United States
Environmental Protection
Prevention, Pesticides
and Toxic Substances
EPA 712-C-96-347
April 1996
&EPA Ecological Effects Test
OPPTS 850.4230
Early Seedling Growth
Toxicity Test

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,
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
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:
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

OPPTS 850.4230 Early seedling growth toxicity test.
(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 40 CFR 797.2800 Early Seedling Growth
Toxicity Test.
(b)	Purpose. This guideline intended for use in developing data on
the toxicity of chemical substances and mixtures ("chemicals") subject
to environmental effects test regulation. This guideline prescribes tests
using commercially important terrestrial plants to develop data on the
phytotoxicity of chemicals. The EPA will use data from these tests in as-
sessing the hazard of a chemical to the environment.
(c)	Definitions. The definitions in section 3 of TSCA and in 40 CFR
Part 792—Good Laboratory Practice Standards (GLP) apply to this test
guideline. The following definitions also apply to this test guideline.
ECX means the experimentally derived chemical concentration that
is calculated to effect X percent of the test criterion.
Germination means the resumption of active growth by an embryo.
Support media means the quartz sand or glass beads used to support
the plant.
(d)	Test procedures—(1) Summary of the test—(i) Root exposure.
In preparation for the test, seeds are planted in the potting containers (or
in cotton or glass-wool plugs supported in hydroponic solution) and, after
germination, seedlings are thinned by pinching the stem at the support
medium surface to the 10 most uniform seedlings per pot. This marks
the start of the test and the time of first application of test chemical. Seed-
lings emerging after this time are also pinched off at the surface. Potting
mixtures of sand or glass beads are subirrigated with nutrient solution.
Chemicals are applied to the plants via nutrient solution or are sorbed
to the support media. Plants are harvested after 14 days and analyzed for
(ii) Foliar exposure. The foliar exposure test is identical to the root
exposure test except that chemicals are applied to plants by either spraying
or dusting the foliage or by exposing the plants to gas in a fumigation
(2) Chemical application—(i) Root exposure. (A) Chemicals that
are soluble in water should be dissolved in the nutrient solution just prior
to the beginning of the test. Deionized or glass-distilled water should be
used in making stock solutions of the test chemical. Sufficient quantities

of each concentration should be made up as needed to minimize storage
time and disposal volume.
(B)	Chemicals that are insoluble in water, but which can be suspended
in an aqueous solution by a carrier, should be added, with the carrier,
to the nutrient solution. The carrier should be soluble in water, relatively
nontoxic to plants, and should be used in the minimum amount required
to dissolve or suspend the test chemical. There are no preferred carriers,
however, acetone, gum arabic, polyethylene glycol, ethanol, and others
have been used extensively in testing herbicides, plant growth regulators,
fungicides, and other chemicals that affect plants. Carrier controls should
be included in the experimental design of the test and tested simulta-
(C)	Water-insoluble chemicals for which no nontoxic, water-soluble
carrier is available should be dissolved in an appropriate volatile solvent.
The solution should be mixed with the sand or glass beads which are then
placed in a rotary vacuum apparatus and evaporated, leaving a uniform
coating of chemical on the sand or beads. A weighed portion of beads
should be extracted with the same organic solvent and the chemical as-
sayed before the potting containers are filled. Solvent controls should be
included in the experimental design and tested simultaneously.
(ii) Foliar exposure. (A) Water-soluble chemicals should be dis-
solved in deionized or glass-distilled water just prior to use. Sufficient
quantities of each concentration should be made up as needed. These solu-
tions should be applied daily (during a normal 5-day work week). Plants
should be placed in an exhaust hood and the chemical applied to the foli-
age. A plastic sleeve may be fitted over the top of the pot, and the foliage
sprayed with specific quantities of test solution at known concentrations.
The plastic sleeve, confining the chemical to plant and pot, facilitates ex-
pression of chemical dosage to quantity per pot area (i.e., micrograms per
square meter). Shoots of control plants should also be sprayed deionized
or distilled water. A miniature compressed-air sprayer mounted on a pen-
dulum and equipped to spray a plant positioned directly beneath the center
of its arc of swing may be used.
(B) Water-insoluble chemicals, existing as solids, may be prepared
for testing by grinding or other reduction to particles of <200 (im diame-
ter. Each day (during a normal 5-day work week) plants should be placed
in an exhaust hood, a plastic sleeve fitted over the top of the pot, and
specific quantities of chemical sprinkled uniformly over the potted seed-
lings. Prior to chemical application, plants should be misted with water
to promote foliar retention of the chemical. Control plants should also be
misted with deionized or distilled water at each treatment date and dusted
with an inert material of the same particle size. Applications are expressed
as quantity per unit pot area (i.e., micrograms per square meter).

