Irradiation as a Quarantine Treatment: Research Protocols (Guideline III)
Irradiation as a Quarantine Treatment for Commodities including Fresh Fruit, Cut Flowers and Durables against Insects, other Arthropod Pests
and Nematodes. Guideline III Multiple Quarantine Treatments.
Multiple quarantine treatments are sequential applications of two or more postharvest treatments, which achieve quarantine security only when both are applied. For irradiation this approach is relevant when a commodity is very susceptible to treatment injury. Multiple treatments are usually proposed after research is completed for each single treatment component and the efficacy data show that no single treatment will provide quarantine security alone. The most tolerant developmental stage and the dose-mortality relationship for each component must be experimentally determined. Multiple treatments may have additive or synergistic effects on pest mortality. Dosimetry and response of the commodity to the treatment must be determined for the irradiation treatment and for the other treatment(s) eg heat; cold, modified atmosphere must be similarly defined. Statistical confidence limits would be based on pest survival for the combined treatments proposed. Treatment injury responses of the commodity, should be reported for all treatments used to provide quarantine security. Chemical treatments are unlikely to be combined with irradiation but where they are residue data will be necessary.
The following components are essential in the development of a multiple quarantine treatment:
i. Determine the highest risk developmental stage of the target pest that would be encountered at the time of treatment for each of the components of the multiple treatment. Internal organisms should be treated inside the commodity to determine the most tolerant life stage. The most resistant stage must be determined for each of the multiple treatments. When the most resistant stage to each component of the treatment is the same, the preliminary tests to determine the treatment doses for confirmatory testing may be performed as a single series of tests. If the most resistant stages are different for each treatment component, a separate series of preliminary tests may be required to determine the treatment doses for each component of the confirmatory test. If previous research indicates that the first treatment(s) in the sequence achieves the necessary quarantine security against certain developmental stages, only the surviving stages need to be tested in subsequent treatments.
ii. Determine pest mortality for the range of treatment doses for each component to predict the maximum number of survivors at the highest doses which can be tolerated by the commodity. Based on results and calculations, propose a multiple treatment schedule which will provide quarantine security. For irradiation, this is the highest minimum of the potential commercial max/min ranges
iii. Confirm that the proposed multiple treatment schedule provides quarantine security against the most resistant developmental stage of the target pest under actual or simulated commercial conditions. In most cases involving treatments that are effective on different developmental stages of the pest, separate confirmatory tests on each developmental stage may be required to ensure that both stages are killed to provide quarantine security. The sequence of treatments used in the confirmatory tests will be the sequence approved for commercial use. Determine pest mortality to treatment variables for each treatment component to predict maximum number of survivors for each treatment. Based on results and calculations, propose a multiple treatment schedule which will provide quarantine security.
It is essential that post-harvest physiology studies be done in conjunction with pest disinfestation testing to ensure that treatments do not result in unacceptable levels of injury to the commodity. These studies should include the production history of the commodity as this can affect its response to irradiation.
The commodity should be subjected to a range of doses, if necessary using different max/min dose geometry within the irradiator, temperatures, and modified atmospheres. Electron beam irradiation response should be compared experimentally with gamma- or x-radiation if it is intended to use that method but previous research had been done with the other methods. Doses should be extended to a level where injury occurs to identify the threshold. This would then become the upper limit permitted for the max-min ratio.
Following are parameters, some or all of which could be used to assess possible injurious effects in the product as a result of irradiation:
colour development or changes
softness of fruit or flower tissue
pH of fruit juice
Brix (total solids) of fruit as a measure of maturity
Ascorbic acid content of fruit
ethylene production patterns with time
CO2 production patterns with time
visible injuries - appearance
citric acid content of fruit
eating quality of fruit.
appearance of cut flowers
Data on these parameters should be collected using sound experimental designs and should be analysed statistically. It should be done a representative range of production areas and repeated over more than one season - ideally 3, taking the form of comparisons with unirradiated fruit otherwise held identically.
Systems Approaches and Good Agronomic Practices
A Systems Approach to quarantine security is the integration of cultivation, preharvest integrated pest management, harvest, postharvest treatment, packaging, storage, handling, inspection, certification, and distribution practices for a host commodity that, collectively, preclude establishment of a pest in an importing country. The objective of using a systems approach is to prevent a mating pair or a fecund female pest from entering the importing country in a shipment of the host commodity. To establish a systems approach, it is necessary 1) to determine what combination of practices reduces pest infestation risk to a level that will meet the above objective, and 2) to determine, and be able to predict, maximum infestation levels of the pest in a shipment of the commodity when it arrives in the importing country. Consistent and documented usage of a standardised set of practices is important to success and acceptance of a systems approach by the importing country. Efficacy of a systems approach should be demonstrated experimentally for one quarantine pest in one commodity at a time. In practice, a system may be set up to manage several pest species at once in a commodity. Systems approaches may be used instead of stand-alone postharvest commodity treatments or pest-free areas (described elsewhere in this protocol). Systems may consist in part of purposeful disinfestation measures, such as irradiation as well as routine operational procedures.
Good agronomic practices which minimise pest incidence are an important component of any disinfestation program but they are especially important with irradiation where mortality of pests will be slow. They may be linked to a systems approach. If high levels of infestation are present in a commodity, live pests will be intercepted at a post-treatment inspection and this will lead to concerns over whether a treatment has been applied correctly. Although tests for irradiation of pests may be available, such as the phenoloxidase test for fruit fly larvae, it is generally more practicable to produce commodities with pest incidence below the level of detection. These production practices can also assist in minimisation of irradiation injury to commodities such as that which is linked to immaturity levels.
