Residue trials to obtain permanent maximum residue limits for crops (Residues)

Crop residue trials are required for estimating the maximum residue level likely to occur in a commodity or crop when a product is used according to good agricultural practice.

Residues remaining on or in a crop commodity from a given method, timing and rate of pesticide application may vary with trial site and climate, and the limits of such variation are important to the establishment of maximum residue limits (MRLs). Other interacting factors of varying importance include growth dilution, ratio of crop surface to mass, volatility of the product, and degree of absorption onto and into the surface of the treated commodity. All of these must be taken into account when designing residue trials that represent a range of agricultural and climatic conditions.

The recommendations for use of the chemical included on the approved label must reflect good agricultural practice. MRLs are based on good agricultural practice and will never, under any circumstances, be higher than can be justified on toxicological grounds. MRLs are legal limits that are used for enforcement purposes to monitor misuse of a chemical. Hence, accuracy in their estimation is important.

1. Objectives

The objectives of crop residue trials are:

• to establish, in accordance with good agricultural practice, the likely residue levels in crops at the time of grazing or harvest
• to determine the rate of decline of residues
• to provide the data to establish a new MRL or amend an existing one
• to provide data to support the proposal of a withholding period that is compatible with good agricultural practice.

This guideline gives general information about the minimum requirements for crop residue trials and assumes that all trials will be conducted in Australia. However, in many cases, considerable residue information may be available from other countries that can be used to reduce the number of residue trials that need to be conducted in Australia. Inclusion of residue data from other countries is discussed in the relevant sections of this guideline.

It is not possible to cover every situation and it is stressed that this guideline is not a mandatory requirement to be rigidly implemented. If your intended residue trial program is atypical, you should consult with the APVMA before commencing work.

Residue guidelines that expand on specific situations or give general information about pesticide residues are published in the regulatory guidelines and are available on the APVMA website.

These include the following guidelines:

• Animal transfer studies
• Failed crops
• Maximum residue limit proposals ‘at or about the limit of analytical quantitation’
• Pastures
• Definition of residues for the purpose of setting a maximum residue limit
• Processing studies
• Stability of residues during storage
• Withholding periods
• Reporting of residue trials
• Crops—post-harvest applications except fumigants and grain protectants
• Fumigants
• Residue analytical method
• Extrapolation of data—isomers
• Grapes

You should consult the guidelines listed above when planning a residue trial. For a situation that is not covered by this, or one of the above, guidelines, you should consult the APVMA.

You can find further information on residue trials in the Food and Agriculture Organization’s Guidelines for conducting pesticide residue trials to provide data for the registration of pesticides and the establishment of maximum residue limits and Manual on the submission and evaluation of pesticide residues data for the estimation of maximum residue levels in food and feed (1997), OECD guidance documents and test guidelines and the APVMA regulatory guidelines.

2. General principles of trial design

Supervised residue trials form the basis for the estimation of MRLs for plant protection products on commodities of plant and animal origin. The trials should be conducted under maximum treatment regime conditions as proposed for the label. The chosen test conditions should predict the highest residues that may reasonably arise and be representative of conditions encountered under normal farming practices. Some of the factors that must be considered when designing a residues trial are described below.

2.1. Trial layout

2.1.1. Selection of sites

Trials should be conducted in major areas of cultivation or production of the crop and should be sited to cover a range of representative conditions in which the pesticide is to be used. Such conditions include climatic variation, seasonal variation, differing soil types and different cropping systems. Areas or sites with conditions that are atypical and not representative should be avoided, unless it is anticipated that normal usage of the pesticide under those conditions will result in higher residues in the crop.

2.1.2. Number of sites

The number of sites in the study depends on the range of conditions to be covered, the uniformity of the crop and the variation in agricultural practices.

Trials should preferably be conducted in different geographic locations rather than establishing multiple trials at a single trial site. Under special circumstances, however, it may also be appropriate to carry out trials under controlled conditions, such as in climate-controlled chambers or greenhouses, so that all the factors that influence residue behaviour can be controlled.

