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SUMMARY

The environmental data requirements for registration of microbial pest control agents in Canada have undergone a series of major revisions since the first proposal was released for public comment 10 years ago. The major revisions, currently numbering five, have evolved to reflect advancements made by the scientific research and regulatory communities in the environmental risk assessment of microorganisms, in general, and microbial pesticides, in particular. The current draft of the guidelines recognizes the difficulties in evaluating the environmental effects (e.g., toxicity and pathogenicity) and fate (e.g., dispersal and persistence) of microbial agents and the difficulties involved in integrating these characteristics into an overall ecological risk assessment. Because these guidelines apply to a wide range of microorganisms (e.g., bacteria, fungi, viruses, protozoa, etc.) that may be used against an equally diverse number of pests (e.g., weeds, insects, plant diseases, etc.), and given the complex interaction between all these different microbial agents and the different environments to which they may be exposed, the environmental data required in support of registration are determined for each microbial agent on a case-by-case basis. The current environmental data requirements are discussed in detail and we provide rationales for each testing requirement and recommend general approaches to testing.

Key words: Microbial, pesticides, environmental, risk, guidelines

INTRODUCTION

Background. In Canada, microbial pesticides are regulated under the

Pest Control Products Act which is administered by the federal department of Health (Health Canada). Section 9 of the Pest Control Products Regulations requires that applicants for registration of pest control products shall provide data to support the registration of their products. In Health Canada, the Pest Management Regulatory Agency is responsible for managing the pesticide regulatory process and registration system.

The Canadian guidelines for registration of microbial pest control agents (MPCAs) and products (MPCPs) were initiated in 1982, with the first regulatory proposal (Agriculture Canada, 1986) released for public comment in 1986. An expanded second draft of the guidelines was produced in 1988 and released as a working paper for a workshop (held in 1989) to discuss key areas of the Canadian approach to regulating microbial pesticides. In 1990, a third draft (Agriculture Canada, 1990) was released again for public comment. Also in 1990, another workshop was held to discuss registration requirements for genetically modified microbial fertilizer and pest control agents.

The fourth regulatory proposal (Agriculture and Agri-Food Canada, 1993) was released for public comment on 25 November 1993. Approximately 65 written responses were received from interested parties in the biotechnology, agriculture and forestry sectors. Scientific issues collated from these written responses were addressed at a technical workshop held at Montebello, Quebec in June 1994. This workshop resulted in the resolution of some issues of scientific contention concerning the environmental data requirements for registration of microbial pesticides.

Currently, the PMRA is in the process of finalizing the registration guidelines for MPCAs and MPCPs. The guidelines, the culmination of over a decade of consultation among technical specialists, manufacturers, users, academics, consultants, regulators and public interest groups, represent a balanced approach for regulating microbial pesticides in Canada.

The following is a brief synopsis of the major developments in the environmental data requirements since initiation of the guideline project. Guidelines specific to research permits for microbial agents are not discussed in this paper.

1st draft. Modified chemical pesticide approach with indicator species used to assess effects on non-target organisms; two-tiered system; fate and effects data both required in Tier I of initial data submission; guidelines applied only to naturally occurring agents and not to genetically engineered or modified microorganisms; pure active ingredient and formulated product testing required; test substance dependent on tier level, non-target test organism (e.g., bird, fish, etc.), and type of microbial agent (e.g., bacterium, fungus, virus, etc.).

2nd draft. Criteria established for selection of appropriate non-target organisms based on relatedness of non-target to target pest species, potential susceptibility to microbial agent, potential for exposure, and overall morphological or physiological similarity to target; numbers of non-target organisms requiring effects testing not specified; continued differentiation between naturally occurring and genetically engineered or modified microorganisms; ecozone concept adopted for classification of microbial agents as either indigenous or non-indigenous to area where formulated product (where the active ingredient is the microbial agent) is intended for use; 15 ecozones specific to Canada; three-tiered system for environmental fate and four-tiered system for environmental effects testing; maximum challenge (hazard) concentration introduced for effects testing in Tier I; formulated product required for effects testing in all four tiers with additional testing of active ingredient required in Tier II; requirement for fate data with indigenous microorganisms moved to Tier II and dependent on results of Tier I effects testing.

