United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA 712-C-96-153
April 1996
&EPA Ecological Effects Test
Guidelines
OPPTS 850.4100
Terrestrial Plant Toxicity,
Tier I (Seedling
Emergence)
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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
(7 U.S.C. 136, etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
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), or call 202-512-0135 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 850.4100 Terrestrial plant toxicity, Tier I (seedling emer-
gence).
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2)	Background. The source material used in developing this har-
monized OPPTS test guideline is OPP 122-1 Seed Germination/Seedling
Emergence and Vegetative Vigor (Tier I) (Pesticide Assessment Guide-
lines, Subdivision J—Hazard Evaluation; Nontarget Plants) EPA report
540/09-82-020, 1982.
(3)	Test objective. This guideline should be used in conjunction with
OPPTS guideline 850.4000, Background—Nontarget plant testing, which
provides general information and overall guidance for the nontarget plants
test guidelines.
(i)	General. Seedling emergence studies are designed to provide
phytotoxicity data on a pesticide. These phytotoxicity data are needed to
evaluate the effect of the level of pesticide exposure to nontarget and ter-
restrial plants and to assess the impact of pesticides on endangered and
threatened plants as noted under the Endangered Species Act. The prelimi-
nary level (Tier I) study evaluates the effect of the maximum exposure
level. Where a phytotoxic effect is noted in one or more plants, further
studies may be required. These studies are required by 40 CFR 158.150
to support the registration of any pesticide intended for outdoor use under
FIFRA, as amended.
(ii)	Objective of seedling emergence test, Tier I. (A) The objective
of the Tier I seedling emergence test is to determine if a pesticide exerts
a detrimental effect to plants during early critical stages in their develop-
ment. The test is performed on species from a cross-section of the
nontarget terrestrial plant population that have been historically used for
this type of testing and, therefore, have known types of responses. This
is a maximum dose test designed to evaluate the phytotoxic effects of
the pesticide quickly at the one dose.
(B) The terrestrial nontarget plant phytotoxicity seedling emergence
test is a greenhouse or growth chamber test. The test organisms are three
required species—corn, soybeans, and a root crop, plus seven other spe-
cies, usually tomato, cucumber, lettuce, cabbage, oat, ryegrass, and onion
(six species of at least four families of dicots and four species of at least
two families of monocots). The soil or plant surface is treated with test
chemical (typical end use product (TEP)) at a concentration comparable
to the maximum label application rate or at a concentration 3x the esti-
mated environmental concentration. Results are reported in grams or
pounds of active ingredient (AI) per acre and are expressed as the percent
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of detrimental effect growth compared to the control after at least 14 days.
Parameters measured include plant height, plant dry weight, and percent
phytotoxicity. The results are used to establish acute toxicity levels to indi-
cate if further testing at a higher tier is necessary.
(b) Test standards. In addition to the general test standards set forth
in OPPTS 850.4000, the following standards for the seedling emergence
studies apply:
(1)	Test substance. Refer to 40 CFR part 158 for information re-
quired on the test substance.
(2)	Species. Each report should include the following information:
(i) Identification of the six dicotyledoneae species and four
monocotyledoneae species with family identification.
(A) The six dicots are to be of at least four different families and
the monocots of at least two families. Soybeans, corn and a dicot root
crop like carrot are the required species. The proposed species and families
are given below and are acceptable for the seedling emergence test:
Table 1.—Species and families acceptable for the seedling emergence test
Family
Species
Common name
Solanaceae	
Lycopersicon esculentum
Tomato
Cucurbitaceae	
Cucumis sativus
Cucumber
Compositae	
Lactuca sativa
Lettuce
Leguminosae 1 	
Glycine max
Soybean
Cruciferae 	
Brassica oleracea
Cabbage
Umbelliferae	
Daucus carota
Carrot
Gramineae 	
Avena sativa
Oat
Gramineae 	
Lolium perenne
Perennial ryegrass
Gramineae 	
Zea mays
Corn
Amaryllidaceae 	
Allium cepa
Onion
1 Innoculation with Rhizobium japonicum is unnecessary
(B) Seeds of plants with a low or variable germination potential
should be avoided for the seedling emergence study. When selecting plant
species other than corn, soybean, and a root crop, the Agency encourages
the use of sensitive plants other than crop plants—weeds, native species,
perennial species, etc. The Agency also encourages testing of more than
10 plant species.
(ii)	Identification of the cultivars of the plant species or assignment
of an identification number to the cultivar used and seed or plant source.
(iii)	Identification of the number of replicates and the number of
plants per replicate per dose.
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(iv) Identification of the date of planting, date of pesticide application
and height of plants at application, and date of phytotoxicity rating or har-
vest and analysis.
(3)	Application levels. One concentration level equal to no less than
maximum label rate should be tested. If it can be determined that the maxi-
mum quantity that will be present in the nontarget area is significantly
less than the maximum label rate, a concentration equal to no less than
3x that maximum quantity may be tested. The phrase "the maximum label
rate" means the maximum recommended amount of AI in the rec-
ommended minimum quantity of carrier such as water to be used per land
area.
(4)	Number of plants. At least three replicates, each with 10 plants,
should be tested per dose level for the seedling emergence tests. Larger
populations and more replicates may be needed to increase the statistical
significance of the test.
(5)	Site. The seedling emergence studies should be conducted under
controlled conditions in growth chambers, greenhouses, or in small field
plots.
(6)	Duration. Seedling emergence should be observed weekly, or
more frequently, for at least 2 weeks after germination.