In general, trials should be carried out over a minimum of two growing seasons for crops with a single, relatively narrow harvest period (such as cereals, tree crops and vines). Separate residue trials should be conducted for cereals and other crops that are grown in more than one season in any one year (for example, wheat grown in winter and spring). For products used in an extended growing and harvesting season, such as vegetables, glasshouse crops and citrus fruit, sufficient data from two different growing seasons may be acceptable, provided the use pattern is the same for each season.

In some circumstances, trials from one growing season may be adequate—for example:

• when negligible residues are present and therefore the MRL may be set ‘at or about the limit of quantitation’
• for post-harvest treatments when there is no expected variation in residues
• when extensive data from overseas uses show a trend that is consistent with one season’s use in Australia
• if sufficient trials are provided from different climatic zones in Australia for a widely grown crop.

2.1.3. Replication

Since variations in residue levels between replicate samples at individual sites are small compared to those from a group of sites, it is usually not necessary to replicate treatments at individual sites. However, it may be useful to have three or four replicates at one individual site to study experimental variation within a site. If an efficacy trial with replicated plots is sampled, residue samples taken from plots receiving ‘identical’ treatments should be analysed separately to provide an indication of within-site variation.

2.1.4. Plots

In general, residue data should not be generated from plots that are too small to be representative. The size of the individual plot will vary from crop to crop, but should be large enough to:

• apply the pesticide under conditions that reflect normal commercial practices
• provide sufficient quantities of representative crop samples for analysis.

A control plot for untreated samples is necessary to determine the limit of quantitation of the method for the crop (see Rates of application). The control plot should be close enough to the treated plot to have identical growing and climatic conditions. However, it has to be sufficiently far away to exclude any contamination from the treated crop (for example, from spray drift, volatilisation or leaching). A sufficient buffer zone (lanes or guard rows) should be left to prevent cross-contamination. In general, the control plot should be placed upwind from the treated plot.

2.1.5. Type or variety of crop and cropping system

The type or variety of crop and the way in which it is grown may influence the residue pattern. Data should be generated on the most commonly used type or variety and cropping system and on the factor or combination of factors most likely to result in the highest residue levels. If more than one variety of crop is commonly grown, then more than one variety should be used in the trials.

2.2. Application of the pesticide

2.2.1. Formulation

The residue trials should use the formulation of the pesticide to be marketed. Before you submit other formulations for registration, you should obtain a limited amount of data from comparative trials (bridging data) to check that residue levels resulting from the formulation change will not be significantly different. Details of formulations used in the trials should be reported. If in doubt, you should discuss the proposed formulation change with the APVMA.

2.2.2. Growth stages of crop

The stages of crop growth at which the pesticide is applied should be clearly identified (using terms such as pre-emergent, post-emergent, mid-season, flowering, fruit set, post-harvest, etc.; or accepted growth stage codes such as the BBCH scale).

Persistence of residues is dependent on the timing of the application, both plant growth stage and season. This is especially true in moderate climate zones, where it has been observed that the residue decline in autumn is generally less than in high summer due to the lower ultraviolet light intensity and lower temperatures.

2.2.3. Method of application

The method of application should reflect the recommendations and directions on the product label. Applications should be made with equipment similar to that used in normal commercial practice. Spray equipment should be calibrated. Other forms of applicators may be used, provided they are readily calibrated and are compatible with normal practice. Care should be taken to ensure uniformity of application and to avoid contamination in neighbouring plots.

2.2.4. Rates of application

Trials must include the maximum rate that is likely to be recommended or 1× and preferably some trials at double the maximum rate or 2× (if phytotoxicity is not a consideration). Trials at the double rate may be substituted for more trials at the maximum label rate. There may be circumstances when trial data may not be required at the double rate, such as with vines or fruit trees.

The double-rate data will give the study future value if there is ever a need to increase the use rate. It is therefore prudent to consider the need for conducting trials at the double rate.

