United States Prevention, Pesticides EPA712-C-96-056
Environmental Protection and Toxic Substances February 1996
Agency (7101)
&EPA Microbial Pesticides Test
Guidelines
OPPTS 885.5000
Background for
Microbial Pesticides
Testing
-------
INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), internet: http://
fedbbs.access.gpo.gov, or call 202-512-0132 for disks or paper copies.
This guideline is also available electronically in ASCII and PDF (portable
document format) from the EPA Public Access Gopher (gopher.epa.gov)
under the heading "Environmental Test Methods and Guidelines."
-------
OPPTS 885.5000 Background for microbial pesticides testing.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.}.
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is OPP guideline 155A-1.
(b) Purpose. Tier II tests are intended to demonstrate whether a mi-
crobial pest control agent (MPCA) is able to survive or replicate in the
environment, thereby indicating which nontarget organism(s) may be ex-
posed to the microbial agent, as well as provide an indication of the level
of exposure. A determination of the environmental expression of an MPCA
includes an evaluation of the growth of the agent when introduced into
a new environment—the way the MPCA may alter its growth habits, take
advantage of new environmental conditions or take advantage of changes
in the existing equilibrium among microbial species (e.g. in a commensal
association, an association in which one species benefits and the other is
unaffected). The expression of a microorganism's presence may result in
its insertion into a new habitat and continued propagation in the new habi-
tat. Terrestrial and aquatic (freshwater and marine) environments are to
be considered, as indicated by results from Tier I, the product type and
the product usage. Advance consultation with Agency personnel is rec-
ommended since requirements may be modified on a case-by-case basis.
The Tier II guidelines consist of descriptions of tests to determine the
environmental expression of an MPCA in a terrestrial environment
(OPPTS 885.5200), in a freshwater environment (OPPTS 885.5300), and
in a marine or estuarine environment (OPPTS 885.5400). If Tier II data
is negative (no adverse effects), no further testing is needed. Tier progres-
sion is fully explained in OPPTS 885.0001.
(c) Definitions. The following definitions apply to OPPTS 885.5200,
885.5300, and 885.5400.
Contained environment. Refers to the use of natural materials from
the proposed use-environment (sediment, soil, plants, and marine/estuarine
liquids) arranged as naturally as possible, and held within a plastic, glass,
or other container to prevent the escape of the microbial agent.
Substance to be tested. A typical end-use product (EP) or the tech-
nical grade of the active ingredient (TGAI) shall be tested.
Observation period. This should be of sufficient duration to determine
survival, growth, or decline (to the limit of detection) of the MPCA in
the appropriate environment unequivocally. Decline of the MPCA should
be followed for a sufficient period of time to detect regrowth of the agent
after a prolonged adaptation period.
-------
Regrowth. The number of microorganisms present may decrease ini-
tially (perhaps to an undetectable level) and then increase (regrowth) to
or above the initial cell density. Observations should continue for a period
of time sufficient to assure lack of regrowth. In addition, efforts to resusci-
tate the MPCA (such as enrichment techniques and/or passage in suscep-
tible hosts) should be attempted to support the conclusion that the micro-
bial agent no longer persists in the environment.
(d) Approach. (1) Environmental expression testing consists of simu-
lated terrestrial and aquatic applications of the MPCA. Terrestrial applica-
tions are conducted in a contained environment (greenhouse) to assess ex-
pression in soil and vegetation. Aquatic applications are conducted in
aquariums to assess expression in water and sediment. The need for terres-
trial, freshwater, or marine environmental expression testing under specific
conditions depends on:
(i) The Tier I tests in which adverse effects were observed.
(ii) The intended use patterns for the MPCA.
(2) Testing is only required to assess environmental expression when
susceptible nontarget species may be exposed. Tier I tests define the need
and the environment to be examined. In some cases, no tests may be need-
ed. For example, if adverse effects are noted with terrestrial plants and
the intended use pattern calls for use in freshwater only, probably no addi-
tional tests would be required. If additional tests are required (e.g. adverse
effects with terrestrial insects and a terrestrial use pattern), the timing (sea-
son of use, time of day for release) and location (climate, soil type) of
usage, as well as doses shown to be effective in Tier I, must be considered
in selecting Tier II conditions for establishing environmental expression
of the test microbe. This simulation of the particular natural environment
reflecting the area of use of the product is necessary in order to achieve
the overall objective of Tier II testing—determining whether the intro-
duced microorganism will survive, multiply, and potentially threaten the
nontarget species. The intent is to define potential exposure of susceptible
nontarget species to the test product.