3rd draft. Centrifugal taxonomic approach introduced as main criterion for selection of non-target organisms; determination of host range of microbial agent additionally required; number of ecozones reduced to 5 with cross-over of some ecozones into northern U.S. states; requirement for fate data with non-indigenous microorganisms also moved to Tier II and dependent on results of Tier I effects testing; challenge concentrations revised in effects testing.

4th draft. Numbers of non-target organisms requiring testing specified; requirements extended to include genetically engineered or modified microorganisms; challenge concentrations revised in effects testing; fate testing for indigenous microorganisms moved from Tier II to Tier III.

Current draft (unpublished). Centrifugal taxonomic approach with consideration to geographical occurrence of non-target organisms in ecozone(s) of intended use of formulated product; challenge concentrations revised in effects testing; numbers of non-targets requiring testing not specified; accept testing with active ingredient or formulated product; accept data on other closely related species to microbial agent; accept published data on microbial agent or closely related species that meet certain minimum standards for validation of data.

The following is a detailed outline of the current environmental data requirements, the rationale for requiring the data, and the regulatory approach to assessing the environmental hazards and risks of microbial pesticides, in general. We do not address data requirements for assessing human health and safety, product characterization or efficacy as they are beyond the scope of this paper. The registration guidelines apply to microbial pest control agents which include bacteria, algae, fungi, protozoa, viruses, mycoplasmae or rickettsiae and related organisms, and not to other biological control agents such as nematodes, predators or parasites.

Approach. A four-level or tier system of information requirements is a feature of the approach to environmental toxicology and fate testing of the microbial pesticides (Figure 1). Initial submissions from applicants for registration must address all testing requirements in Tier I. The first tier requires acute toxicity testing on up to seven broad taxonomic groups of non-target organisms. The second tier requires environmental fate (persistence and dispersal) as well as additional acute toxicity testing of MPCAs while the third and fourth tiers require chronic toxicity and experimental field testing of toxicity and/or fate, respectively. If initial Tier I testing indicates no significant hazard, then further testing will not usually be necessary. However, if results of concern are observed in Tier I, then the appropriate information from Tier II may be required, with progression to higher tiers as necessary.

In Canada, the country has been divided into five distinct ecozones representing the major agricultural and forestry land areas where microbial pesticides are most likely to be applied. Ecozones are defined as large and very generalized ecologically distinctive areas based on the interplay of landform, water, soil, climate, flora, fauna and human factors. Ecozones are based mainly on natural diversity and therefore they cut across political boundaries separating the provinces and territories and extend into northern U.S. states. The boundaries between ecozones are viewed by the PMRA as transitional areas, rather than distinct lines of demarcation. Ecozones are used by the PMRA to determine the extent and the nature of environmental testing required for both registration and experimental field trials. For example, a microorganism is considered indigenous if it has been isolated from, or is known to occur in, the ecozone(s) of intended use. Conversely, a non-indigenous microorganism is one which has not been isolated from, or is not known to occur in, the ecozone(s) of intended use. Genetically engineered microorganisms, or GEMs, include microorganisms that have been modified through in vitro manipulation of genetic material. They are generally classified by the PMRA as non-indigenous microorganisms.

ENVIRONMENTAL TOXICOLOGY REQUIREMENTS

Environmental toxicology testing is required by the PMRA to predict possible adverse effects of MPCAs, and added ingredients (formulants) in MPCPs, on such broad groups of non-target organisms as birds, mammals, fish, arthropods, non-arthropod invertebrates, microorganisms, and plants. Adverse effects produced on these non-targets may be expressed in terms of infectivity, pathogenicity/toxicity, or hypersensitivity.

Rationale. When an MPCA is applied, large numbers are often spread over biotic and abiotic components of the intended (target) area or environment. The MPCA can also be expected to disseminate to off-target areas, or neighbouring environments, during and following application via drift, surface runoff, and possibly through leaching. As a result, exposure of non-target organisms to the MPCA may be greater than under natural conditions in terms of numbers of non-target organisms exposed, number of different species exposed, and concentration of the MPCA to which individual non-targets are exposed.