The maximum application rate is usually specified as grams or kilograms of active ingredient per hectare, although other units are appropriate depending on the label instructions and type of crop treated (for example, grams or kilograms per tree, or grams or kilograms per meter of canopy diameter). It should also be specify whether the application rate changes during the growing season. Spray concentrations should be specified in grams or kilograms of active ingredient per 100 litres of spray volume for fruit trees, vines and orchard situations in general. If a rate is specified in an orchard situation, an appropriate conversion factor should be provided such as the spray volume per hectare or number of trees per hectare.

If two or more rates of application are included in the trial, care should be taken to avoid cross-contamination.

2.2.5. Number and timing of applications

The number of treatments and the intervals between applications should reflect the closest use to harvest and the maximum use of the product. Generally, it is the final application that has the greatest influence on the magnitude of residues at harvest. The growth stages at each application spray should be specified. If multiple applications of a product are recommended, the maximum number and the minimum interval between treatments should be studied. In some cases, the use pattern may recommend different doses for different situations (for example, ‘for control of Alternaria spp. apply 300 g/ha at 7–8 day intervals; for preventative treatment apply 500 g/ha with an interval of 10–14 days’).

2.2.6. Additional pesticides

No pesticides other than the one being analysed should be applied to the control or test plots before or during the sampling period. However, the use of other products may be necessary to maintain the health of the treated and untreated plants. In that case, only those pesticides that do not interfere with the residue analysis may be used. The additional products used should be noted and, where possible, advice from the analyst should be sought before use.

2.3. Residue decline studies

A number of the supervised residue trials submitted for registration of a product should be residue decline studies, which show how the residue levels change with time. The APVMA recommends that at least 50 per cent of the trials be decline studies. Residue decline studies are important when:

• a systemic pesticide or slow-acting formulation is applied and maximum residues occur over an extended period after application
• the chemical is applied when the edible portion of the crop has formed, especially if it is applied close to harvest
• residues may occur at the earliest possible harvest or grazing period and harvesting or grazing may take place over an extended period.

The degradation or decline of residue levels may be due to one or more of the following factors:

• physical removal (for example, by washing or volatilisation)
• chemical degradation or metabolism in or on the plant
• apparent disappearance due to growth dilution.

Samples to establish the residue decline should be taken as soon as the spray has dried and at regular intervals thereafter. The choice of sampling intervals will depend on the persistence of the chemical and the anticipated period between treatment and harvest or grazing. If multiple applications are recommended, a sample taken before the final application may be of value in determining the rate of degradation or decline between sprays. Sampling on at least four occasions, up to and including harvest, is recommended and it is important that the plot size is large enough to allow sampling at each interval. The proposed withholding period should be one of the sampling points (for further information, refer to the Withholding periods guideline).

The weather conditions during the period of the study should be recorded and the age and growth of the crop at each sampling interval should also be noted (for further information, refer to the Reporting of residue trials guideline).

In a normal decline trial, samples are taken from a single treated plot at appropriate intervals after application, up to and including harvest. Alternatively, it is possible to carry out a ‘reverse decline trial’, in which the product is applied to several plots at intervals corresponding to the sampling points and samples are taken from all the plots at the same time (at maturity or normal crop harvest). A reverse decline trial is recommended when the withholding period is uncertain and the crop is growing or maturing rapidly over the proposed treatment period. Under such circumstances, the residues present may be affected by the growth stage of the crop at treatment.

In cases where the product is applied at a particular stage of crop growth and harvest does not occur for several weeks or months after application, residue decline studies are required to address a failed crop situation (for further information, refer to the Failed crops guideline).

