United Slalas
Environmental P'OlecHon
flgancy
Prevention Posllcicfes
and TovlcSubslanws
(71011
EPA 712-0-36^364
Apill 1996
&EPA Ecological Effects Test
Guidelines
OPPTS 850.4250
Vegetative Vigor, Tier II

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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 Slates 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 tor 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 (NTJS) and ihe 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 lo minimise variations among the testing procedures
that must be performed to meet the data requirements ot the U S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodcniicidc 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.cpa.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@epamdil.epa.gov.
To Submit Comments: Interested persons arc 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 Roctrd By modem dial 202-512-1387, telnet and ftp-
tedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0135 for disks
or papei copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopherepa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 850.4250 Vegetative vigor, tier II.
(a)	Scope—(I) Applicability. This guideline ts 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-
monr/ed OPPTS test guideline are 40 CFR 797.2750 Seed Germination/
Root Elongation Toxicity Test and OPP guideline 123-1 Seed Germina-
tion/Seedling Emergence and Vegetative Vigor (Tier II) (Pesticide Assess-
ment Guidelines, Subdivision J—Hazard Evaluation; Nontarget Plants),
EPA report 540/09-82-020, 1982.
(b)	Tier II—vegetative vigor dose response tasting. This guideline
should be used in conjunction with OPPTS guideline 850.4000, Back-
ground—Nontarget Plant Testing, which provides general information and
overall guidance for the nontarget plants test guidelines.
(I) Objective, (i) 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 Group D guide-
lines of the 850 series.The terrestrial nontarget plant phytotoxicity tests
are laboratory tests that evaluate the acute toxicity of pesticides to 10 crop
plants. These studies evaluate the effects of multiple dosage levels on plant
growth, using less than the maximum label rate with dosages in a geo-
metric progression of no more than twofold, and with subtoxic (
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caying vegetation provides nutrients essential for the aquatic food chain.
Hedgerows, woodlots, and other similar nontargct areas provide food and
cover to mammalian and avian species.
(iii)	Nontargct plant phytotoxicity data arc routinely used to conduct
ecological risk assessments for the registration and reregistration of pes-
ticides. These data are also useful in our assessment of potential hazards
to endangered/threatened plant species listed by the Department of Interior,
Fish and Wildlife service.
(iv)	Tier II tests are initiated following a determination that a greater
than 25 percent adverse effect occurred in the Tier I vegetative vigoi study
for one or more plant species. If less than a 25 percent detrimental effect
or response to all tested species is noted in the vegetative vigor study,
no higher tier testing is ordinarily required. Under FTFRA, if the pesticide
is a known phylotoxicant, terrestrial plant testing begins with Tier II.
Unique Agency concerns arising from Special Review issues, pesticide
contamination cases, 6(a)(2) incidents (adverse effects reporting), pub-
lished literature, or other public sources may result in requests for addi-
tional tests.
(c) Test methods—(1) Test facility/location/test conditions. Dose
response vegetative vigor tests can be conducted in the greenhouse or in
small field plots. Report soil type and texture, soil Kj values, soil K>IC
values, and soil pH. Environmental conditions during the test should be
recorded daily—light intensity, air temperature, humidity, photoperiods,
thcrmopcriods, watering schedules and methods (rainfall if field test), and
pest conditions are to be recorded.
(2)	Test substance. Refer to 40 CFR pail 160 lor test substance re-
quirements. Use ot TEP instead of TGAI is preferred for all nontarget
plant phytotoxicity tests, using the TEP with the highest percent active
ingredient and/or the one most commonly used.
(3)	Controls/solvents/additives and other pesticide treatments.
Pesticide treatments other than the test pesticide should be avoided. Me-
chanical, cultural, and biological pest control methods are suggested. If
solvents or other pesticides are used m the test, the registrant must show
that the solvent/pesticide is not toxic to the test species and that no syner-
gistic or antagonistic interactions with the test pesticide exists (additional
test data). To demonstrate solvent or adjuvant activity, a separate set of
conuol plants, (set aside for this purpose at the beginning ot the experi-
ment) can be treated with the solvent/pesticide using the highest dosage
A negative control is still required. If the solvent/pesticide controls and
the negative control are contaminated with the test chemical, the study
should most likely be repeated. If the solvent control is contaminated with
the lest chemical and the negative control is not (and visa versa), the study
may not be invalid it zero percent toxicity occurred in the negative control
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and at the lowest dose tested. Within a given study, all test organisms
including the controls should be from the same source. To pievent bias,
a system of random assignment of the test plants to test and contiol gioups
is required. If adjuvants are recommended on a TEP label, a representative
adjuvant within each class (anionic, ionic, nonionic, etc.) must be used
in the test.
