherwise (e.g. turnips, beets) they should be bagged

separately.
Quantities:

Fodder beets, Sugar beets Potatoes

Codex Code No AM 1051 VR 0596 VR 0589

12 plants 12 tubers (the sample should weigh at least 2 kg - where necessary, take a larger number to produce a 2 kg sample)

10

Residue Trials in Crops

Other root crops e.g. carrots, red beet, Jerusalem artichoke, sweet potato, celeriac, turnip, sweee, parsnip, horseradish, salsify, chicory, radish, scorzonera Leeks, Bulb onions Spring onions

Group 016

12 roots. (The sample should weigh at least 2 kg - where necessary, take a larger number to produce a 2 kg sample).

VA 0384 VA 0385 VA 0389

12 plants 24 plants. (The sample should weigh at least 2 kg - where necessary, take a larger number to produce a 2 kg sample). 12 bulbs from 12 plants. (The sample should weigh at least 2 kg -where necessary, take a larger number to produce a 2 kg sample).

Garlic, Shallots

VA 0381 VA 0388

Brassica vegetables, leafy vegetables, stalk and stem vegetables, legume vegetables and fruiting vegetables: Take the sample from all parts of the plot, leaving 1 metre at the jedges and ends of rows. The number of sampling points depends on the sample size of the crop (see below). Sample items of crops such as peas or beans protected from the spray by foliage and also from parts exposed to the spray. To provide a representative sample of the raw commodity, adhering soil may have to be removed. This may be done by brushing and if necessary gentle rinsing with cold running water. Do not trim except for the removal of obviously decomposed or withered leaves. Details of any trimming should be recorded.

Quantities: Codex Code No Group 010

Large brassica crops e.g. cabbage, cauliflower, kohlrabi Broccoli Brussels sprouts

12 plants

VB 0400 VB 0402

1 kg from 12 plants 1 kg from 12 plants. Buttons to be taken from at least two levels on each plant. 12 fruits from 12 separate plants.

Cucumbers

VC 0424

11

Residue Trials in Crops

Gherkins, courgettes, squash

Group 011

12 fruits from 12 plants. (The sample should weigh at least 2 kg - where necessary take a larger number of fruit to produce a 2 kg sample)

Melons, gourds, pumpkins, watermelons Egg plants (Aubergines) Sweet corn

Group 011 VO 0440 VO 0447

12 fruits from 12 separate plants 12 fruits from 12 separate plants 12 ears. (The sample should weigh at least 2 kg - where necessary take a larger number of items to produce a 2 kg sample. 12 items. (The sample should weigh at least 0.5 kg - where necessary take a larger number of items to produce a 0.5 kg sample) 24 fruits from small-fruiting varieties, 12 from large fruiting varieties. From 12 plants in all cases. (The sample should weigh a minimum of 2 kg - where necessary take a larger number of items to produce a 2 kg sample) 12 plants

Mushrooms

VO 0450

Tomatoes, Peppers

VO 0448 VO 0051

Endive, Lettuce Spinach, Chicory leaves (also, at immature stages during decline studies, endive and lettuce) Kale Small-leaf salad crops e.g. cress, dandelion, corn salad Peas, Phaseolus beans e.g. Franch, kidney, runner

VL 0476 VL 0482, VL 0483 VL 0502 VL 0469

1 kg from 12 plants

VL 0480 Group 013

2 kg from 12 plants sampled from two levels on the plant. 0.5 kg from 12 plants (or sites in plot) 1 kg (fresh green or dry seed as appropriate)

Group 014

12

Residue Trials in Crops

Pulses e.g. dried broad beans, beans, lentils, soya beans Celery Asparagus, Rhubarb Globe artichoke Fodder crops

Group 015

12 plants 12 sticks from 12 separate plants. (The sample should weigh a minimum of 2 kg - where necessary take a larger number of sticks to produce a 2 kg sample). 12 heads 2 kg from 12 separate areas of plot. (Crops harvested mechanically can be sampled from the harvester as it proceeds through the crop).

VS 0624 VS 0621 VS 0627 VS 0620 Groups 050, 051,052 Group 023

Oilseed e.g. rape seed, mustard seed, poppy seed 3.5.3 Grasses Cereals: -

If the plot is small, cut the whole yield. If the plot is large but mechanical harvesting is not carried out, cut not less than twelve short lengths of row chosen from all over the plot. Cut stalks 15 cm above the ground and remove the grain from the straw. Care should be taken to avoid contamination when mechanical methods are used to separate the parts of the crop. The operation is best done in the laboratory. If the plots are harvested mechanically, take not less than twelve grab samples of grain and/or straw from the harvester at uniform intervals over the plot. Codex Code No Group 020

Quantities:

Cereal grains e.g. wheat, barley, oats, rye, triticale and other small cereals; maize (off the cob), rice, sorghum Straw of the above crops

1 kg

Group 051

0.5 kg

Residue Trials in Crops

13

Maize straw, fodder and forage

AF 0645

12 plants. (Cut each stem into three equal lengths (with leaves attached). Take top portion from stems 1 to 4, middle portion stems 5 to 8 and bottom portion from stems 9 to 12, thus that parts of all 12 stems are included in the sample). 12 plants. (Cut each stem and subsample as in previous item, retaining any cobs present on the appropriate portions of stem)

(forage)
AS 0645

(mature plants excluding cobs)

(fodder)

Green or silage maize

Group 051

Maize cobs

Group 051

12 ears. (The sample should weigh at least 2 kg - where necessary, take a larger number of ears to produce a 2 kg sample).