2.4. Guide to sampling

Generally, the ‘portions’ that make up the field sample are selected randomly, systematically or from pre-determined parts of the plot, depending on the situation. In sampling, the factors that control the residue distribution over the entire plot need to be considered. In certain circumstances—for example, when there is likely to be some within-plot variation such as orchards or vines—samples from each plot should be selected at random from the high, middle and lower parts of the tree or vine and bulked for analysis. The field sample should be representative of the treated plot and the individual ‘units’ must be typical of those taken at harvest—immature or unripe samples, for example, are not representative of a commercially viable commodity. The samples should also be as close as possible to the normal harvested crop in relation to trimming or cleaning (outer or wrapper leaves removed, or adhering soil removed). It is useful if the number of units per composite sample is recorded by the analytical laboratory together with the residues analyses.

Table 1 shows the commodity to be analysed and sampling procedure outlining the quantity of sample to be taken, the edible portion of the commodity for particular crop groups and the portion of the commodity to which the MRL applies.

Information on residues in the edible portion of the commodity is also necessary for chronic intake estimates for crops for which the MRL does not necessarily reflect the residues in the edible part of the crop (bananas, pineapples, mangoes, etc.). Residue analyses of the edible portion of the crop should be conducted on representative samples.

Similar principles apply when collecting single units to determine a variability factor for acute intake estimation. Single-unit analyses should be conducted if the applicant wishes to determine a variability factor from trial data, rather than use default factors that have been determined by the Joint FAO/WHO Meeting on Pesticide Residues. A minimum of 100 single units should be analysed to be statistically valid.

In summary, the following points should be considered:

• When taking a sample at harvest, avoid taking undersized or diseased commodities or crop parts at a stage when they would not normally be harvested.
• Sample the parts of the crop that normally constitute the commercial commodity.
• Take samples in a way that is representative of the normal harvesting practice.
• Take care not to remove surface residues during handling, packing or preparation.
• Take and weigh the required weight of samples in the field and do not subsample.
• Store samples under freezer conditions to avoid any deterioration of the residues.
• Record the number of units per composite sample together with the analysis.

Control or untreated samples should be taken before the treated samples, to avoid contamination from handling. The control samples should be of a similar quality to the treated samples. For further detail, refer to the Food and Agriculture Organization’s Manual on the submission and evaluation of pesticide residues data for the estimation of maximum residue levels in food and feed.

3. Number of trials required for registration

It is impossible, and could be misleading, to indicate precisely the number of trials that need to be conducted to establish an MRL. However, experience indicates that a minimum number of datasets (trials) are required. The minimum number of datasets is provided in the Suggested number of crop trials (Australian and overseas data section. The datasets should be spread across representative cropping areas or geographic locations in Australia and may also include trials conducted in other countries, if the use pattern and good agricultural practice in those countries are considered to be similar to Australia’s. In the Suggested number of crop trials (Australian and overseas data) section, the representative states for particular crops are indicated by crop yield. The basic requirements for the number of trials are determined partly by whether the crop is a major or minor crop, is a significant export commodity or forms a significant proportion of the diet, and also by the degree of risk involved in extrapolating data from trials to commercial practice. The numbers indicated are the minimum numbers required for normal situations. In situations that are considered unusual, more or fewer trials may be appropriate, and the APVMA should be consulted prior to developing a trial program.

4. Negligible residues

Residues are considered to be negligible if the levels in the harvested produce are below the limit of analytical quantitation (see the Maximum residue limit proposals ‘at or about the limit of quantitation’ guideline). This is often the case with pre-emergent and early post-emergent applications of herbicides, applications of non-systemic insecticides and fungicides to fruits prior to flowering, and some seed treatments.

When no detectable residues are found, it is often due to the properties of the chemical, the type and timing of application and the rate of application. This result can be confirmed by considering studies of the plant’s uptake, metabolism and distribution of the compound.

In situations where metabolism studies and method of treatment indicate that negligible residues are expected, all the trials may be carried out within one growing season. However, if, contrary to expectations, detectable residues are found in preliminary trials, then trials over a second growing season may be requested.

If no quantifiable residues occur under maximum treatment regime conditions, no further trial results are required if conditions of use are changed to be more favourable (that is, the chances of residues remaining in the commodity are reduced by a modification in the use pattern).