(4)	Equipment, (i) All equipment used in conducting the test, includ-
ing equipment used to prepare and administer the test substance, and
equipment to maintain and record environmental conditions, should be of
such design and capacity that tests involving this equipment can be con-
ducted in a reliable and scientific manner. Equipment should be inspected,
cleaned, and maintained regularly, and be properly calibrated. Acceptable
types of sprays and foliar spray methods are found 111 paragraph (f)(5)
of this guideline. Computerized belt sprayers are also acceptable.
(ii) The application equipment used 111 testing products in small field
plot studies should be designed to simulate conventional farm equipment
using the basic components of commercial application equipment in the
design of the small-plot equipment. For example, nozzle types, sizes, and
arrangements on small plot sprayers can be identical to those used by
growers on commeicial ground sprayers. Specific details as to descriptions
of equipment design, adjustment, and opeiation should be piovided in test
reports.
(5)	Dosages. (1) At least five dosages should be tested.
(ii)	The dosages should include a subtoxic (
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(v)	If a pesticide (FIFRA) is applied more than once within a year
01 giowing season, each rate and the interval between applications should
be indicated. If products arc applied in a tank mixture or arc applied seri-
ally, rates and intervals, as appropriate, should be reported with identifica-
tion and formulation for each product.
(vi)	If conducting tests under TSCA, the lest chemical is applied daily
with each watering for the duration of the study.
(6) Plant test species, (i) At least three replicates, each with 10
plants, should be tested per dose level. Larger populations and more rep-
licates of certain plants with low germination may be needed to increase
the statistical significance of the test.
(ii)	Healthy plants must be used Pesticide treated seeds should be
avoided. The Agency should be consulted prior to test initiation if seed
treatments other than steam, a weak hypochlorite solution (recommended
by Environment Canada), captan, or thiram are used. Captan and thuam
seed treatments are the only approved pesticide seed treatments
(noninteractive with most other pesticides). Steam sterilization ot soil and
seeds is the recommended piocedure for killing pathogens, fungi, and in-
sects on seeds and in soil media. Some methods used to remove seed treat-
ments include a weak methanol solution, detergents, and hypochlorite solu-
tion rinsing. When unapproved pesticide seed treatments are used in a
study, it is the responsibility of the laboratory conducting the icm to show
that no synergistic or antagonistic interactions occur between or among
the various pesticides in the test.
(iii)	Ten plant species must be tested. The following plant species
and groups are recommended:
(A)	Dicotyledoncae. Six species of at least four families, one species
of which is soybean (Glxane max) and a second of which is a root crop
(B)	Monocotylc
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(7)	Support media and pesticide dosing method, (i) Plants may be
grown in pots using a sterilized standardised soil that consists of primarily
sandy loam, loamy sand, loamy clay, or clay loam soil that contains up
to 3 percent organic matter Glass beads, rockwool, and 100 percent acid
washed sand are not recommended.
(ii)	Test methods and protocols for hydroponic tests should be submit-
ted to the Agency tor review prior to test initiation.
(iii)	The pesticide is sprayed onto the plant toliage ensuring even and
thorough contact with plain surfaces.
(8)	Test containers. Test containers should be nonporous to prevent
absorption of the test material. Do not use peat or clay. Containers should
be thoroughly cleaned prior to use. A dichromatc solution should not be
used to clean containers.
(9)	Test parameters, (i) Carbon dioxide level should be maintained
at 350 ±50 ppm.
(ii)	Relative humidity should approach 70±5 percent during light pe-
riods and 90 percent during dark periods.
(iii)	Irradiation measured at 1 m from the source, at
350 + 50 jlE/m2 sec at 400 to 700 nm.
(iv)	Photoperiods of 16 h light and 8 h darkness.
(v)	Day/night temperatures at 25/20±3 °C.
(vi)	Half strength modified Hoagland nutrient solution may be used
as nutrient media.
(10)	Watering methods. Bottom watering of test containers is pre-
ferred, however, top watering under the foliage can also be used to prevent
washing pesticide off foliage.
(11)	Plant density. The numbei of planus per pot is left to the discre-
tion of the laboratory conducting the test, for example: One or two corn,
soybean, tomato, cucumber, or sugarbeet plants in a 6-inch container, three
rape, or pea plants in a 6-inch container, and a maximum of six onion,
wheal, or other small grains in a 6-inch container. The test conditions
should approximate those optimal conditions for the species and varieties
tested (cool and warm season plants should be tested separately).
(12)	Test duration/end-points, (i) The test material is applied 2 to
4 weeks after the plants have emerged from the soil. During the minimum
14-day observation period following chemical application, the plants are
observed every 7 days (or more frequently) for visual phyiotoxicity, total
dead plants, and shoot height. At the end of the study, final shoot heights
and phytotoxicity measurements are taken prior to harvesting of the above
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and below-ground portions for dry weight measurement (root dry weight
and root length measurements are optional depending on the chemicals
mode-of-action information, incident reports, literature, etc.)- It abnormal
symptoms occur, the observations should be continued until the plant dies
or fully recovers. The individual test species responses to environmental
conditions, the mode-of-action of the pesticide, and the speed of uptake
of the pesticide by the test plants arc factors that affect study duration.