Grasses, forage and animal feed: - Cut with shears at normal harvest height (usually 5 cm above the ground) the vegetation from not less than twelve areas uniformly spaced over the entire plot, leaving 1 metre at the edges of the plot. - Record height of cutting and avoid soil contamination. - Crops which are harvested mechanically can be sampled from the harvester

as it proceeds through the crop. Quantities: Codex Code No Group 050,

051

Green foragelsilage crops of

1 kg

alfafa, clover, pea and bean forage, vetch, sainfoin, lotus, soya bean fodder and forage, rye forage, fodder cereals, sorghum forage Dry hay of the above crops
Sugarcane

Group 050,
051

0.5 kg

GS 0659

Select whole canes from 12 areas of the plot and take short (say 20 cm) sections from all parts of the length of the canes. Care is necessary owing to the rapid changes which normally occur in cane juices. If required, 1 litre

14

Residue Trials in Crops

samples of juice should be taken and frozen immediately and then shipped in cans. 3.5.4 Seeds Oil seed: Use essentially the same technique as for cereals, taking samples of mature seed from at least twelve parts of the plot. Where the sample is harvested by hand, seed should normally be sent to the laboratory in the pod. Where mechanical harvesting is used, only the seed should normally be supplied. Cotton seed (Codex Code No. SO 0691): - Pick the cotton at the normal stage of harvesting. Take 1 kg, with or without fibre. Peanuts (Codex Code No. SO 0697): - Collect at the normal stage of harvesting. Take 1 kg. Sesame seed, rape seed (Codex Code Nos. SO 0700, SO 0495): - Collect the pods when they have reached the stage of maturity at which they are normally harvested. Take 0.5 kg. Sunflower seed, safflower seed (Codex Code Nos. SO 0702, 0699): Where the sampling is done by hand select ripe heads. Where it is done mechanically submit the seed to the laboratory. Take 12 heads or 1 kg of seed.

Coffee and cacao beans (Codex Code Nos. SB 0716, 0715): - Take samples in a manner reflecting common practice, quantity kg. - The freshly harvested produce is not normally required. 3.5.5 Herbs and Spices; Tea Leaves; Hops; Beer Take samples in a manner reflecting common practice. The freshly harvested produce is not normally required for tea although herbs, such as parsley and chives, should be sampled fresh. In the case of hops, both fresh and dried cones should be supplied.

Residue Trials in Crops

15

4.

SAMPLING PROCESSED COMMODITIES Where a commodity is normally processed between harvest and marketing, for example by milling, pressing, fermentation, drying or extraction, data may be required on the processed crop or its products. Details of the processing method should be supplied with the samples together with storage and handling histories. In such cases, the trials should be planned to provide samples with appropriate residue levels so that the fate of residues can be studied during the processing. Sample separately any cleanings, husks or by-products which could be used for animal feed.

5.

SAMPLING STORED COMMODITIES Supervised trials of post-harvest treatments of stored products should be carried out over a wide range of storage facilities and the sampling technique must be carefully chosen if valid samples are to be obtained. Procedures for taking valid samples from most commodities in storage units are well established. Such procedures are acceptable in sampling for pesticide residue analysis and may be used if adequate references are given. The sampling procedures are usually designed for three kinds of storage conditions.

5.1

Sampling from Bulk Obtaining a representative sample from a (large) bulk container (e.g. of cereal grains) is difficult: if possible, samples should be taken at frequent intervals from the stream during transfer into another container. A probe sample is not representative but may be acceptable if: it is possible to reach every part of the storage container; and a larger number of individual samples are taken before mixing and reducing to produce a final sample. Pesticide residues are normally higher in the dust fraction and this should be recognised in the sampling procedure.

5.2

Sampling Bagged Commodities Sampling of the commodity within a bag must be random. A representative sample from a large stack of bags can be obtained only if every bag is accessible. This is not always possible in practice and the alternative is to obtain a sample from a number of randomly chosen bags by probing. Since pesticide treatments are often directed to the surface of the bag, selective sampling to show the effect of the position of the bag in the stack and the penetration of the pesticide into the bag may be necessary.

5.3

Sampling Fruit and Vegetables in Packing Houses Where post-harvest treatments are applied to fruit and vegetables in packing houses, an adequate number of samples must be taken to determine the range of

16

Residue Trials in Crops

residue levels resulting from variations in the treatment process. The effects on residue levels of concentration, temperature, duration of treatment, drying (after dip treatments) and subsequent handling may need to be considered. 6. SAMPLE SIZE REDUCTION Ideally, the field sample should be submitted intact for analysis. The requirements of the analyst should not influence the sampler to take a smaller sample than is necessary for a valid field sample. In practice, a valid field sample is often much larger than the sample needed by the analyst and cannot be handled economically, especially if freezing and long transport are involved. In such cases, a reduction in the size of the field sample is desirable. Nevertheless, sample size reduction should only be undertaken using techniques which have first been agreed with the analyst. For samples consisting of small units, such as cereal grains or even small fruit, there is little difficulty in valid sample reduction. The normal procedure of mixing, quartering and rejection of opposite quarters until the desired reduction is achieved is satisfactory. With samples of medium sized products such as beans and peas in the pod, there is an increased risk of losing sample validity by sample reduction. However, the random selection of the required number of units to make up the laboratory sample from a well-mixed field sample can be a satisfactory procedure. The problem of maintaining sample validity during sample size reduction is greatest with large fruit and vegetables such as cabbages or melons. In these situations, there is little alternative to shipment of the whole field sample to the laboratory, particularly since the number of items required for an acceptable laboratory sample is often the same as that required for the field sample. 7. SAMPLE PACKING AND STORAGE Once packed and labelled, samples may be stored or immediately sent to the Residue Laboratory according to the nature of the sample, the stability of the residue and the kind of study undertaken. It is important that the packing and shipment are done so that the samples arrive as soon as possible (normally within 24-36 hours) after being taken and without change of any kind: e.g. deterioration, physical damage, contamination, loss of residue, or change in moisture content. Frequently, storage and shipping should be under deep-froze conditions.

7.1

Packing

7.1.1 Containers
Individual samples should be placed in suitable containers, e.g. heavy polyethylene bags, and then put inside additional heavy paper bags and, where necessary, frozen or refrigerated as soon as possible after sampling according to the nature of the chemical involved. Polyethylene bags alone may become brittle in contact with dry ice and therefore risk breakage and subsequent loss of sample. Residue Trials in Crops 17

Avoid other plastic containers, or plastic-lined caps, unless made of "Teflon" or other inert plastic which does not interfere in the analytical method; laboratories have frequently experienced such interferences and PVC bags should be avoided. If cans are used, they should first be checked to demonstrate the absence of materials such as oil films, lacquers or resin from soldered joints that could interfere with analyses. Glass containers should be used for liquid samples and should be thoroughly cleaned and rinsed with one or more suitable pesticide-free solvents such as acetone, isopropyl alcohol or hexane, and dried before use. Pesticides can migrate to the walls of a container and be adsorbed; hence even a glass container, after the sample is poured out, should be rinsed with solvent if the extraction is not made in the container itself. In summary, any type of container or wrapping material should be checked before use for possible interferences in the analytical method and at the limit of determination of the analysis. Fasten boxes securely with strong twine, rope or tape.