5. Multiple formulations

Applicants seeking to register more than one formulation type of the same active constituent may need to generate residue data for the additional formulation types. The number of trials required to register additional formulations of a chemical will depend on the use pattern and the relative risks involved. The factors to consider include mode of application, timing of application and crop growth stage, and formulation type.

Slow-release formulations (microencapsulated, controlled release) normally require a full set of residue trials. As these formulations are designed for controlled release of the pesticide, the residues incurred cannot be compared to those resulting from a single treatment with ‘normal’ fast-acting formulations.

The data required for other types of formulations will be determined on a case-by-case basis. In general, applications made prior to crop emergence will require less residue data than formulations applied directly to growing plants. In some cases, the number of trials required may be reduced by conducting side-by-side or equivalence residue trials in which the two formulations are applied using the same rates and pre-harvest intervals. If the residues from the new formulation are less than or equal to those from the registered formulation, no additional studies will be required. However, if the new formulation results in greater residues, a full set of residue trials will be required, unless existing studies already characterise the fate of higher residues.

6. Changes to existing use patterns

In the following discussion, it is assumed that the original residue data used to set the existing MRL were of a quality to meet contemporary standards and are well documented.

When applying for a change in use pattern or registration, the effect of the changes on the residue behaviour may be based on argument, although thorough justification for the argument must be submitted. A justification could be that existing trial data show that relevant residues do not occur, or are unlikely to occur, under the new use pattern. The following sections discuss some of the common changes to use pattern or registration.

6.1. Changes in formulation

The discussion in the multiple formulations section also applies here. In principle, if there is a significant change in formulation new residue trials are necessary. If the formulation change does not involve a change in application method, it may be sufficient to carry out a comparative trial on one crop for each of the major crop groups listed on the label. If the label claims cover a wide range of crops, data should be generated for representatives of the major crop groups (leafy crops, root crops, soft fruits, tree fruits, cereal grains, etc.). The trials should be carried out on the crops expected to show the highest levels of residue or the greatest variation in residues. Representative crops in the major crop groupings are summarised in Table 3. If the residues found in a crop are not comparable for each type of formulation, trials for each crop listed on the label may be required.

6.2. Changes in application rate

In order to achieve maximum treatment regime conditions, the trials must be carried out using the highest rate of application. An increase or reduction in the rate of application of up to 25 per cent is accepted as comparable, provided all other conditions of use remain identical.

6.3. Changes in number of applications

In order to encompass the maximum treatment regime, the trials must be carried out using the maximum number of applications, or the maximum number of consecutive applications, that may be made to the crop. It is generally the last application before harvest that contributes most significantly to the level of residue in the harvested commodity. Applications prior to flowering are generally of lesser importance. In the case of relatively stable active ingredients, the residue results can be assumed to be comparable if the number of applications after flowering is increased by not more than 25 per cent (for example, 4 + 1 or 8 + 2 applications), provided there is no change in the interval between applications.

If an increase of more than 25 per cent is proposed, data may be required. Existing residue data may be used to support proposed reductions in the number of applications recommended on the label, unless a reduction in the MRL is requested.

6.4. Changes in application method

Different application methods, such as spraying, dipping, flooding, dusting, misting and granule spreading, will not, as a rule, produce comparable residue results and must therefore be tested separately. In some situations, however, there may be some comparability (for example, low volume application and dilute spraying).

6.5. Changes of timing of application; changes in withholding period

The stage of development of the crop and the intervals between applications are important factors influencing the level of residues. When setting an MRL, the maximum treatment conditions are the determining factors. An application at a later stage of crop growth may encompass residues from treatment at an earlier stage of crop development. Similarly, an application at a shorter interval before harvest may encompass residues from treatment at an application at a longer interval before harvest.

With proposed changes in the withholding period, a change of 25 per cent or less can be assumed to be comparable. This depends, however, on the interval between the last treatment and the withholding period. If the withholding period is to be extended, comparative data would not be required unless a change in the MRL is requested.