Selection of non-target test organisms. Selection of non-target organisms is dependent mainly on the known characteristics, the host specificity, and the proposed use pattern(s) of the MPCA. As a result, the number and species of non-target organisms to be tested may differ between MPCAs.

Consideration should first be given to the characteristics of the MPCA and any known adverse effects associated with the MPCA. For example, if the MPCA is known or suspected to be a toxin producer or a pathogen, selection of test species should consider the most likely host species. If there is no evidence that an MPCA is a pathogen, but it has closely related species (perhaps in the same genus) that are known to be pathogens, then selection of test species should again consider the most likely host species. When selecting the most likely host species, a centrifugal taxonomic approach should be considered to identify the Canadian test species most closely related to a known host. In this approach, the first group of non-target organisms selected are those with close phenetic (i.e., phenotypic and/or genotypic) and/or phylogenetic (i.e., evolutionary) relatedness to the target pest organism followed then by testing on more distantly related organisms. For example, the initial non-target organisms selected for testing would be species from the same genus as the target organism, then testing would be expanded to include non-target species from the same family, order, etc. as the target. For genetically modified microorganisms (i.e., MPCAs modified through classical mutational techniques such as chemical mutagenesis, conjugation or transduction or through in vitro or genetic engineering techniques), selection of test species is based upon the characteristics of both the parental microorganism(s) and the modified genetic material.

The non-target test organisms selected for testing should also include taxonomically related species which meet one or more of the following general criteria:

are known, or are suspected of being able, to be infected by the MPCA;

are susceptible to pathogens closely related, taxonomically, to the MPCA; and

are morphologically, physiologically or biochemically similar to the target, i.e., possess traits that are known to be significant in host choice/acceptance.

If there is no reason to suspect that the MPCA, or its close relatives, are pathogens or capable of producing toxins, then test species should be selected based upon the intended use area and use pattern of the MPCA. Special consideration should be given to non-target species closely related to the target host that are known or believed to occur in the ecozone(s) of intended use. If there are any non-target species that are obviously expected to be exposed to high concentrations of the MPCA (e.g., utilize the affected target host as a food source), then these species should also be considered for testing.

Finally, representative species from some or all seven broad groups of non-target organisms (birds, mammals, fish, arthropods, non-arthropod invertebrates, microorganisms, and plants) require testing with the MPCA.

Test substance. The formulated product, or MPCP, is the desired test substance in all toxicology tests. However, due to the high dosages or challenge concentrations recommended in Tier I and Tier II (and to a lesser extent in Tier III) tests, the PMRA recognizes that the use of the MPCP may not always be feasible. Therefore, the technical active ingredient (TAI), or technical product, of the MPCA should be employed for all exposures in Tier I, II and III tests. In addition, any substances (i.e., adjuvants or formulants) intended to enhance the virulence or toxicity of the MPCA should be tested along with the TAI. In Tier IV (actual or simulated) field testing, the MPCP should be used for all exposures. If the TAI of the MPCA and MPCP are identical, however, Tier IV testing may also be carried out with the TAI of the MPCA.

Routes of exposure and test concentrations. For most Tier I toxicity tests, the PMRA has determined that non-target organisms should be exposed to a single high dose of the MPCA known as the maximum hazard or maximum challenge concentration (MCC). The MCC is calculated according to the: i) type of non-target test organism; ii) route of exposure expected in the environment; and iii) concentration of the MPCA in the TAI. In the case of testing on birds, the MCC is dependent on the route of exposure, as follows:

Oral = MPCA Concentration in TAI (units MPCA/mL) X 5.0 mL/kg Body Weight X Weight of Bird (kg)

Pulmonary = MPCA Concentration in TAI (units MPCA/mL) X 0.2 mL/kg Bird Body Weight X Weight of Bird (kg)

Intravenous = MPCA Concentration in TAI (units MPCA/mL) X 0.5 mL/kg Bird Body Weight X Weight of Bird (kg)

Intraperitoneal = MPCA Concentration in TAI (units MPCA/mL) X 2.0 mL/kg Bird Body Weight X Weight of Bird (kg)

