United Slates
Environmental Projection
Prevention Pesticides
end Torfc Substances
EPA 712-G-8&-363
April 1996
SEPA Ecological Effects Test
<¦> Guidelines
OPPTS 850.4225
Seedling Emergence,
Tier II
'•Public Draft"

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,
Chaptei 1, 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
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimise 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. 26011 and the Federal Insecticide, Fungicide and Rodcnticidc 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:
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, 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 "

OPPTS 850.4225 Seedling emergence, tier II.
(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 40 CFR 797.2750 Seed Germination/Root
Elongation Toxicity Test and 797 2800 Early Seedling Growth Toxicity
Test and OPP 123-1 Seed Germination/Seedling Emergence and Vegeta-
tive Vigor (Tierll) (Pesticide Assessment GuidftJines, Subdivision J—Haz-
ard Evaluation; Nontaiget Plants) EPA leport S-ft)/09-82-020, 1982.
(b)	Tier II—Seedling emergence dose response testing. This guide-
line 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.
(1) Objective, (i) The terrestrial nontarget plant phytotoxicily tests
are laboratory tests that evaluate the acute toxicity of pesticides to 10 plant
species. These studies evaluate the effects of multiple dosage levels on
plant growth, using less than the maximum label rate with dosages in a
geometric progression of no more than two-fold, and with subtoxic
food chain. Hedgerows, woodlots, and other similar nontarget areas pro-
vide food and cover to mammalian and avian species.
(iii) Noniarget plant phytotoxicity data are routinely used to conduct
ecological risk assessments for the registration and rcrcgistralion 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. Tier II (Definitive Studies) tests are initiated
following a determination that a greater than 25 percent adverse effect
occurred in the Tier I (Initial Screening Study) seedling emergence study
for one or more plant species. It less than a 25 percent detrimental effect
or response to all tested species is noted in the seedling emergence study,
no highei tier testing is ordinarily required. Under FIFRA, if the pesticide
is a known phytotoxicant, terrestrial plant testing begins with Tier LI.
Unique agency concerns arising from 6(a)(2) incidents (adverse effects re-
porting), Special Review issues, pesticide contamination cases, published
literature, or other public sources may result in requests for additional tests.
(c) Test methods—(1) Test facility/location/test conditions. Dose
lesponse seedling emergence tests can be conducted in the greenhouse or
in small field plots. Report soil type and texture, soil Kj and ICuc values
and pH. Environmental conditions during the test should be recorded
daily—light intensity, air temperature, humidity, photopenods,
thermoperiods, watering schedules and methods (rainfall if field test), and
pest conditions.
(2)	Test substance. Refer to 40 CFR part 160 for test substance re-
quirements. Use of TEP instead of TGAI is preferred for all noniarget
plant phytotoxicity tests, using the TEP with the highest percent A1 and/
or the one most commonly used.
(3)	Controls/solvcnts/additives and other pesticide treatments.
Pesticide treatments other than the lest pesticide should be avoided. Me-
chanical, cultural, and biological pest control methods are suggested. It
solvents or other pesticides are used in the test, the registrant must show
that the solvcnt/pcsiicidc is nol 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
control plants (set aside for this purpose at the beginning of 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 arc contaminated with the test chemical, the study
should most likely be repeated. It the solvent control is contaminated with
the test chemical and the negative control is not (and visa versa), the study
may nol be invalid if 
is icquiied. If adjuvants are recommended on a TEP label, a representative
adjuvant within each class (ionic, anionic, nonionic, etc.) must be used
in the test.
(4)	Equipment. (1) 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.
(ii) The application equipment used in 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 commercial ground spiayeis Specific details as to descriptions
of equipment design, adjustment, and operation should be provided in test
(5)	Dosages, (i) At least five dosages should be tested.
(ii)	The dosages should include a subtoxic (
(v) If conducting tests under TSCA, the test 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 thiram
seed treatments are the only approved pesticide seed treatments
(nonmteractive with most other pesticides). Steam sterilization of soil and
seeds is the recommended procedure for killing pathogens, fungi, and in-
sects on seeds and in soil media. Some methods used to remove seed treat-
ments include rinsing with a weak methanol solution, detergents, or hypo-
chlorite solution. When unapproved pesticide seed treatments are used in
a study, it is the responsibility of the laboratory conducting the test to
show that no synergistic 01 antagonistic interactions occur between or
among the various pesticides in the test.
(in) Ten plant species must be tested. (A) The following plant species
and groups are recommended:
(/) Dicotyledoneae: Six species of at least four families, one species
of which is soybean {Glycine mar) and a second of which is a root crop.
(2) Monocotyledoncae: Four species of at least two families, one spe-
cies of which is corn (Zea mays).
(B) Of the 10 test plants, 3 must be tested: corn (Zea mays), soybean
(Glycine mat), and a root crop such as carrot (Ducas carrotta), onion
(Allium cepa), beet (Beta vulgaris), or sugarbeel {Beta vulgaris). The other
seven test species might include: tomato (Lycopersicon esculentum), cu-
cumber (Cucumis saliva), lettuce (Lactuca saliva), cabbage (Brassica
oleracea), oat (-4vena saliva), and perennial ryegrass (Lolium perenne)
Substitution of other test species (other crops, weeds controlled, native
plants, perennials, woody species) when species sensitivity to the test
compound is known ahead of lime is encouraged. Endangered or threat-
ened species as determined by the Endangered Spccic-s Act of 1973 (Public-
Law 93-205) may not be used without permission from the Fish and Wild-
life Service.
(7)	Support media and pesticide dosing method. Seeds may be ger-
minated in pots using a sterilized standardized soil that consists primarily
of sandy loam, loamy sand, loamy clay, or clay loam soil that contains
up to 3 percent organic matter. The use of 100 percent acid washed sand
or hydroponic methods (glass beads, rockwool, etc.) are not recommended.