6.6. Use situation (outdoor, under glass, in store, under plastic)

The results of outdoor trials are not normally comparable with the results of trials carried out in other situations. In non-outdoor situations such as under glass, under plastic, in climate-controlled chambers or in stores, the difference in climatic conditions generally results in higher residues than those found in outdoor testing. Therefore, a full testing program is necessary for each of these situations. However, if an MRL is already set for indoor use, data for outdoor use will not be required.

6.7. Simultaneous changes in several trial parameters

The 25 per cent rule for purposes of comparability applies only when one of the parameters of the use pattern is changed. If more than one parameter is changed at the same time, the effects may be cumulative, or may cancel each other out. If more than two trial parameters are changed at the same time, it is not possible to assume comparable residue behaviour with any sufficient degree of certainty. Residue trials will therefore be required.

7. Comparable residue behaviour in different crops

In some cases, the morphology or growth habit, growing season and agronomic practices applied to one crop may be similar to other crops. For such crops, it is possible to extrapolate the MRLs based on the same use pattern from crop to crop. Guidance for possible crop groupings within which an extrapolation might be possible is given by the Codex Classification of foods and animal feeds, a summary of which has been reproduced in Table 3.

7.1. Inference of group or subgroup maximum residue limits

A group MRL applies to all the individual crops in the group, so the maximum use pattern used to generate the residue data for the major crops must be similar. Group MRLs will therefore only be established if good agricultural practice for the crops in the group is comparable. Minimum data requirements apply only if comparability can be established between production areas (similar climate, methods of growing, seasons of production, etc.). Some groups are further classified into subgroups, which are primarily indicative of form and growth habit. The subgroups are based on differences such as the size of the commodity, the surface of the crop (curly leaf versus straight leaf, or hairy surface versus smooth surface versus wrinkled surface) and the edible portion of the crop.

If a group contains crops that are considered animal feed commodities, additional data may be required for that portion of the commodity used as an animal feed (for example, fodder, forage or stubble).

7.2. Residue situations

The comparability of residue behaviour in different crops is dependent on a series of influencing factors that can affect the residues and can vary under a different set of conditions. In situations when data cannot be transferred from one crop to another, a full trial program will be required for each individual crop, in the same manner as for establishing a new MRL. For example, a group MRL will not normally be considered if the individual crop residue levels vary by more than a factor of 5.

The factors that affect the residue data requirements for an extrapolation include:

• similarity of use pattern (number and timing of applications)
• similarity of growth habit, form of crop and size of edible commodity
• range of residue levels in different crops from a proposed use pattern.

We will consider extrapolations on a case-by-case basis. Residue data from other countries may be used to assist in setting a group MRL and for extrapolation from one crop to another. Possible extrapolations are given in Table 3. For more information, you can contact the APVMA.

8. Suggested number of crop trials (Australian and overseas data)

Table 2 is a guide to the number of crop trials needed for registration of a pesticide. The number of trials may be reduced depending on factors, such as:

• timing of application (how close application of the product is to harvest of the crop)
• method of application
• total number of applications and re-treatment intervals
• persistency of the product applied and resulting residues in the crop
• physicochemical characteristics of the active constituents and product formulation.

All uses will be considered on a case-by-case basis and applicants are advised to contact the APVMA before considering the generation of any residues data.

The following numbers of trials are suggested:

• major crops: 8–12 trials
• major-minor crops: 6 trials
• minor-major crops: 4–6 trials
• minor crops 2 trials.

The figures in parentheses in Table 2 indicate the numbers of trials required to set a group MRL; refer to Table 3 for the commodities that are required for a particular crop group.

Reductions in trial numbers will be considered by the APVMA for crops where experience shows that agronomic practices and growth habit lower the risk of finite residues being present in the edible part of the crop. (Note: Codex requires a minimum of 8 trials to be conducted according to good agricultural practice before an MRL can be considered.)