In the case of testing on fish or on aquatic and terrestrial invertebrates (arthropod and non-arthropod), where the MPCA is applied directly to soil or water, the MCC is calculated as follows:

10⁶ viable units of the MPCA per g of soil or water; or

1000 times the expected environmental concentration of the MPCA, immediately following a direct application at the maximum label rate to a 15-cm layer of soil or water;

Other routes of exposure include the diet whereby an MPCA is added to artificial food (i.e., active units of the MPCA per gram of feed) to a concentration equivalent to the maximum concentration found in the target pest (i.e., active units of the MPCA per g of target). Alternatively, an artificial diet can be substituted with a diet of target organism which has been maximally infected with the MPCA. In cases where it may be difficult to determine the MCC in the target, test organisms can be fed a diet treated with an application of the MPCA equivalent to 100 times the maximum label rate. For routes involving topical exposures, non-targets can also be treated with 100 times the maximum label rate of the MPCA.

For the treatment of non-target plants, the MCC should be equivalent to spraying the plant to runoff at the maximum rate of application proposed for the product label. For seed treatments, seeds should be thoroughly drenched with the MPCA. For other routes of exposure involving direct application to soil or water, the MCC should be as indicated above for other terrestrial and aquatic non-targets.

Tier testing. Consultation with the PMRA after each tier of testing is recommended as results from lower tier tests will determine the need for testing at higher tier levels.

Tier I. Test organisms in Tier I should be exposed to an MCC of the MPCA so as to optimize (maximize) chances for either infection or toxic/pathogenic effects. The MCC, test substance, and route of exposure are expected to vary for the different broad groups of non-target organisms requiring testing.

Tier II. Generally, Tier II toxicology testing will be required for MPCAs that either demonstrate a wide host range or produce significant adverse effects on non-target organisms of environmental or economic importance in Tier I. In some cases, an adverse effect on a single important species in Tier I, e.g., honey bees, may be sufficient to trigger Tier II testing, whereas, in other cases adverse effects on a number of non-target species (presumably of lesser importance) may be needed to trigger Tier II testing.

Tier II toxicology testing is to be carried out on any non-target species adversely affected by the MCC of the MPCA in Tier I tests. The MPCA concentration used to challenge test organisms in Tier II should be lower than the MCC used in Tier I testing.

The results of Tier I environmental toxicology tests will also determine the need for, and extent of, environmental fate testing in Tier II. If required in Tier II, fate studies will only apply to MPCAs that are non-indigenous to the ecozone(s) of intended use.

The environmental risk or hazard of a non-indigenous MPCA may be considered a concern if:

the Tier II environmental toxicology studies indicate a wide host range and/or significant effects are noted on environmentally or economically important non-target organisms; and

the environmental fate studies indicate that the MPCA is persistent and/or dispersive.

Tier III. Tier III testing is reserved for non-indigenous MPCAs which produce significant adverse effects on non-target organisms in Tier II testing. Indigenous MPCAs are exempt from testing at Tier III even if adverse effects are noted on test organisms in Tier II. If Tier III testing is required, a reduced, but representative, number of the test species adversely affected by the MPCA in Tier II should be selected for testing.

There are two elements to the testing requirement in Tier III. The first is a definitive toxicity test in which the dose-response relationship (e.g., LC50, ED50) between the MPCA and test organism is determined, and the second is a chronic or life-cycle study in which the long-term effect(s) of low-level exposure to the MPCA is assessed.

Tier IV. Tier IV (simulated or actual) field testing of indigenous and non-indigenous MPCAs is required when adverse effects are produced on non-target organisms in Tier II. Tier IV studies are to involve small-scale field testing (terrestrial or aquatic) to determine whether adverse effects can be produced under operational conditions of use. Testing with the MPCP is required and exposure concentrations should be equivalent to the maximum recommended application rate proposed for the product label.