(C) Chemicals existing in gaseous form at normal ambient tempera-
tures and pressures can be generated as needed or stored under pressure.
The bottled gas may be 100 percent chemical or may be mixed with an
inert carrier, such as nitrogen, to known concentrations. Chemicals of con-
trolled or measured concentrations should be metered into the exposure
chamber, uniformly mixed about the plants, and exhausted through an out-
let port.
(3)	Range-finding test, (i) A range-finding test should be conducted
to establish if definitive testing is necessary and to establish the concentra-
tions of test substance used in the definitive test for each species.
(ii)	The recommended procedure is to expose newly germinated seed-
lings to a series of widely spaced concentrations of test chemical and as-
sess effect as growth reduction. Seeds (approximately 30) should be plant-
ed directly in containers filled to within 2.5 cm of the top with quartz
sand or glass beads. If a hydroponic system is used, the seeds should be
planted in plugs of cotton or glass wool supported at the top of the solu-
tion. When 50 percent of the seeds have germinated the seedlings should
be thinned (by pinching) to the 10 most uniform per pot and exposed
to a widely spaced concentration series (i.e., 0.01, 0.1, 1.0, 10,
100, 1,000 mg/L) of test chemical. The lowest concentration in the series,
exclusive of controls, should be at the chemical's detection limit. The
upper concentration, for water-soluble compounds, should be the saturation
concentration. If the anticipated fate of the chemical is soil or soil water,
and the mechanism of concern is root uptake, the chemical should be ap-
plied in nutrient solution to the root support media (or coated on sand
or glass beads for nonwater soluble chemicals). With a chemical whose
anticipated mode of exposure to plants is surface deposition by atmos-
pheric transport, or irrigation water, the appropriate testing method may
be foliar application allowing subsequent movement into the rooting zone
with watering. Effect is assessed as growth reduction.
(iii)	Alternatively, the seed germination/root elongation test may be
used to establish the appropriate concentration range for testing.
(iv)	No replicates are required and nominal concentrations are accept-
able unless definitive testing is not required.
(v)	Definitive testing is not necessary if the highest chemical con-
centration tested results in less than a 50 percent reduction in growth or
if the lowest concentration tested (analytical detection limit) results in
greater than a 50 percent reduction in growth.
(4)	Definitive test, (i) The purpose of the definitive test is to deter-
mine the concentration response curves and the EC 10s and EC50s for each
of the species tested with the minimum amount of testing beyond the
range-finding test.