7.1.2 Shipment of Samples Non-perishable commodities containing residues that are known to be stable over the period required to reach the laboratory can be shipped in a nonfrozen state but samples should be protected against any effects which might cause degradation or contamination. Where samples need to be frozen, use shipping containers of polystyrene foam, if available, as they are excellent for this purpose. If not available, use two cardboard boxes of slightly different size with insulation between. Proper insulation is essential to ensure samples arriving at the residue laboratory still frozen. Sufficient dry ice must be used for some still to remain when received at the residue laboratory. This usually requires a minimum of one kg of dry ice per kg of sample. For journeys lasting more than two days, two kg of dry ice or more per kg of sample may be required. Poorly insulated containers require more dry ice. Use caution in handling dry ice (gloves and ventilated work area). Packages must of course comply with transport regulations. Frozen samples must never be allowed to thaw, either before or during shipment. They must be shipped under conditions that permit their arrival at the residue laboratory still solidly frozen. Advise consignee by telegram or telex the full details of shipment of samples, including shipping document numbers and flight numbers, so that delay in delivery to the laboratory is avoided. When samples have to be shipped across national boundaries, quarantine regulations must be observed and appropriate permits obtained well in advance of dispatching samples. 7.2 Labels and Records Label each sample with the appropriate sample identification. The label and ink should be such that the writing will not be illegible if it becomes wet. Attach the
18 Residue Trials in Crops

label securely so that it cannot become loose during shipment and place the label so that it will not become wet from condensation. Complete the residue data sheets clearly and accurately with all the requested trial details. Failure to do this may mean that data will not be acceptable. The completed sheets should be protected by enclosing them in protective polythene bags and they should be sent with the sample. Duplicate sheets should be kept by the sender. Use a label on the outside of the shipping container showing the following: "Perishable Goods: Deliver immediately upon arrival" and "This material is not fit for human consumption". 7.3 Sample Reception and Handling Immediately upon arrival of the samples, the Residue Laboratory personnel should: - Verify that the copy of the residue form is included with the samples. - Check and report on the condition of the samples. - Check to see that the samples match the details of the residue form. - Check the residue form for accuracy (especially the rate and interval data) and verify that the information is complete. - Check the residue form to determine whether any special treatment or testing is indicated. If there are any deviations of any consequence, or the residue form is not received or is incomplete (in such a way that a proper comparison is not possible), then the samples should be stored in the simplest form that will preserve the residue and the crop. The trial organiser should then be contacted immediately to determine how to proceed.

Note: it is dangerous to put packages containing dry ice into deep freeze. 7.4 Storage Samples should be analysed as quickly as possible after collection before physical and chemical changes occur. If prolonged storage is unavoidable, it is usually preferable to extract the sample, remove most or all of the solvent and store the extracts at a low temperature, preferably at or below -20C. This removes the residue from contact with enzymes which might degrade the pesticide and also prevents further possibility of residues being "bound" in the tissue. Do not store homogenised samples for analysis unless an adequate check has been made on the stability of the residue. Studies of the stability of residues in samples, over the time and at the temperature of storage, should be carried out with representative pesticides and

Residue Trials in Crops

19

substrates. When there is doubt about the stability of residues in storage, spiked control samples should be held under the same conditions as the samples or extracts. Light degrades many pesticides; it is therefore advisable to protect the sample and any solutions or extracts from needless exposure. Samples other than water should ordinarily be stored in a freezer, preferably at -20C or below. Even then, physical and chemical changes may occur either in the sample or in the residues sought. Extended storage in freezers can cause moisture to migrate to the surface of the sample then to the freezer coils, slowly desiccating the sample. This effect may be of importance if water content affects the subsequent analysis and can affect the calculated residue concentration. Water samples should be stored slightly above freezing to avoid rupture of the container as a result of freezing. 8. REPORTING ON FIELD ASPECTS OF RESIDUE TRIALS All the data relating to the treatment and history of the residue trials should be recorded. It is usually convenient to record these data in standard form and essential items for specific trials may be drawn from the following list. These refer to the supervised trial, field sampling and shipment of sample to the laboratory. Further data on the chemical analyses will be provided by the analyst. Model report forms are attached. (See Appendix 1.) 8.1 General Information on the Supervised Trial - Pesticide (active ingredient and trade name). - Formulation. - Trial number and type (field/glasshouse/other). - Commodity (1). .2 Variety. Test locations (country and site). Soil characteristics, pH, physical and chemical properties. Name(s) and signature(s) of the person(s) responsible for the trial and for collecting the sample.

Application Data for Field Trials Crop planting or sowing date. Description of plot plan/crop layout/cropping system.

(1) 20

Plot size or number of plants per plot/unit area.

Number of plots per treatment. See Codex Classification of Foods and Animal Feeds (Part 4 of the Guide to Codex Recommendations Concerning Pesticide Residues). Residue Trials in Crops

Target pest or disease (if any). Method of application and equipment. Number of applications and application date(s). Application details (e.g. overall, banded). Dose rate - active ingredient/ha weight/volume of formulation/ha - applied dilution Climatic conditions during and after applications, preferably for the whole period of the trial. Other pesticides applied to trial plot with relevant details as above. Cultural treatments before, during and after application - include irrigation and fertilizer information. Growth stage at (last) treatment.