(c)	Seedling emergence protocol The following protocol has been
developed to provide guidance in the performance of pesticide plant hazard
evaluation testing: Seeds may be germinated in pots using a standardized
soil. Three replicates should be set up using at least 10 seeds per pot.
The seeds may be surface-sterilized. The soil or support medium should
be sprayed or otherwise treated with a known quantity of the chemical,
typically the maximum label dosage. The test conditions should approxi-
mate those optimal conditions for the species and varieties considered. The
seeds should be incubated for at least 14 days. The seeds are observed
after 10 and 14 days, and seedling emergence is recorded as the number
of emerged seedlings. Shoot height, shoot dry weight, and visual
phytotoxicity are also recorded. Root dry weights are also reported for
root inhibiting pesticides. See paragraph (g)(1) of this guideline for further
guidance.
(d)	Reporting. In addition to the information required in OPPTS
850.4000, the test report should include the following information.
(1)	The number of seeds tested and the number emerged per dosage
level for each replicate.
(2)	Descriptions of the appearance and the growth and development
of the seeds and emergent plants, indicating any abnormalities and expres-
sions of phytotoxicity.
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(3)	Tabulation of the results indicating the percentage effect level for
each species as compared to untreated control plants.
(4)	Data on dry weights and heights, or other growth parameters are
required to be submitted.
(e)	Tier progression. If the results of the seedling emergence test
have indicated an adverse effect greater than 25 percent on one or more
plant species, the seedling emergence tests at the Tier II level are required
(see OPPTS 850.4200). If less than a 25 percent detrimental effect or re-
sponse is noted for the seedling emergence test, no additional testing of
the respective tests at higher tiers is ordinarily required. The Agency, after
review of the data, may require certain additional tests to determine a more
definite nondiscernible effect level.
(f)	Data reporting. (1) The registrant's report on preliminary seedling
emergence studies should include all information necessary to provide:
(1)	A complete and accurate description of the laboratory/greenhouse/
field treatments and procedures.
(ii)	Sampling data and phytotoxicity rating.
(iii)	Data on storage of the plant material if so performed.
(iv)	Any chemical analysis of the plant material as to chemical con-
tent,
(v)	Reporting of the data, rating system, and statistical analysis.
(vi)	Quality control measures/precautions taken to ensure the fidelity
of the operations.
(2)	Each laboratory/greenhouse/small field plot seedling emergence
report should include the following information:
(i) General. (A) Cooperator or researcher (name and address),test lo-
cation (county and state; country, if outside of the United States), and
date of study.
(B)	Name (and signature), title, organization, address, and telephone
number of the persons responsible for planning/supervising/ monitoring.
(C)	Trial identification number.
(D)	Quality assurance indicating control measures/precautions fol-
lowed to ensure the fidelity of the phytotoxicity determinations; record-
keeping procedures, and availability of logbooks; skill of the laboratory
personnel; equipment status of the laboratory or greenhouse; degree of ad-
herence to good laboratory practices; and degree of adherence to good
agricultural practices in maintaining healthly plants.
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(E) Other information the registrant considers appropriate and relevant
to provide a complete and thorough description of the test procedures and
results.
(ii)	Test substance (pesticide). (A) Identification of the test pesticide
AI including chemical name, common name (ANSI, BSI, ISO, WSSA),
and company developmental/experimental name.
(B)	AI percentage, plus any inerts and adjuvants in test material.
(C)	Solvent used to dissolve and apply the pesticide if the pesticide
is insoluble in water or other intended carrier.
(D)	Dose rates in terms of AI per area of land or of leaf (if leaf-
area-index is provided).
(E)	Dose rates in terms of the maximum label rate, or if the registrant
has shown that the maximum quantity that will be present in the nontarget
area is the maximum label rate, the dose equal to or no less than three
times that maximum environmental quantity (environmental quality cal-
culations).
(F)	Method of application including equipment type.
(G)	Number of applications.
(iii)	Site of the test. (A) Site description of the seedling emergence
and vegetative vigor studies such as the type of growth chamber, green-
house, or field plot.
(B)	Location of the test site.
(C)	Climatological data during the test (records of applicable condi-
tions for the type of site, i.e., temperature, thermoperiod, rainfall or water
regime, light regimen—intensity and quality, relative humidity, wind
speed).
(D)	Plant density and container types.
(E)	Cultural practices such as cultivation, pest control, and irrigation
practices (frequency of watering and method used—overhead vs. bottom
watering).
(F)	Substrate characteristics (name/designation of soil type and its
physical and chemical properties, including pH and percent organic mat-
ter).
(iv)	Results. (A) Reporting of percent emergence, plant height, plant
dry weights, root dry weights, root length, dead plants, or other growth
parameters that may have been measured to ascertain toxic effects of the
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pesticide upon the plants with dates of observation (Root measurements
are only needed if the test chemical is a root inhibitor).
(B)	Phytotoxicity rating (including a description of the rating system)
for each plant or population in the test.
(C)	Statistical analysis of the results including an environmental or
effective concentration effect (EC) value. (Note, for Tier I, there will be
only a percent effect level at a specific concentration which is then com-
pared to 25 percent of the growth (mass or rate] of the control.)
(v) Evaluation, (i) For Tier I studies, determination as to whether
Tier II studies would be required due to phytotoxic effects noted in one
or more of the tested species.
(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1)	Truelove, B., ed., Research Methods in Weed Science. Southern
Weed Science Society. Auburn Printing, Auburn, AL. (1977).
(2)	[Reserved]
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