The registrant is expected to extend die duration of the study as dictated
by these factors. All dead plants should be measured, weighed, and ob-
served and included in the statistical analysis.
(ii)	Observations should include all variations, either inhibitory or
.stimulatory, between the treated test organisms and the untreated control
test organisms. Such variations may be phytotoxic symptoms (chlorosis,
necrosis, and wilting), formative (leaf and stem deformation) effects, and/
or growth and development rales. Observations should include the stage
of development and dates when adverse results occurred and subsided or
recovered. Any lack of effects by the pesticide should also be reported.
(iii)	Include actual counts, weights, and other measurements for each
plant, replicate and variable. Uniform scoring procedures should be used
to evaluate the observable toxic responses. Such data should include the
actual values used to determine any percentages of effects. Raw data
(chromatographs, field reports, and analysis data) may also be included
to substantiate the basic data that are required.
(d) Reporting requirements—(1) General. Refer to 40 CFR
160 185. subpart J, for reporting requirements. Report should include name
of laboratory or test location, personnel information, test substance infor-
mation, test procedures, materials, methods, results, and analysis of data
m tabular summary form Statistical methods must be described. Any
available information on mode-of-action (biochemical) and resultant plant
effects should be included. Either the metric system or the U.S. Standard
Measures (to preclude extensive conversion to the metric system) may be
used in test reports. The two systems cannot be mixed (e.g., grams per
square feet). The English language must be used in all test reports; English
translations must be provided with foreign language reports.
(2) Test report. The test report should include the following intorma-
tion:
(i)	Severity of phytotoxicity (percent or rating), abnormal changes in
growth and development, and/or abnormal changes in plant morphology
as compared to the untreated control.
(ii)	Tabulation of the results indicating the percentage effect level for
each species as compared to untreated control plants for each test param-
eter (height, weight, etc.).
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(iii)	Data on plant dry weight, plant shoot height, root dry weight,
root length/volume, number of dead plants, height or other growth param-
eter;. are recommended test endpomts.
(iv)	Report the aetual dates of the .studies including dates of initiation
(planting, transplanting, and cultural practices), applications, observations,
and harvest
(v)	Electronic transfer of test data on disc is encouraged to reduce
review time
(2) Statistics. When test results such as efficacy, phytotoxicity, or
yield indicate adverse effects on crops and other nontargct test organisms,
statistical analysis is required to evaluate the responses. The statistical
analysis should consist of:
(i)	The tabulation of data for the most sensitive endpoint (plant height,
plant dry weight, visual phytotoxicity, etc.) tor each plant species tested
at each treatment level for each test.
(ii)	The determination of 25 and 50 percent detrimental effect levels
(EC25, EC50) and the 95 peicent confidence limits, where possible, for
each (Probit Analysis, Bruce/Versteeg).
(iii)	The estimated NOEC (or EC05) and LOEC (Williams Test).
(e)	Special test requirements. In addition to the data required by
this guideline, data from other tests may be required by the Agency for
making judgments regarding safety to nontargct plants such as additional
lest species, life-cycle tests, and monitoring studies. Such data will be re-
quired where there are special concerns identified in the literature, 6(a)(2)
(adverse effects reporting) or incident action, a unique use pattern, or a
unique chemical property
(f)	References. The following references should be consulted tor ad-
ditional background material on this test guideline.
(1)	Boutin, C. et al. Proposed Guideline For Registration Of Chemical
Pesticides Nowarget plant testing and evaluation. Tech. Rpt. Series No.
145, Canadian Wildlife Service, Environment Canada, pp. 1-91 (1993).
(2)	Bruce, R.D. and D J. Versteeg. A Statistical Procedure For Model-
ing Continuous Toxicity Data. Environmental Toxicology and Chemistry
1 V. 1485-1494 (1992).
(3)	Gulley, D.D. et al. Toxstat. Release 3.0. University of Wyoming,
Laramie, WY (1989).
(4)	EPA. Nontargct Plants: Seed Germination/Seedling Emergence—
Tiers I and II. EPA 540/9-86-132 (1986).
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(5)	EPA. Nontarget Plants: Target Area Testing. EPA 540/9-86-130
(1986).
(6)	Stephan, C.E. Methods for calculating an LC50. In F.L. Mayer
and J.L. Hamelink, eds., Aquatic Toxicology and Hazard Evaluation, STP
634, American Society for Testing and Materials, Philadelphia, PA, pp.
65-84(1977).
(7)	Truclove, B., (cd.) Research Methods in Weed Science. Southern
Weed Science Society, Auburn Printing Inc., Auburn, AL 36830 (1977).
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