For the application of fumigants, aerosols, smokes or fogs in glasshouses or stores, the procedure of the application and the disposition of fixed equipment/ generators should be described. Any anomaly occurring during the application or during the postapplication period (e.g. doors or windows opened) should be reported. Dosage rates should be expressed both per unit area and per unit volume. 8.3 Application Data for Post-harvest Trials - District, number, volume and area of the trials site. Description of the store including total capacity at time of trials, type of ventilation and state of hygiene. Details, if available, of other recent pesticide treatments in the store. Description and quantities of products and details of packing conditions (whether in sacks, boxes, bales, tins or in bulk). Formulation(s) used. Rates, methods and dates of application. Temperatures and humidities in the storage area during and shortly after applications of the pesticide and the mean temperature and moisture content within the stored product between time of treatment and sampling.

8.4

Sampling Data Growth stage at sampling normal harvest date. Method of sampling. Sampled part(s). Number of units in sample, if relevant (e.g. lettuce, pome fruit).

Residue Trials in Crops

21

Sample weight and preparation (trimming/washing/other if common practice in preparing the commodity) (2). Control or treated. Date of sampling with time interval between last application and sampling. Storage conditions before shipment. Date shipped. Method of packaging.

(2) See Portion of Commodities to which Codex Maximum Residue Limits Apply and which is Analysed. (Part 6 of the Guide to Codex Recommendations Concerning Pesticide Residues.) 9. ANALYTICAL METHODS FOR RESIDUES IN CROPS AND PLANT PRODUCTS Only when a decision has been made on whether or not specific metabolites and/ or degradation products should be included in the definition of the residue from a particular pesticide can the residue analytical procedure(s) be established. (See PART 1 of these guidelines.) The design of suitable analytical procedures and criteria for their applicability and performance are outside the scope of this Section. It is advantageous, however, if the parent compound, metabolites and conjugates can be converted to a single compound. Such a "total residue" approach reduces the number of chemical entities to be determined and improves the efficiency of the analysis. Knowledge of other characteristics of the active ingredient and metabolites such as solubility in water, vapour pressure, partition coefficient in water / n-octanol, hydrolysis rate at pH 5, 7 and 9, and rate of photolysis may also be of help in designing the residue analytical method. The Codex guidelines on Good Practice in Pesticide Residue Analysis (Guide to Codex Recommendations Concerning Pesticide Residues, Part 7) give guidance on various aspects of residue analysis. The Codex Recommendations for Methods of Analysis of Pesticide Residues (Guide to Codex Recommendations Concerning Pesticide Residues, Part 8) provide a series of recommendations for methods suitable for the determination of residues of a wide range of pesticides in a

10.

REPORTING RESULTS OF RESIDUE ANALYSES

This aspect of pesticide residue analysis depends very much on the requirements of the organisation demanding the analytical information and it is difficult to lay down strict rules for reporting, or even on the accuracy required. It is recommended that both analyst and user of the information fully appreciate the capability of the methods used and the interpretation to be placed upon the data produced before the work is started. Valid interpretation of residue data depends on a knowledge of how the various factors contribute to the variability in results. Thus in validating the method of determination, a sufficient number of analyses must be carried out to show the extent of the errors involved, and the standard deviation should be calculated.

22

Residue Trials in Crops

All the analytical data obtained from the analysis of samples, including where relevant the levels of the parent compound and the main metabolites, should be provided, and not just a summary or an average figure. It should be clearly stated how the residues are calculated and expressed. If necessary, explanatory notes on erratic results should be given. For most commodities the residues of the pesticide and its metabolites will be expressed on the basis of the whole product as it moves in commerce or is prepared for marketing. In view of the wide range of moisture contents which occur in most fresh grasses and animal feeds (excluding hay and straw), residues in such commodities should be expressed on a "dry weight" basis. This implies that the commodity is analysed for pesticide residues as received and that the moisture content is determined using a separate analytical sub-sample by a standard method relevant to the commodity. The residue content is then calculated as if it were wholly contained in the dry matter. Residues data should be supported by: 1. a full description of, or adequate reference to, the analytical method used, including apparatus and reagents; 2. data on the specificity of the method used; 3. data on the limits of determination of the analytical method for the commodity in question; 4. adequate recovery data at levels (which should be stated) corresponding to those found in practice; 5. a statement on whether or not the results presented have been corrected for blanks (untreated controls), recoveries, or both; 6. an indication of whether a pre-treatment of the sample, e.g. washing, peeling, removing soil or any other methods of preparing, has occurred before analysis. This should be stated together with the residue level found; 7. names of personnel involved in this phase of the study. It is useful to include examples of key spectra and chromatograms. An example of the type of form that might be used for reporting analytical results is to be found in Appendix 2. 11. FURTHER READING Burke, J. and McMahon, B., Analysis of Food for Residues of Pesticides, FDA ByLines, No. 4, January 1977. Cochrane, W.P. and Whitney, W., The Canadian Check Sample Programme on Pesticide Residue Analysis: Reliability and Performance. Pesticide Residues, Pergamon Press 1979. Car, M., Internal Laboratory Quality Control in the Routine Determination of Chlorinated Pesticide Residues. Pesticide Residues, Pergamon Press 1979.

Residue Trials in Crops

23

Sherma, J., Manual of Quality Control for Pesticides and Related Compounds in Human and Environmental Samples, USA Environmental Protection Agency, EPA 600/1 76 017, February 1976. Telling, G.M., Good Analytical Practice in Pesticide Residue Analysis, Proc. Analyt. Div. Chem. Soc., January 1979. Guidelines on Analytical Methodology for Pesticide Residues Monitoring, Federal Working Group on Pest Management, Washington DC 20460, June 1975. Pesticide Analytical Manual, Volume 1, US Department of Health, Education and Welfare, Food and Druq Administration.

Figure 1. Methods of Systematic Sampling

24

Residue Trials in Crops

APPENDIX 1

REPORT ON PESTICIDE RESIDUE TRIAL. PART A. FIELD REPORT 1 Please type or use block capitals 1. RESPONSIBILITY
1 3

page

Company or Organisation Name and Address

YEAR Trial identity or number

4 2

Person (s) responsible for (include signature)

a. Trial design.................................... .................................... b. Application...................................... c. Sampling......................................... d. Analysis.........................................