(3) Data should be developed which would indicate the dynamics of
population fluctuation (i.e. growth and/or survival curves) of the micro-
organism. The experimental design should consider parameters such as
inoculum size, potential for blooms or regrowth, and physical parameters
such as relative humidity, pH, and temperature.
(4) The following Table 1. summarizes need for testing under this
guideline.
-------
Table 1.—Summary of Environmental Expression Testing as Determined by Tier I Test Results
and Use Patterns
Tier I Test with Positive Results
(Test Species)
OPPTS 885.4050 and .4100
Avian testing — mallard
Avian testing — quail
OPPTS 885.4150
Mammalian testing
OPPTS 885.4200, .4240, and .4280
Fish testing — freshwater spp
Fish testing — estuarine/marine spp
OPPTS 885.4240 and .4280
Aquatic invertebrate testing — freshwater spp
Aquatic invertebrate testing — estuarine/marine spp
OPPTS 885.4300
Terrestrial plant testing
OPPTS 885.4300
Aquatic plant testing — freshwater spp
Aquatic plant testing — estuarine/marine spp
OPPTS 885.4340 and .4380
Terrestrial insect testina
Proposed
Terres-
trial
NA 1
T2
T
F
EM
F
EM
T
F
EM
T
Use Patterns 1
Freshwater
F3
NA 1
F
F
EM
F
EM
NA 1
F
EM
NA 1
or MPCA
Estua-
rine/Ma-
rine
EM 4
NA 1
NA 1
NA 1
EM
NA 1
EM
NA 1
NA 1
EM
NA1
1 NA: Based on results of Tier I tests and the proposed use pattern, adverse effects are not ex-
pected. However, the Agency may require such tests on an individual basis.
2 T: Tests to determine expression in a terrestrial environment are necessary.
3 F: Tests to determine expression in a freshwater environment are necessary.
4 EM: Tests to determine expression in an estuarine or marine environment are necessary.
(e) Applicability. This section outlines the general test standards that
apply to the terrestrial, freshwater, and aquatic studies. Applicants for reg-
istration should also comply with the specific test standards established
for the particular test being conducted. In the case of conflict between
general and specific test standards, the latter shall govern.
(f) Test standards. Data satisfying the provisions of OPPTS Series
885, Group E should meet the general test standards in OPPTS Series
835—Fate, Transport, and Transformation Test Guidelines, and OPPTS
Series 840—Fate and Transport Field Studies Test Guidelines, with the
following exception: The most specific available methods for the identi-
fication and quantification of the MPCA should be used. The methods
used should be consistent with those in OPPTS 885.0001.
(g) Reporting and evaluation of data—(1) Results, (i) Data col-
lected to determine whether the MPCA is able to survive, persist, or rep-
licate in the environment under Tier II test conditions is best expressed
in the form of a population growth or decline curve for the MPCA, al-
though other applicable methods of presenting the data may be used.
-------
(ii) Test reports should also contain the information designated in the
following list, modified as necessary to be applicable to the microbial
agent and environment being tested. This information should be given in
sufficient detail to adequately define potential impact on growth character-
istics of the test organism. In general, the amount of information and kind
of data that should be reported in Tier II testing are very similar for any
of the environments being examined and are, where applicable, as follows:
(A) Type of system and components used.
(B) Source of components.
(C) Equilibrium period before addition of MPCA.
(D) Temperature, light requirements, humidity, or moisture content,
pH of the medium, oxygen requirements.
(E) Type of formulation (EP or TGAI).
(F) Application rate or dose level.
(G) Identity of test substance and composition of formulation.
(H) Characteristics of water in aquatic systems.
(I) Method of application.
(J) Sampling amount, schedule, and sensitivity of detection.
(K) Tabulation/graphs of population dynamics.
(L) Discussion of test results.
(2) [Reserved]
(h) Tier progression. If the results of Tier II expression testing in
simulated, contained environments show that the MPCA will survive and
persist in the environment under study, thereby exposing nontarget orga-
nisms to the MPCA, further testing on the Tier III level is required. If
the results are negative (the microbial agent does not survive or persist),
no further testing is required. Unequivocal determination of negative re-
sults may be difficult to prove. Diligent efforts using enrichment tech-
niques may be needed to prove the results are negative and that progres-
sion to Tier III is not required.
------- |