United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA 712-C-96-163
April 1996
&EPA Ecological Effects Test
Guidelines
OPPTS 850.4150
Terrestrial Plant Toxicity,
Tier I (Vegetative Vigor)
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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 850.4150 Terrestrial plant toxicity, Tier I (vegetative vigor).
(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.).
(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. Vegetative vigor 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 vegetative vigor test, Tier I. (A) The objective of
the test is to determine if a pesticide exerts a detrimental effect on plants
during early critical stages in their development. 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 quick-
ly evaluate the phytotoxic effects of the pesticide at the one dose.
(B) The terrestrial nontarget plant phytotoxicity vegetative vigor test
is a greenhouse or growth chamber test. The test organisms are the re-
quired three 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 plant surface is treated with test chemical
(TEP) at a concentration comparable to the maximum label application
rate or at a concentration 3x the estimated environmental concentration.
Results are reported in grams or pounds of active ingredient (AI) per acre
and are expressed as the percent of detrimental effect on growth compared
to the control after at 14 days. Parameters measured include plant height,
plant dry weight, and percent phytotoxicity. The results are used to estab-
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lish acute toxicity levels to indicate 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 vegetative vigor stud-
ies 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. 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 accept-
able for the laboratory/greenhouse vegetative vigor test:
Species for use in terrestrial nontarget plant phytotoxicity vegetative vigor 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
Perrenial ryegrass
Gramineae 	
Zea mays
Corn
Amaryllidaceae 	
Allium cepa
Onion
1 Innoculation with Rhizobium japonicum is unnecessary.
(ii)	Seeds of plants with a low or variable germination potential
should be avoided for the vegetative vigor 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 the testing of more
than 10 plant species.
(iii)	Identification of the cultivars of the plant species or assignment
of an identification number to the cultivar used and seed or plant source.
(iv)	Identification of the number of replicates and the number of
plants per replicate per dose.
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(v) 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
the maximum label rate should be tested. If it can be determined that the
maximum 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 vegetative vigor tests. Larger popu-
lations and more replicates may be needed to increase the statistical signifi-
cance of the test.
(5)	Site. The vegetative vigor test may be performed in a growth
chamber, greenhouse, or in small field plots.
(6)	Duration. The effects on vegetative vigor should be observed
weekly, or more frequently, for at least 2 weeks following pesticide appli-
cation. Some slow uptake pesticides may require a longer test duration.
If abnormal symptoms occur, the observations should be continued until
the plant dies or fully recovers.
(c)	Vegetative vigor—foliar spray protocol. The following protocol
has been developed to provide guidance in the performance of pesticide
plant hazard evaluation testing: The foliar spray can be applied by any
acceptable method using laboratory, greenhouse, or field-grown plants.
The plants should be 4 to 6 weeks postemergent in order to gain young
foliage. The emerged plants are typically sprayed with the maximum label
dosage; three replicates. Detrimental effects are to be reported as severity
of phytotoxicity (percent or rating), abnormal changes in growth and de-
velopment, and/or abnormal changes in plant morphology as compared to
untreated controls. Direct measurements of plant height, plant dry weight,
and number of dead plants are reported as compared to the controls. Root
dry weights are also reported for root inhibiting pesticides.
(d)	Reporting. In addition to the information required in OPPTS
850.4000, the test report should include the following information.
(1)	Tabulation of the results indicating the percentage effect level for
each species as compared to untreated control plants.
(2)	Data on dry weights and heights, severity of phytotoxicity, or
other growth parameters are required to be submitted.
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(e)	Tier progression. (1) If the results of the vegetative vigor test
has indicated an adverse effect greater than 25 percent on one or more
plant species, then vegetative vigor tests at the Tier II level are required
(see OPPTS 850.4200).
(2) If less than a 25 percent detrimental effect or response is noted
for the vegetative vigor test, no additional vegetative vigor testing is ordi-
narily 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 the Tier I vegetative
vigor study 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)	Results of any chemical analysis of the plant material.
(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 vegetative vigor 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.
(E)	Other information the registrant considers appropriate and relevant
to provide a complete and thorough description of the test procedures and
results.
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(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 3x that
maximum environmental quantity (provide maximum environmental qual-
ity calculations).
(F)	Method of application including equipment type.
(G)	Number of applications.
(iii)	Site of the test. (A) Site description of the vegetative vigor study
such as the type of growth chamber, greenhouse, or test 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 regime—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 plant height, plant dry weights, root
dry weights, root length, number of dead plants, or other growth param-
eters that may have been measured to ascertain toxic effects of the pes-
ticide 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.
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(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. (A) 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.
(B) [Reserved]
(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, AL, Auburn Printing (1977).
[Reserved]
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