2. IDENTITY OF TRIAL Active 6 Class of ingredient (s) pesticide or (common name) agricultural use Trade name(s)
or

8 Formulation Conc'n Type in SI units Comm/ Exper'l

Code number s)

crop/commodity 9 Type

location 12 Country/ Region

10 Variety/ cultivar 11 Codex commodity classification

13 Site or Map ref. (include address)

14 Pests/ diseases

Residue Trials in Crops

25

REPORT ON PESTICIDE RESIDUE TRIAL. PART A. FIELD REPORT. 3. GENERAL INFORMATION ON THE TRIAL. 15 Crop production system or lay-out. e.g. commercial orchard/ glasshouse; crop planting date; age of crop; guard rows; SOIL TYPE Trial identity or number

page 2

plot data

16 Plot dimensions in International units

17

19

Crop spacing
20

Number of plots per treatment (replicates)

18 21

Number of plants per plot (if relevant) Number of rows per plot (if relevant)

Number of control plots

22 Previous year's pesticide treatment 23 Other pesticides applied to the plot (Rates and times) during trial.
24

Cultural treatments e.g. irrigation, fertilisers 25 Summary of climatic conditions. e.g. temperature (C) rainfall wind sunlight (attach details if available) 1. before application. (96 hours)

2. during application.

3. after application (up to sampling)

26

Residue Trials in Crops

REPORT ON PESTICIDE RESIDUE TRIAL. PART A. FIELD REPORT.

page 3

4. APPLICATION DATA. 26 Method/Equipment Type of application e.g. spray to run-off, band, overall. volume applied 27 Dose rate a.i. /ha 28 Dilution or spray conc'n in SI units 29 Numbers of applications

or number

30 Dates of applications 31Growth stage at last treatment Internationally recognised scales if available 5. SAMPLING 32 Control / Treated (delete as applicable) 33 Sampled part of crop 35 Method of 34 Growth stage at sampling

Sampling

36 No of samples per plot 37 No of units in primary sample 39

dates

38

Sample weight and treatment

40 intervals (davs) last treatment/ sampling sampling/freezing sampling/receipt in laboratory

sampling freezing receipt in laboratory

Residue Trials in Crops

27

APPENDIX 2 REPORT ON PESTICIDES RESIDUE TRIAL. PART B. ANALYTICAL REPORT

Person(s) responsible for the analysis

IDENTITY OF SAMPLE
Crop commodity

(Please type or use BLOCK capitals) Sample identity or number

Pesticide(s) used
on samples

CONDITIONS AND TREATMENT OF SAMPLE(S)

Date(s) of receipt in laboratory Date(s) of analysis

Method of storage and

condition of sample(s) Portion of sample(s) to be analysed

ANALYSIS Method of analysis

(or reference) and/or modifications Extraction: Clean-up

Method of determination
and expression of residue

Recoveries
Limit of determination

RESULTS
Dosage Rate Interval (Treatment to sampling) Residue* (Not corrected for

recovery or control)
Control (including standard deviation)

Other information

e.g. stability of residues under storage conditions:

* give mean values, range and number of analyses

28

Residue Trials in Crops

Part 4 Metabolism Studies and Supervised Residue Trials in Animals

INTRODUCTION Uptake of pesticides by animals, leading to residues in foods of animal origin, can occur following either direct application of the pesticide to the animal or ingestion of feed containing residues of pesticides. In the first of these situations the application of the pesticide is deliberate and can therefore be controlled by the farmer. In recognition of the fact that residues in foods of animal origin can result from residues in feed, and that such animal feeds enter into commerce, the Codex Committee on Pesticide Residues (CCPR) has recommended maximum residue limits (MRLs) on agricultural commodities specifically grown for, or occasionally used as, animal feed. The residues in food of animal origin (meat, milk, eggs) resulting both from the ingestion of feeds containing pesticide residues and from direct application of the pesticide to the animal, have also been evaluated by the Joint FAO/WHO Meeting on Pesticide Residues (JMPR) and suitable values for MRLs have been recommended where appropriate. In order to obtain the necessary data to estimate maximum residue levels, studies are needed when pesticide residues are present in the feed of farm animals, or when pesticides are applied directly to farm animals. There is a need for internationally accepted guidelines on the experimental design, procedures and reporting of such studies. The purpose of these Guidelines is: (1) (2) to indicate acceptable techniques which may be followed in order to secure valid experimental data appropriate to the above objectives; and to promote the establishment of harmonized procedures to facilitate international acceptance of the data obtained.

Initial studies using radiolabelled chemicals are essential to identify the metabolites present in animal products intended for human consumption and hence to define the nature of the residue. If residues are found, it may be necessary to follow these studies with a "cold" feeding study to quantify the level of residues involved. These studies must be carefully planned, conscientiously executed, carefully evaluated and intelligently interpreted to ensure that the decisions taken are meaningful and that they reflect the practical situation resulting from approved uses of the chemical. Where appropriate, the studies should be carried out according to Good Laboratory Practice (GLP) standards. It may be helpful to consult registration authorities regarding specific details of protocols. 2. DESIGN OF STUDIES It is customary to use radiolabelled compounds in metabolism studies on animals to identify the nature of the residues in animal products. Quantitative measurements of these residues are then made using unlabelled compounds. Usually the most important studies are those involving ruminants and poultry. Normally lactating cows are used but, in the interests of economy, goats are acceptable alternatives. In the case of poultry, chickens are the animals of choice. Except in special cases, it is not necessary to carry out metabolism studies with pigs since information on Metabolism/Residue Trials in Animals 1

metabolism in a monogastric animal is available from studies with rats. If metabolism in the rat is different from that in the cow, goat and chicken, pig metabolism studies may be necessary. Where feeding studies are required, they are usually carried out using the parent pesticide which is normally the residue of greatest toxicological significance on the treated crop. In cases where this is not so, the animal transfer studies may be carried out using a metabolite or a realistic crop residue mixture. Discussion with Registration Authorities before the studies are started is advisable. Where external treatment of animals is needed, this is best carried out with the formulated product in a manner which reflects the proposed use.