Non-target organism testing

Birds. Non-target avian species are likely to be exposed to the MPCA most commonly through the oral route via feeding of infected food (e.g., insects) or through the pulmonary (respiratory) tract via spray drift or aerosolization. Therefore, an avian oral test and an avian pulmonary test will be required for all microbial pesticides. For Tier I and Tier II tests, the MPCA is to be administered to the gut by oral gavage or intubation and to the respiratory tract by intranasal or intratracheal instillation. The diet is considered to be an appropriate route of administration for Tier III and Tier IV tests.

An injection (i.e., intravenous or intraperitoneal) test may be substituted altogether for the pulmonary test, if the microbial dosing preparation is sufficiently free from exogenous protein and other contaminating substances that would otherwise confound the test. Although this route is environmentally unrealistic, it provides a maximum hazard challenge by bypassing the bird's primary defence mechanisms.

Test Species. Testing should be conducted on one avian species, preferably bobwhite quail (Colinus virginianus) or mallard duck (Anas platyrhynchos). Other avian species, however, would also be acceptable for testing, especially altricial species.

Testing Requirements. Tier I acute oral tests should consist of the MCC administered orally by gavage to each test bird daily for five successive days.

Tier I acute pulmonary tests should consist of intratracheal instillation of the MCC daily for five successive days. Pulmonary studies conducted using the alternative inhalation (aerosolization) method should also expose birds to the MCC daily for five consecutive days. If the injection route of administration is substituted for the pulmonary test, the test should consist of the MCC administered as a single dose by intraperitoneal (for fungi and protozoa) or by intravenous (for viruses and bacteria) injection.

For Tier II acute oral and acute pulmonary tests, the birds should be dosed once at the MCC. The relevancy of effects produced by the intraperitoneal or intravenous injection route of administration will be considered on a case-by-case basis and, thus, the applicant may not need to repeat these tests in Tier II, particularly if the dosing regime is the same as in Tier I.

Wild mammals. The toxicology data required by the PMRA to evaluate hazard to human health and safety are usually adequate to indicate hazard to wild mammals. Under certain conditions, however, these data may not be sufficient to assess the potential hazard to wild mammals. For example, if there is considerable variation in the sensitivity of different mammalian species to the effects of the MPCA, and if wild mammals are expected to be heavily exposed to the MPCA under operational conditions of use, then additional testing may be required. If testing beyond Tier I is required, the testing approach should be similar to that utilized for birds, adapted appropriately for mammalian test methods.

Fish. In testing the effects of an MPCA on non-target species of fish, two routes of exposure which may occur in the wild should be considered: (1) suspension in the test water (aqueous exposure); and/or (2) dietary, either in the form of diseased target pests (hosts) or incorporation of the MPCA in standard fish feed.

Test species. Testing should be performed on one cold water fish species, preferably rainbow trout (Oncorhynchus mykiss) or a species of salmon such as Chinook (Oncorhynchus tshawytscha), coho (Oncorhynchus kisutch) or Atlantic (Salmo salar) salmon. If rainbow trout is selected in Tier I and adverse effects are noted, additional testing on a salmon species is required.

In cases where estuarine or marine waters are likely to be impacted and the MPCA is capable of surviving in water with a salinity 10, one estuarine or marine fish species such as the sheepshead minnow (Cyprinodon variegatus) should be tested.

If the MPCA is intended to control nuisance fish (e.g., sea lamprey or gizzard shad), effects testing on other non-target fish species will be required. Selection of appropriate numbers and species of fish should be based on the centrifugal taxonomic approach and take into account species that occur in drainage basins in the areas (i.e., ecozones) of intended use. Consideration should also be given to species which are likely to prey upon or scavenge the diseased target host.

Testing requirements. For Tier I, test fish should be exposed to the MCC of the MPCA for the duration of the study period. During testing, fish should be periodically examined for the incidence of any adverse effects. If any adverse effects are observed, then a period of recovery should be instituted immediately on half of the replicates in order to determine whether the effects are reversible. The dissemination, replication, and survival of the MPCA should also be determined in representative tissues, organs, and fluids.