Test methods and protocols tor hydroponic tests should be submitted to
the Agency tor review pnor to test initiation. Depending on the intended
application method, the pesticide is either thoroughly mixed into the soil
(incorporated to label or TSCA specified soil depth) or applied to the soil
surface using a properly calibrated spiayer
(8)	Test containers. Test containers should be nonporous so that the
test material is not absorbed. Do not use clay or peat containers. Containers
should be thoroughly cleaned prior to use. A dichromale 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 400 to 700 nm at 1 m from the source,
at 350±50 nb/m2 sec.
(iv)	Photoperiods ot 16 h light and 8 h darkness.
(v)	Day/night temperatures at 25 °C/20 °C ± 3 °C.
(vi)	Half-strength modified Hoagland nutrient solution may be used
as nutrient medium.
(10)	Watering methods. Bottom watering ot test containers is pre-
ferred in order to prevent washing the chemical through the soil during
watering. The laboratory should assess the potential for leaching of the
pesticide based on solubility and K
root formation (based on modc-of-action information, incident reports., lit-
erature). Individual test species responses to cultural conditions, mode-ot-
action o1' the pesticide and speed of uptake of the pesticide by the test
plants are factors that affect the interval between pesticide application and
final observations tor adverse effects. The study length should be extended
as dictated by these factors. All dead plants are to be measured, weighed
and observed and included in the statistical analysis.
(li) Observations should include all variations, either inhibitory or
stimulatory, between the treated and the untreated organisms. Such vari-
ations may be phytotoxic symptoms (chlorosis, necrosis, and wilting),
formative (leaf and stem deformation) effects, anchor growth and develop-
ment 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. Include actual counts,
weights, and other measurements for each plant, rcplicaic and variable.
Uniform scoring procedures should be used to evaluate the observable
toxic responses. Such data should include the actual values used to deter-
mine any percentages of effects. Raw data (chromatography Held reports,
and analysis data) may also be included to substantiate the basic data that
are required
(13) Minimum seed germination standards. The following mini-
muni acceptable USDA seed germination (control) standards for vegetable
crops (as described in the Federal Seed Act Regulation. 7 CFR parts 201 -
202) and other available standards tor agronomic crops will be used: Field
corn (85%), pop com (75%), sweet corn (75%), carrot (55%), onion
(70%), tomato (75%). field-garden bean (70%). pea (80%), pepper (55%),
beet (65%), buckwheat (60%), cabbage (75%), lettuce (55%), mustard—
all types (75%), soybean (75%), sugarbeet (55%), small grains—wheat,
oats, barley, rice (80%), ryegrass (75%), vetch (75%), alfalfa—clover
(70%), tape (75%). Refer to legulation for additional vegetable crops.
(d) Reporting requirements—(I) 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
in tabular summary form. Statistical methods must be described. Report
the actual dates of the studies including dates of initiation (planting, trans-
planting, and cultural practices), applications, observations, and harvest.
Information on mode-of-action (biochemical) and resultant plant effects
should be included Either the metric system or the U.S standard measures
may be used in test reports. The U.S. standard measures may be used
to preclude extensive conversion to the metric system. The two systems
should not be mixed (e.g., grams per square feet). The English language
is to be used in all test reports. English translations must be provided with
foreign language leports.