2.1

Studies with Radiolabelled Compounds (Metabolism Studies)

The purpose of these studies is to identify the nature of the residue in the edible tissue of livestock, milk and eggs. Animal metabolism studies are required whenever a pesticide is applied directly to livestock, animal premises are to be treated, or residues occur in crops or crop parts used for feed. A livestock metabolism study should primarily identify the compounds for which analytical methods and residue data must be generated. It should also indicate the distribution of residues in tissues, eggs and milk, and whether residues are stored and accumulated. A livestock metabolism study should also lead to the elucidation of the efficiency of extraction of the various components of the residue so that extraction and residue-release procedures can be developed as part of the analytical methods. For these studies, the position of radiolabelling must be chosen so that the label is not easily lost by metabolic transformation. For example, ring labelling is preferred for aromatic and cyclic compounds. Where the molecule is complex and more than one large fragment may be formed in animals, it may be important to carry out more than one study with samples of the pesticide labelled in different parts of the molecule. Alternatively a chemical labelled in more than one position may be used. However, this may mean that results are difficult to interpret and this approach should only be adopted after careful consideration of such problems. For each position of the radiolabel, at least one ruminant (cow or goat) and/or three chickens should be dosed or treated with the chemical.

2.1.1 Oral Administration Studies

In the case of oral administration, the dosage should be at least equivalent to the daily intake estimated from feed containing residues at the level of existing or proposed MRLs. However, if the estimated intake is low and the identification of metabolites consequently difficult, then a higher standardized daily dose is recommended. In this case, a dose equivalent to 10 mg/kg in the total feed ration (10 ppm in the diet) is usually sufficient. Ruminants can be conveniently dosed twice a day at each milking whereas it may be better to dose chickens only once daily. The dose should be administered to the animals in the least stressful manner. Successful procedures for administration are outlined in Appendix 1. 2 Metabolism/Residue Trials in Animals

During the study, the animals under test should be suitably housed: cows maintained indoors in a stall, goats in a metabolism crate and chickens in battery cages. The animals should be housed in this way for a few days before dosing commences. This allows an acclimatization period to overcome any effect on milk yield or egg production as a result of moving the animals. During the acclimatization period, samples of milk and eggs can be taken to establish background levels of radioactivity. When the animals are acclimatized, dosing should be started. Ruminants are preferably dosed for three days and chickens for 14 days; in no case should either be dosed for less than three days. This normally allows total radiochemical levels in milk and eggs to reach or be close to plateau levels. Establishing a plateau level in milk and eggs is an indication that levels in tissues may also be close to a plateau level. 2.1.1.1 Sampling and Analysis Samples of excreta (see Appendix 1) and milk or eggs should be collected for analysis during the dosing period. The animals should be slaughtered 24 hours after the final dose and samples of tissues taken for analysis. The following tissues are essential. Ruminants. Meat (leg and breast), fat (renal and subcutaneous), liver, and kidneys. Chickens. Meat (leg and breast), with overlying skin and any associated fat, liver, and abdominal fat. The following analyses of the samples are usually required. (1) Levels of radioactivity in excreta. It is difficult and unnecessary to make this fully quantitative but total recoveries in the range 60-80% of the applied dose indicate that the stated dose has been administered and that the chemical is readily excreted. (2) It may also be useful to identify metabolites in urine and/or faeces as these will usually indicate metabolites which may be present in tissues, milk and eggs. (3) Levels of radioactivity in tissues, milk and eggs. (4) Identification of residues in tissues, milk and eggs. Identification or characterization of residues present above 0.05 mg/kg should normally be attempted. Only occasionally will identification of residues below this level be needed, for example if a low dose rate has been selected. 2.1.2 Dermal Treatment Studies If a previous oral administration study has been carried out, dermal treatment using the radiolabelled chemical may not be necessary. This is because oral ingestion of a dermally applied chemical, as a result of the animal grooming itself, is a major route of uptake and metabolites may be satisfactorily characterized in the oral study. However, if the chemical is to Metabolism/Residue Trials in Animals 3

studies have been done, dermal studies are usually needed. When treatment is needed, this should simulate the proposed use pattern of the chemical and the following ways of applying formulated radiochemicals have been found useful. An area equivalent to 25% of the surface area of a cow can be painted with the formulation at four times the normal application rate. Similarly, formulated products can be applied with a brush to the skin and base of the feathers on the breast, abdominal area and upper leg of a chicken. Following treatment, the animal should be allowed to lick itself or groom normally. This is important because much of the residue found in normal use may well be derived from ingestion rather than dermal penetration. (In special cases, e.g. treatment along the backbone of an animal, grooming may not result in a major oral uptake.) Samples of milk and eggs can be collected for a period until slaughter. At this stage, tissues should be taken from below the treated area of the animal as well as those listed under Oral Administration Studies (2.1.1.1). Analyses of tissues, milk and eggs are required to determine the levels of radioactivity and the identity of residues. 2.2 Studies with Unlabelled Compounds ("Cold" Studies) If residues have been identified in animal products in the radiochemical studies, and if the crop on which it is proposed to use the pesticide is an important part of the animal diet, feeding studies should be carried out using the normal technical grade pesticide. Similarly, treatment studies using commercial formulations will be necessary where a pesticide is to be applied to the skin of an animal. These studies will provide the quantitative data on the basis of which maximum residue limits (MRLs) can be proposed for animal products. 2.2.1 Oral Administration Studies Separate oral administration studies are required for a ruminant and poultry whenever residues occur in items of feed for these animals. Lactating cows and laying chickens are normally the animals of choice. Similar studies with pigs may also be required if accumulation of residues in tissues is likely, if residues occur at a significant level in major components of pig feed, or if the metabolism of the pesticide in pigs is significantly different from that in cows and poultry. Often the parent pesticide is the residue of concern and it should be incorporated into the diet to simulate, as far as practicable, a normal residue. In other cases, a metabolite or metabolite mixture may be needed for the study. For a definition of the residue of concern, see FAO Plant Production and Protection Paper No. 20 Pesticide Residues in Food, 1979. The feeding study should include a control group, a group dosed with the expected level of intake (1x) and a group dosed with an exaggerated level (3-5x; occasionally a higher level (10x) may be needed). The latter will allow an estimate of what will happen if the normal level is exceeded, will indicate whether residues are proportional to intake and will provide additional data if new uses of the product are introduced.