If Tier II environmental toxicology testing is indicated, then the route(s) of exposure that produced the greatest impact on test organisms in Tier I should be tested. For Tier II aqueous exposure studies, test fish should be exposed for 5 days to the MCC of the MPCA. For Tier II dietary studies, test fish should be fed the MCC for 5 days. All fish should be monitored for any adverse effects observed in Tier I studies. Regardless of the exposure route chosen for Tier II testing, the test fish should be monitored for an additional 25 days following dosing or until adverse effects are observed.

Arthropods. Test arthropods should be exposed to the MPCA in a manner consistent with the route of exposure and greatest degree of susceptibility under natural environmental conditions. The route of exposure selected for testing is based mainly on whether the non-target arthropod is terrestrial or aquatic:

Terrestrial Arthropods. The MPCA should be administered topically; or in the diet; or as a combination of these two routes of exposure.

Aquatic Arthropods. The MPCA should be administered as a suspension in the water (aqueous exposure); in the diet; or as a combination of these two routes of exposure.

Test Species. Non-target arthropod species selected for testing should be representative of groups that will be exposed to the MPCA under actual conditions of use, and that have some important relationship with the target pest species. Particular consideration should be given to arthropods established in the ecozone(s) of intended use, and to those "beneficial" species with broad environmental and/or economic importance, such as bees (Apis mellifera [L.] [Hymenoptera]), insects used in biological control programmes (e.g., Apanteles spp. [Hymenoptera]), and predatory orchard mites (e.g., Neoseilus fallacis [Garman] [Acari], Phytoseiulus persimilis [Acari]).

A list of terrestrial and aquatic arthropod taxa, from which representative species may be selected for testing, is provided in the guidelines.

Testing Requirements. For Tier I topical exposure tests, the non-target arthropod should be treated with the MCC for 5 successive days and then observed for an additional 16 days. For Tier I exposure or dietary tests, non-target arthropods should be exposed to, or fed, the MCC of the MPCA (or MPCP) for at least 21 days, or until mortality in the control group increases to a significant level.

For Tier II environmental toxicology tests, arthropod species adversely affected in Tier I should be exposed to, or fed, the MCC of the MPCA for 5 days and then observed for the equivalent period of time required for manifestation of adverse effects in Tier I tests. For Tier II topical exposure tests, susceptible arthropod species from Tier I should be treated once with an application of the MPCA equivalent to the maximum label rate.

Although Tier III chronic (life cycle) toxicity testing may be triggered by the results of Tier II acute studies, the PMRA recognizes that testing beyond the acute toxicity level may not be possible for some arthropod species.

Non-arthropod invertebrates. If the MPCA is intended to control invertebrates such as annelids (e.g., Lumbricus terrestris) or molluscs, then effects testing is required on non-target non-arthropod invertebrates. Applicants are required to follow the centrifugal taxonomic approach when selecting test species.

If the MPCA is not intended to control non-arthropod invertebrates, but may be closely related to a known pathogen to this group of organisms, then the MPCA should be tested on potentially susceptible non-arthropod invertebrate species which may be exposed during operational use of the MPCA. If the MPCA does not resemble any known or suspected pathogen to non-arthropod invertebrates, effects testing may still be required if exposure is a concern.

Microorganisms. For most MPCAs, effects testing on non-target microorganisms will not be required. However, the PMRA has determined that in cases where the biology, ecology, and proposed use pattern(s) of the MPCA indicate a potential for adverse effects on environmentally and/or economically important microbial species or microbially mediated biogeochemical processes, effects testing on microorganisms or microbial processes may be required. Testing on non-target microorganisms and/or processes will mainly be reserved for MPCAs intended to control pest microorganisms or processes. The requirement for microorganism testing is determined by the PMRA on a case-by-case basis.

Plants. MPCAs intended to control pest plants (i.e., herbicidal MPCAs), and any MPCPs which may contain phytotoxic and/or phytopathogenic MPCAs, must be tested on non-target plants to determine their plant host range. MPCAs that are taxonomically similar to known plant pathogens with very narrow host ranges may only need to be tested on a limited number of potentially susceptible plant species. MPCAs that are similar to a wide range of known plant pathogens, however, may require testing on a greater number of plant species in order to determine its complete plant host range. Knowledge of the mode of action may also assist in determining the extent of testing needed for potential plant pathogens.

