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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