Metabolism/Residue Trials in Animals

The calculation of the expected ingestion level (1x) should take into account all the residues on various feed materials which may form part of the animal's diet. The composition of animal diets will vary from country to country but the only information readily available pertains to the USA and is given in the current EPA Guidelines (subdivision 0: Residue Chemistry, Oct. 1982, pp 40-57). As an example, in choosing the dosage level based on total rations the proportions in the diet of the feed items bearing the residue must be considered. For example, dried citrus pulp may in some circumstances constitute up to 20% of the total ration (dry weight basis) of dairy cows. If a residue of 5 mg/kg of a given pesticide were likely on dried citrus pulp, the total diet (dry weight basis) should be fortified at the 1 mg/kg level to reflect the expected level of intake (1x). If additional residues occur on other feed items which could also be fed in combination with citrus pulp, the contribution from these feed items should also be added. However, there are still problems in selecting the expected ingestion level (1x). The components of the diet will never all contain residues at the maximum residue level. Nor in fact will each component always consist only of treated material. In addition, if a wide range of produce is treated, the sum of all the components will give a total well above 100% of an animal's diet. As an example from the EPA table, if alfalfa, almonds and apples are treated, the resulting hay, hulls and pomace could amount to 130% of an animal's feed intake. Clearly a sensible judgment of the expected ingestion level (1x) must be made and it is suggested that the probable contribution from the typical residue level on a range of feed components should be calculated. For these studies, the number of animals in each treated group should be at least 3 for larger animals (cows) and 5-10 for poultry (laying chickens), although the control groups may be smaller. Cows should be in mid-lactation, producing an average milk yield, and chickens should be in full egg production before dosing is started. If it is considered desirable to study the decline of residues after the cessation of dosing, additional animals should also be treated. To administer the required dose, a portion of the animal's feed (normally the concentrate ration) may be treated with the chemical in a suitable solvent (e.g. corn oil) and the required amount fed to individual animals. For chickens, it may be more convenient to fortify the total feed at the required level and allow the birds to eat ad libitum. It is not recommended that animals are dosed daily with capsules containing the required dose. Animals are normally dosed for 28 days. Before starting the experiment, analytical checks should be made to ensure the consistency of level and stability of the chemical in the feed over this length of time. 2.2.1.1 Sampling and Analysis

It is useful to analyse milk and eggs on two occasions before dosing to determine background levels. Samples should also be analysed at least twice weekly during the feeding period. This enables the development and establishment of plateau levels to be checked. Milk samples from individual animals should be analysed separately.

Metabolism/Residue Trials in Animals

Animals should be slaughtered within 24 hours of the end of the feeding period and the following samples should be taken for analysis: Cows, pigs. Meat (fore and hindquarter and diaphragm muscle) (1 kg). Fat (subcutaneous and renal) (1 kg). Liver and kidney (entire organ or 1 kg).
Chickens. Meat with overlying skin and any associated fat (composite sample

of leg and breast) (0.5 kg). Liver (entire organ) and all abdominal fat. All samples from individual animals should be analysed for the dosed chemical (normally parent pesticide) and any major metabolites with toxicological significance (see FAO Plant Production and Protection Paper No. 20). Analytical methods should be able to determine residues at levels of 0.01 - 0.05 mg/kg but lower limits of determination may be needed for whole milk. For fat-soluble compounds, where residues can also be expressed on a fat basis, it is desirable to determine the fat content of the milk.
2.2.2Dermal Treatment Studies

Studies of the dermal treatment of animals, which are required for cows, pigs, chickens and other species when direct treatment is proposed, often present special problems. The most common methods of application are by dipping or spraying. The formulated product should be applied in the recommended manner or one closely simulating it. Residue data reflecting exaggerated rates of use (x 2) are desirable provided such application rates do not cause ill effects in the animals. The concentration of pesticide in the working dilution is the primary consideration in dips and sprays. When dips are used instructions are needed for recharging and maintaining a constant concentration in the dip tank and for the disposal of spent dips. Factors which may affect the deposition of residues on the animals should be taken into account in planning the study, e.g. the state of the animal's coat, the maximum number of treatments, the duration of the animal's stay in the dip tank, nozzle type, pressure, or delivery rate of sprays, and the amount of solution to be applied per animal for "pouron" or other specialized treatments. When the application of pesticides is by automatic devices (i.e. photoelectric or treadle-actuated sprays) or backrubbers, they should be used to give maximum treatment. Samples of milk and eggs should again be taken at intervals after treatment. At the anticipated withholding interval, the animals should be slaughtered and samples taken for analysis as described under Section 2.2.1.1. Extreme care should be taken in collecting these samples to ensure that residues on the skin of the animals are not transferred to the meat. If the withholding interval between treatment and slaughter in normal practice has not been defined, consideration should be given to the inclusion of additional animals to monitor the residues present at different intervals after treatment. Where treatments will be repeated, the possibility of stepwise accumulation should be studied.
6 Metabolism/Residue Trials in Animals

3.

STORAGE OF SAMPLES Samples should be labelled unambiguously in waterproof ink and frozen within a few hours of collection. The samples should be stored in airtight containers at or below-20C. Samples should be analysed as quickly as possible after collection, before physical and chemical changes take place. If prolonged storage of residue samples is unavoidable, it is essential to carry out a separate study to confirm the stability of the residue over the storage period. Extended storage in freezers can cause moisture to migrate to the surface of a sample and then to the freezer coils, thus slowly desiccating the sample. This may result in an increase in the calculated residue level. Milk samples should be stored in partially filled glass, plastic or aluminium bottles. Glass bottles, although fragile, may have advantages where the residue may be adsorbed on to a plastic surface. Prolonged storage of milk is highly undesirable as the emulsion becomes broken on thawing. It is then virtually impossible to obtain a representative sample.

4.