MPCAs not intended to control plants (i.e., non-herbicidal MPCAs) should also be tested for potential adverse effects on those non-target plants expected to be contacted during operational uses. However, MPCAs which do not resemble any known plant pathogen would not be expected to have phytopathogenic properties and, therefore, would require little, if any, plant testing.

Test species. Selection of non-target plant species depends on whether the intended use pattern of the MPCA is terrestrial or aquatic:

For MPCAs intended for terrestrial uses (where direct aquatic exposure is not anticipated), representative test species should be selected from up to 12 terrestrial plant families listed in the guidelines.

For MPCAs intended for forestry applications (where direct aquatic exposure is anticipated), species of Pinaceae and Salicaceae, and representative species from six aquatic plant families listed in the guidelines should also be tested.

For non-herbicidal MPCAs that are closely related (taxonomically) to known phytopathogenic microorganisms, plant species susceptible to the known pathogens should be selected as a starting point to assess the phytopathogenicity of the MPCA. If pathogenic effects are noted with any of these species, further testing would be required, utilizing the centrifugal taxonomic approach, with the affected plant species as the new starting point.

For MPCAs applied in aquatic use patterns, or in terrestrial use patterns where aquatic exposure is anticipated, testing on representative species from six aquatic plant families listed in the guidelines is required. Additional testing is required on at least one representative species from each of the algal families Chlorophyceae (green), Cyanophyceae (blue-green), and Bacillariophyceae (diatom).

Testing on marine plant species is not ordinarily required unless, as a result of the use pattern, a potential exists for exposure of marine plants.

Testing requirements. For Tier I testing, non-target plants should be treated with the MCC of the MPCA at the time of most likely susceptibility or at the normal stage of maturity that the plants would be exposed under actual conditions of use. When the optimum conditions for penetration, infection, and disease development are known or suspected, it is important, particularly for herbicidal MPCAs, to simulate these conditions rather than those known to be optimum for plant growth and development. In many cases, however, the optimum environment may be similar. Test plants should be observed regularly until normal harvest or death, or, as in the case of perennials, at periodic intervals for at least the time required to adversely affect the target plant pest.

In Tier II, testing on both the germinating seed (i.e., seed emergence and root elongation testing) and young vegetative growth (i.e., vegetative growth and vigour testing) stages is required for certain terrestrial non-target plants. Plants should be tested with a concentration of the MPCA or MPCP that is equivalent to the maximum rate of application proposed on the product label.

Tier IV testing on terrestrial non-target plants should be conducted on small-scale field plots with the MPCP applied at the maximum label rate and under conditions that are optimal for disease development. Tier IV testing on aquatic non-target plants is not required for small-scale terrestrial field trials.

ENVIRONMENTAL FATE REQUIREMENTS

Environmental fate testing is intended to demonstrate whether an MPCA is capable of surviving or replicating in the environment to which it is applied. Environmental fate tests further provide an indication of which non-target organism(s) may be exposed to the MPCA as well as provide an indication of the extent of exposure.

Approach. The extent of environmental fate testing, if required for registration, is based mainly on the nature of the MPCA, i.e., whether it is indigenous or non-indigenous to the ecozone(s) of intended use. The need for fate testing is dependent on whether adverse effects on non-target organisms were noted in Tier I or Tier II environmental toxicology tests.

If results of Tier I (for non-indigenous and genetically engineered MPCAs) or Tier II (for indigenous MPCAs) environmental toxicology studies indicate that the MPCA has a narrow host range (i.e., the MPCA affects only the target pest) or that it produces adverse effects on a limited number of closely related (taxonomically) species, other than those of environmental and/or economic importance, then the likelihood of hazard to unrelated organisms not tested would likely be sufficiently low to not require environmental fate testing.

If results of Tier I (for non-indigenous and genetically engineered MPCAs) or Tier II (for indigenous MPCAs) environmental toxicology studies indicate that the MPCA has a broad host range or that it produces significant adverse effects on a number of non-target species, including those of environmental and/or economic importance, then the likelihood of environmental hazard would be of sufficient magnitude to warrant laboratory fate (pure culture and microcosm) testing for non-indigenous and genetically engineered MPCAs and environmental fate testing (small-scale field trials) for indigenous MPCAs.