(2)	Test report. The test report must include the following informa-
(i)	The number of seeds sown and the number emerged per dosage
level for each replicate compared to controls.
(ii)	Descriptions of the appearance and the growth and development
of the emergent plants, indicating any abnormalities and expressions of
(lii) Tabulation of the results indicating measured differences in emer-
gence, height, dry weight, phytotoxicity, and total number of dead plants
for treated plants compared to control plants (individual counts/mcasure-
ments may be combined for each replicate tor statistical purposes).
(iv) Electronic transfer ot test data on discs is encouraged to reduce
review time.
(3)	Statistics. Statistical analysis is required to evaluate the responses
when test results such as efficacy, phytotoxicity, or yield indicate adverse
effects on crops and other nun target test organisms, The statistical analysis
should consist of:
(i)	The tabulation of data for the most sensitive endpoint (plant height,
plant dry weight, vibual phytotoxicity, etc.) for 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 percent confidence limits, where possible, for
each (Probit Analysis, Bruce/Veisteeg).
(111) The estimated NOEC (or 1X05) and LOF.C (Williams Test).
(e) Special test requirements. In addition to the data required in this
guidance, data from other tests may be required by the Agency for making
judgments regarding safety to nontarget plants such as additional test spe-
cies, lifc-cyclc tests, and monitoring studies. Such data will be required
where there are special concerns identified in the literature, 6(a)(2) or inci-
dent action, a unique use pattern, or a unique chemical property.
(0 References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1)	Boutin, C et al. Proposed Guideline For Registration Of Chemical
Pesticides. Nontarget plant testing and evaluation. Tech. Rpt. Series No.
145, Canadian Wildlife Service. Environment Canada, pg. 1-91 (1993)
(2)	Bruce, R.D. and D J Versteeg. A Statistical Procedure For Model-
ing Continuous Toxicity Data. Environmental Toxicology and Chemistry
II 1485-1494 (1992)

(3)	Gulley, D.D. et al. Toxstat. Release 3.0. University of Wyoming,
Laramie, WY (1989).
(4)	EPA. Noniargct Plants: Seed Germination/
cedlingEmergencc—Tiers I «md II. EPA 540/9-86-132 (1986).
(5)	EPA. Noniargct Plants: Target Area Testing. EPA 540/9-86-130
(6)	Stephan, C.E. Methods tor 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)	Truelove, B., (ed). Research Methods in Weed Science. Southern
Weed Science Society, Auburn Printing Inc., Auburn, AL 36830 (1977).