REPORTING OF RESIDUE TRIALS Before commencing these studies, a protocol should be drawn up and agreed by all parties contributing to the study. At the completion of a study, a full report should be produced to include the following:

4.1

In-life Part of the Study This should give details of: - the type (age, breed, strain), number, housing and weights of the animals used. the preparation of the dose and the timing and means of administration or application. the feeding of the animals and any unusual behaviour or health effects noted. the collection of milk and eggs during the study, also urine and faeces in the radiochemical study. the slaughter and sampling of the animals at the end of the feeding period. the labelling of the samples. the names of the personnel involved in this phase of the study.

In the planning stage of a study, it is useful to consult Sections 8 and 9 of Part 3 of these Guidelines for further information on sampling, analysis and reporting. 4.2 Analysis of the Samples This should report on: - purity of the test chemical and specific radioactivity (if appropriate).

Metabolism/Residue Trials in Animals

- stability checks and determination of the levels of chemical in the doses or applications. Analysis to check the uniformity of feeding levels where appropriate. - stability of the chemical in the samples or in appropriately "spiked" samples and also the extractability from samples. receipt, storage, preparation and extraction of the samples. analyses performed and both the identification of products (where appropriate) and levels of residue found*. names of personnel involved in this phase of the study.

* It is useful to include examples of key spectra and chromatograms.

Metabolism/Residue Trials in Animals

APPENDIX 1 SUGGESTED PROCEDURES FOR DOSING ANIMALS WITH RADIOLABELLED CHEMICALS Goats Goats are very sensitive and can easily cease lactation. Milk supply is often better in the summer months. The doses of radiochemical can be administered as follows. A small quantity of powdered food material (e.g. 100-200 mg of cow concentrate pellet) is inserted into a hard gelatin capsule. A solution of the radiochemical in a volatile solvent (50% of a daily dose in 50-100 ul of solvent) is added and the solvent allowed to evaporate. The lid of the capsule is sealed in place. The capsule is wrapped in a leaf which it is known that the goat likes, and this is offered to the goat. Additional leaves are then offered to ensure total ingestion of the sample. This procedure is carried out at the morning and afternoon milking. Cows If cows are used, an alternative to capsule dosing as described for goats is to apply a solution of the radiochemical in a suitable solvent to a sample (500 g) of the feed concentrate pellets contained in a plastic bucket. Additional untreated pellets (200-500 g) are mixed with the material in the bucket and the cow is allowed to eat this. It is convenient to apply half the required daily dose to the pellets and administer this at the morning milking. A second dose is given at the afternoon milking. Before starting the study, a check should be made to see that the cow will readily eat the concentrate pellets. Chickens A total daily dose is inserted into a gelatin capsule filled with powdered chicken food as described for goats. After sealing, one capsule is administered each day to the chicken by force feeding. Pigs (a) A capsule containing the dose is prepared as for goats. This is mixed with a small amount of the pig's food. The dosed food is readily eaten by the animal and extra food is given to ensure proper swallowing of the dose. (b) Some experts prefer to hold the pig securely, open its mouth and insert a stomach tube. The radiochemical is fed through the tube in solution and the dose is washed down with additional amounts of water or oil.

Metabolism/Residue Trials in Animals

Part 5 Lower Practical Analytical Levels for the Determination of Residues of Pesticides

The continuing availability of improved clean-up systems and more sensitive and selective detectors has enabled residue chemists to measure smaller and smaller residues in many different samples. However, the measurement of very low levels of residues may not be essential in some circumstances. The residue chemist is frequently involved in measuring residues in samples in order to establish or to monitor compliance with maximum residue limits (MRLs) of chemicals present in or on commodities moving in international trade. The measurement of residues in soil and water to assess the environmental fate of chemicals is also frequently important. In these cases residue methods should be sufficiently sensitive to establish and monitor the MRL and to determine residues likely to be present in a crop or an environmental sample; they need not necessarily be so sensitive as to be able to determine residues two or more orders of magnitude lower than the MRL. Methods developed to measure residues at very low levels usually become very expensive and difficult to apply, and can also lead to technical problems in specifying precisely the limit of determination of the method. However, it may be acceptable to define a lower practical level (LPL) to be determined in any sample. This would have the advantage of reducing the technical difficulty of obtaining the data and would also reduce costs. The following proposals for LPLs in various samples could be useful in enabling the residue chemist to devise suitable methods. For registered active ingredients with agreed MRLs the LPL can be specified as a fraction of the MRL. For analytical convenience this fraction will vary and should be as follows: MRL (mg/kg) 5 or greater 0.5 to 5 LPL (mg/kg) 0.5 0.1 to 0.5 according to MRL

When the MRL is set at the limit of determination of the analytical method, the LPL will also be at this level. In the case of new active ingredients, where the chemical is not yet or only recently marketed, MRLs will not have been established. It is therefore more difficult to establish LPLs. Two situations can occur: (i) Als without particular toxicological or ecotoxicological hazard On the basis of the experience of the Company concerned (e.g., use rates, preliminary residue data) an estimate of a possible MRL should be made at an early stage and, on this basis, a provisional LPL can be derived as above. For practical reasons there should in general be no need to measure below the following levels. Edible crops, juices, wine Beer and other beverages containing added water, dry soil Milk Water 1 LPLs-Lower Practical Analytical Levels 0.05 mg/kg 0.02 mg/kg 0.005 mg/kg 0.002 mg/kg 1

These limits are normally achievable using commonly available equipment and procedures. The specified limits would allow safety to be addressed in a costeffective manner.
(ii) Als with some potentially hazardous aspect

The registrant company has to determine adequate LPLs for the types of samples where the toxic effect may be mediated, based on careful consideration of all the facts available and perhaps in discussion with Registration authorities. When establishing LPLs, the validity of the analytical procedures should be checked during the development and use of the method according to the following criteria: - the normal overall recovery of the method, determined by fortifications of blank samples, should normally be between 70 and 110% at the recommended LPL. Recoveries outside these limits may be acceptable for occasional batches of analyses. The decision on aceptability should be left to the experience of the supervising analyst; - the repeatability of the method should be checked by analyses (six for example) of blank samples fortified at the proposed LPL. The relative standard deviation of the results should normally be no more than 20% but may be slightly greater at the lowest residue levels. Its reproducibility should also be checked wherever possible.

LPLs-Lower Practical Analytical Levels