Non-host or Non-Infestable Concepts
Many commodities are not hosts of quarantine pests under field conditions. Some commodities that are hosts are not infested by the pest at some stages of maturity. Certain cultivars of known hosts may not be infested. If the non-host or non-infestable condition can be clearly defined and monitored on a routine basis, the commodity might be able to be harvested and shipped without a quarantine treatment. A host is defined as a commodity, which is infested under normal field conditions (including greenhouse conditions) which an organism can use to successfully complete its life cycle. For the purpose of this document, non-host refers to a plant species that the pest does not infest under normal field conditions or that the pest infests but fails to complete its life cycle. The non-infestable concept refers to either a cultivar of a known host or a stage of maturation of a host that the pest does not infest. This is particularly useful where a host may have multiple quarantine pests.
An evaluation of the literature, pest interceptions, and other evidence can usually determine if a specific commodity is a host for the pest of concern. Table 1 identifies the scientific parameters used in the Pest Risk Assessment to determine the host status.
If the commodity is determined to be a host, then some type of risk mitigation will be required. An evaluation of the literature, pest interceptions, and other evidence can usually determine this, or research might be used to identify a condition under which the commodity is non-infestable. No mitigation or research is required if the assessment determines that the commodity is not a host. It might not be possible to determine whether a commodity is a host because evidence is lacking or questionable. Regulatory authorities in the importing country would then consider the commodity to be a host until additional information became available. The following research procedures can help in determining host status when it is questionable. Tests of this nature would only be initiated on pest/commodity combinations where doubt as to host status existed:
Perform laboratory cage tests under optimum pest conditions. If cage tests result in successful infestation defined as development through more than one generation, field confirmation using the growing commodity should be done for a field pest. Ensure that there are adequate numbers of pests, fruits, and replicates to provide meaningful statistical results. Puncture (damage) some commodities to mimic normal field injury. Commodities from different growing areas, cultivars, seasons or times of the year may need to be tested. The fruit commodity should generally be of market quality and this should be defined. Determining the factors conferring non-infestability and the level of those factors is desirable.
A commodity cannot be considered non-infestable if infestability is demonstrated in the field. However degree of infestability may be considered as a component of a Systems Approach
A pest-free period is a time of the year during which a commodity can be exported with minimal risk of infestation from an area in which a pest of regulatory concern is found. The reproductive stage of the pest is not present during this period. The pest-free period is applicable to species of pests that have no overlapping generations during the season in which the commodity is grown and harvested. The development time of a generation of the pest during the season in which the commodity is harvested or the daily flight activity must be predictable. An effective method of detecting the pest must be available. Detection of the reproductive stage of the pest ends the pest-free period. Field monitoring may be an integral part of this concept.
The SPS Agreement formulated under the auspices of the World Trade Organisation provides overall guidance for negotiation of market entry of commodities subject to phytosanitary quarantine constraints. The format of a proposal for assessment of a disinfestation treatment depends on the recipient country and should be prepared on the advice of that country. It should be supported by full records of irradiation treatments including timings, dosimetry results to an approved Standard and any other relevant parameters. If possible a submission should be supported by skilled technical advocates to ensure that there are no misunderstandings. It may also be advantageous to enlist the assistance of importers and retailers in the recipient country to counter the effects of producer lobbyists seeking to minimise competition.
Because mortality from irradiation often occurs late in the life cycle of a pest when it is treated at low doses, any proposed treatment should be supported by "Quality Control" or "Pest Management" programs which ensure that any pest infestations are below the level of detection at any inspection at the time the disinfestation treatment is applied. This is usually feasible because for many pests domestic markets will not normally tolerate infested produce. An additional supporting facet would be a means of determining whether the pest had been irradiated should it be detected at pre- or post-entry inspection e.g. the phenol oxidase test for fruit flies.
The following tables of data should be included with a submission:
Development times for stages of the pest in culture medium used for naked insect tests, range of times and/or mean or modal times ± S.E.
Development times for stages of the pest in each variety of the fruit or flower proposed for export, range of times and/or mean or modal times ± S.E.
Treatment mortality or survivors of each stage of each pest species in each cultivar at 5 evenly spaced doses resulting in 5 -100% survival. Additional doses or test numbers at doses near 100% if appropriate.
Dose-response regression analyses on each stage of each pest species in each cultivar. These should include representative appropriately transformed mortalities with 95% fiducial limits, regression line parameters, parallelism-test results and if applicable, comparisons of regression lines. They should meet all of the assumptions intrinsic to the analysis model.
Alternatively, an ANOVA of response data at one or more doses comparing responses of stages, with Least Significant Difference values could be given.
The size range of fruit should be given (size or weight) for any in fruit tests.
Results of tests to predict the dose needed for the required quarantine security e.g. 99.5%, 99.99% or 99.9968% on the most tolerant stage in the cultivar in which it is most tolerant (or highest risk stage).
Results of large-scale trials. Replicate number; Date treated; Fruit size range; Stage of pest; Proportion present; Number of control fruit; Number of treated fruit; Number of pupae from controls; Number of pupae from treated; Number of adults from controls; Number of adults from treated; Number of adults from controls; Estimated mortality of treated individuals.
Results of treatment injury testing giving the cultivar, maturity, ripeness and where the commodity was grown.
Dosimetry data for all treatments and initial calibration of the irradiation source and treatment geometry in accordance with an accepted international standard. Include source, target dose, dose rate, max/min dose, max/min ratio, mean dose, distribution of dose, temperature at treatment, RH of atmosphere, medium (in fruit, packing material, naked insects), Absorber density, atmosphere (nitrogen, if ambient whether ventilated, elevated or depressed levels of oxygen, nitrogen, carbon dioxide or other gases present as atmospheric modifiers.
Documentation of source characteristics including strength, geometry and rate of delivery.