United States Prevention, Pesticides EPA712-C-96-335
Environmental Protection and Toxic Substances February 1996
Agency (7101)
&EPA Microbial Pesticide
Test Guidelines
OPPTS 885.4300
Nontarget Plant Studies,
Tier
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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."
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OPPTS 885.4300 Nontarget plant studies, Tier I.
(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 154A-22.
(b) Test standards. In addition to satisfying the applicable general
test standards outlined in OPPTS 885.0001, this study should meet the
following standards:
(1) Test substance. The actual form of the material to be regarded
as the test substance is discussed in section OPPTS 885.0001. In addition,
any substances used to enhance virulence should be tested along with the
test substance.
(2) Dose levels. One concentration level equal to no less than the
maximum label rate shall be tested. The phrase "the maximum label rate"
means the amount of active ingredient in the recommended quantity of
carrier, such as water to be used per land area or applied directly to the
surface of a 15-cm or 6-in column of water.
(3) Test species. The number of species tested depends on the simi-
larity of the microbial pest control agent (MPCA) to known plant patho-
gens. A rationale for selection of the species to be tested must be provided
to the EPA.
(i) Animal-controlling MPCAs. (A) When the pesticide is intended
to control animals, including insects, the plants to be tested should include
six species of Dicotyledoneae of at least four families and four species
of Monocotyledoneae of at least two families. These species should be
selected from the plants of most important commercial value (Table 1.
under paragraph (e) of this guideline).
(B) For MPCAs that have aquatic uses or may be expected to dissemi-
nate to, and survive in, aquatic ecosystems, additional aquatic plants must
be tested to include: Selenastrum capricornutum (a freshwater green alga),
Lemna gibba (duckweed), Skeletonema costatum (a marine diatom),
Anabaena flos-aquae (a blue-green bacterium), and a freshwater diatom.
(C) EPA will consider requests for waiver of part or all of this re-
quirement if it can be shown that the MPCA occurs naturally in the area
of intended usage and the level used does not exceed the naturally occur-
ring concentration.
(ii) Plant-controlling MPCAs and MPCAs similar to known plant
pathogens. When the pesticide is intended to control plant growth and
development (microbial herbicides), or is otherwise closely related to a
plant pathogen, in addition to testing the range of plants identified for
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animal-controlling MPCAs, testing must be performed on all plants of eco-
nomic importance (horticultural/agronomic) or known to be beneficial to
maintenance of the ecosystem that have any reasonable likelihood of serv-
ing as hosts. This selection of additional plant species should be based
upon a survey of plants closely related (same genus or, if not available,
same family) to the target plant and a survey of known hosts of pathogens
closely related to the microbial herbicide.
(4) Controls. Both positive and negative controls should be included
in the test protocols.
(i) Negative (untreated) controls should be as pest-free as reasonably
possible. In addition, in the case of MPCAs that are readily disseminated
(wind, insects, etc.), it may be necessary to conduct tests such that negative
controls and treated plants are grown in separate geographic locations or
in separate contained greenhouses under identical environmental conditions
so that reliable negative controls can be maintained. Alternatively, the neg-
ative control may be treated with a nonphytotoxic chemical pesticide
known to provide effective control of the microbial pesticide. Since it is
sometimes difficult to detect adverse effects, such as delayed maturation
or loss in vigor, growth, quality, yield, or stand, it is important to analyze
untreated controls using a sensitive, specific analytical method to deter-
mine whether or not MPCA infection has occurred.
(ii) Positive controls are required for microbial herbicides, or for
MPCAs similar to known plant pathogens, in order to ascertain that envi-
ronmental conditions are such that penetration, infection, and disease de-
velopment are likely to occur in a susceptible host. The positive control
should be selected to closely resemble the subject MPCA in terms of tax-
onomy and optimal conditions for infection and disease development, if
known. In the case of a MPCA not intended for herbicidal use, the positive
control may consist of a known plant pathogen, with taxonomic character-
istics similar to the MPCA and its susceptible host. In the case of a micro-
bial herbicide, however, the positive control should consist of the target
pest weed and the microbial herbicide.
(5) Environmental test conditions. When the optimum conditions
for penetration, infection, and disease development are known or sus-
pected, it is important, particularly for microbial herbicides, 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.
(6) Application of MPCA. The test plants should be exposed to the
MPCA by whatever route of exposure would be expected by the proposed
use pattern. This route of exposure should be supplemented by other routes
of exposure if indicated by the mode of transmission of typical pathogens
of the test plant or, for microbial herbicides, if indicated by the mode
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of transmission of similar plant pathogens. In some cases, wounding of
plants or simulation of (or actual) insect vectors-might be appropriate. In
other cases, seed treatment, root (soil) application, or foliar spray might
be the most appropriate method.
(7) Timing of application. Plant test species should be treated at the
time of most likely susceptibility (this may be known for microbial herbi-
cides) or at the normal stage of maturity when application to target areas
is initiated.
(8) Observations. Plants should be observed weekly or more fre-
quently until normal harvest or death, or, in the case of perennials, at regu-
lar intervals for at least 2 years. If no obvious adverse effects are evident
after these observation periods, the roots, foliage, fruit, vascular tissues,
etc. should be analyzed for the presence of the organism using sensitive,
specific methods. It is important to complete such analyses because obvi-
ous disease development in perennials may take several years and asymp-
tomatic plants may serve as sites for proliferation and survival of the orga-
nism, thus providing a reservoir of the organism in the environment.
(c) Reporting. In addition to the information specified in OPPTS
885.0001, the test report shall contain the following information.
(1) Rationale for selection of the species tested.
(2) Description of the growth chambers, greenhouse, or other type
of test facility including containment provisions and monitoring devices
for maintaining proper environmental conditions.
(3) Temperature and humidity ranges, including any significant devi-
ations encountered in course of the experiment.
(4) Photoperiod and lighting.
(5) Any abnormal adverse or beneficial effects in treatment and/or
control groups, including dates and times the effects were observed.
(6) Methods for any statistics used for analysis of results.
(d) Tier progression. If any adverse effects resulting from infection
occur or analyses indicate that asymptomatic infection has occurred, test-
ing at Tier II (Environmental Expression, OPPTS 885.5000, 885.5200,
885.5300, and 885.5400) is required as specified in 40 CFR 158.740. In
some cases, a subchronic test may serve to better understand the effects
observed at the Tier I level and might alleviate the need for Tier II testing.
(e) Plant species for testing. The following Table 1. lists plants of
commercial value (see paragraph (b)(3)(i)) of this guideline).
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Table 1.—25 Major Agricultural Crops
Listed by Monetary Listed by Production
Value Weight
corn corn
soybeans alfalfa/hay
wheat wheat
cotton soybeans
tobacco sorghum
sorghum sugarcane
potatoes sugarbeets
oranges potatoes
barley oats
rice oranges
grapes grapes
peanuts apples
apples cotton
sugarbeets grapefruit
sugarcane peanuts
tomatoes plums
lettuce sunflower
oats barley
strawberries peaches
beans tobacco
onions beans
peaches cucumbers
almonds rice
sunflower rye
brocolli peas
NOTE: Depending upon the predicted use pattern, certain forest tree spe-
cies, ornamental trees and shrubs, and weed species may need testing.
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