The results of environmental fate tests are used to:

judge whether concentrations of the MPCA at the time of application will produce a significant impact on non-target organisms; and

determine whether quantities of the MPCA will persist for an extended period of time above background concentrations thereby allowing for continued or subsequent impact on non-target organisms.

Test substance. The form of the MPCA to be tested is dependent on the type of study being conducted and is specified in each testing requirement of the guidelines.

Tier progression. If results of environmental fate tests indicate that the MPCA is capable of surviving and persisting in the environment of intended use and susceptible non-target organism(s) are likely to be exposed, then further testing may be required as shown in Figure 1.

Laboratory studies. Laboratory studies are intended to determine:

the optimum and range of factors required for the growth and subsistence of the MPCA;

the persistence, multiplication, and dispersion of the MPCA under various environmental conditions and in various environments of intended use (e.g., terrestrial, freshwater, estuarine/marine); and

if applicable, the ability of any modified genetic material in a GEM to transfer to other organisms or persist in the environment.

Pure culture, or in vitro, testing is necessary to define the various physical and chemical factors that are required for the growth and subsistence of the MPCA. Although studies with pure cultures may not provide a wholly accurate representation of the growth and persistence of the MPCA in the environment, the data obtained will assist in delineating the limits for growth and subsistence.

Microcosm studies are used to assess the biotic and abiotic interactions of the MPCA in environmental media (e.g., soil, vegetation, leaf litter, water and sediment) to which it is to be applied or exposed. In these studies, the ability of the MPCA to grow and persist in an environment more representative of the area of release is determined. Additionally, these studies can be used to provide an estimate of the ability of any genetic material introduced into an MPCA to transfer to other organisms or to persist ex vivo in the environment.

Field studies. As discussed above, field studies may be required for an MPCA that has a broad host range (i.e., affects more than just target organisms) or that produces significant adverse effects on environmentally or economically important non-target organisms.

Field studies on the environmental fate of the MPCA (including any modified genetic material in the case of a GEM) must be conducted using the MPCP. Field studies should address such aspects of environmental fate as:

growth or subsidence (to environmentally acceptable levels);

persistence;

dispersion; and

transfer and persistence of modified genetic material, if applicable.

Small-scale field studies should be limited to small plots with a total area of less than 10 ha/ecozone. In order to assess the fate of the MPCA under actual conditions of use, field studies must be carried out in the ecozone(s) of intended use. Progression to larger scale field studies may be required, but are not be undertaken until the data from both small-scale environmental fate studies and environmental toxicology studies have been reviewed by the PMRA.

CONCLUDING REMARKS

The purpose of this paper was to provide a brief history and a detailed overview of the data requirements for registering and assessing the environmental hazards and risks of microbial pesticides in Canada. The guidelines provide general guidance to applicants on the test endpoints required in support of registration rather than prescribe specific test methods or protocols to address each test endpoint. For effects testing, the PMRA currently recommends applicants use test methods that are appropriate to the pesticidal mode of action (e.g., toxicity or pathogenicity), or other relevant characteristic, of the microbial agent.

We feel this current guideline approach allows flexibility for expert judgement and adjustment to new developments in microbial testing. Moreover, the PMRA does not prescribe specific test methods for microbial pesticides primarily because of the absence of standardized and internationally recognized protocols for both environmental fate and effects testing of microorganisms. Until such protocols are developed and accepted by the scientific research and regulatory communities, general, rather than specific, guidance will be provided to prospective registrants of microbial pesticides.

REFERENCES

Agriculture Canada (1986) Memorandum to Registrants, R-1-229, Guidelines for the Registration of Microbial/Biological Pesticides.

Agriculture Canada (1990) Memorandum to Registrants, R-90-03, Guidelines for Registration of Naturally Occurring Microbial Pest Control Agents.

Agriculture and Agri-Food Canada (1993) Regulatory Proposal, Pro93-04, Registration Guidelines for Microbial Pest Control Agents.

Figure 1. Environmental toxicology and environmental fate testing tiers for microbial pest control agents.