(ii)	At least five concentrations of chemical, exclusive of controls,
should be used in the definitive test. For each species tested the concentra-
tion range should be selected to define the concentration-response curve
between the EClOs and EC90s. Test chemicals should be added to the
hydroponic or nutrient solution or coated on the support media for the
root exposure test, or sprayed, dusted, or gassed directly on the foliage
in the foliage exposure tests.
(iii)	Control pots should be included in the experimental design and
should be used in each run. In addition, a carrier control should also be
used for those chemicals that need to be solubilized.
(iv)	If plants are to be grown hydroponically, seeds should be planted
in plugs of cotton or glass wool supported in the tops of the containers.
When sand or glass beads are used, the recommended planting procedure
is to fill the potting containers to within 2.5 cm of the top and to sow
seeds directly on the support medium. After 50 percent of the seeds have
germinated, the seedlings should be thinned to the 10 most uniform per
(v)	Alternative planting methods may be required when the chemical
is highly volatile. An impervious barrier of polyethylene film, a modifica-
tion of the double pot method, a glass plate, or other appropriate apparatus
should be used to prevent volatilization from the root zone. Seeds should
be germinated in the dark at 25 °C and seedlings with radicle lengths
in the median range transplanted into the potting containers. The seedlings
should be positioned such that their roots are exposed to the support media
while the shoots pass through holes in the barrier. A ring of nontoxic,
inert, pliable putty should be used to seal the holes around the stems. Con-
trol pots should be handled identically to the test pots except there is no
exposure to the test chemical. This transplanting procedure, without the
volatilization barrier, is also recommended when the test chemical is ad-
sorbed to the support medium.
(vi)	The test consists of one run for each of the recommended plant
species or selected alternates. The duration of a run should be at least
14 days from the time that 50 percent of the seeds have germinated. For
a particular chemical, a run is defined as exposure of the plant species
to five concentrations of the chemical in a minimum of three replicate
pots (10 plants per pot), with appropriate controls, followed by weight
and height determinations and analysis.
(vii)	All abnormalities (visible effects of the chemicals on plant
growth and morphology including stunting of growth, discoloration, chlo-
rosis and/or necrosis of the leaves, or morphological abnormalities) should
be recorded. Observations of plants should be made daily (during a normal
5-day work week).

(viii)	A randomized complete block design is recommended for this
test with blocks delineated within the chambers or over greenhouse bench-
es and randomization of treatment occurring within the blocks. If, because
of the use of very large pots, there is inadequate space within chambers
for blocking, total randomization within chambers is acceptable.
(ix)	Irradiation measurements should be taken at the top of the plant
canopy and the mean, plus a maximum and a minimum value, determined
over the plant-growing area. These measurements should be taken daily
and should be taken at least at the start and finish of the test. If the test
is conducted in a greenhouse facility, hourly measurements of irradiation
should be recorded and presented as daily total irradiance plus representa-
tive hourly curves for clear sky conditions and cloudy days.
(x)	Temperature and humidity should be measured daily at the top
of the plant canopy during each light and dark period.
(xi)	Measurements of carbon dioxide concentration should be made
at the top of the plant canopy (of chamber-grown plants) on a continuous
(5) Analytical measurements—(i) Chemical. Stock solutions should
be diluted with glass-distilled or deionized water to obtain the test solu-
tions. Standard analytical methods, if available, should be used to establish
concentrations of these solutions and should be validated before beginning
the test. An analytical method is not acceptable if likely degradation prod-
ucts of the chemical, such as hydrolysis and oxidation products, give posi-
tive or negative interference. The pH of these solutions should also be
measured prior to use.
(ii) Numerical. Mass and length of roots, shoots, and entire plants
(root and shoot) should be measured for the definitive test. Means and
standard deviations should be calculated and plotted for each treatment
and control. Appropriate statistical analyses should provide a goodness-
of-fit determination for the concentration-response curves.
(e) Test conditions—(1) Test Species—(i) Selection. (A) Test plants
recommended for the definitive test include:
(7) Lycopersicon esculentum (tomato).
(2)	Cucumis sativus (cucumber).
(3)	Lactuca sativa (lettuce).
(4)	Glycine max (soybean).
(5)	Brassica oleracea (cabbage).
(6)	Avena sativa (oat).

(7)	Lolium perenne (perennial ryegrass).
(8)	Allium cepa (common onion).
(9)	Daucus carota (carrot).
(10)	Zea mays (corn).
(B) Other species, of economic or ecologic importance to the region
of impact, may also be appropriate and selected for testing.
(11)	Seed selection. Information on seed lot, the seed year or growing
season collected and germination percentage should be provided by the
source of the seed. Only untreated seed (not treated with fungicides,
repellants, etc.) taken from the same lot, and year or season of collection
should be used in a given test. In addition, all seed of a species used
in a test should be of the same size class, and that size class which contains
the most seed should be selected and used in a given test. Any seed which
is damaged should be discarded.
(2) Facilities—(i) Apparatus. (A) Greenhouses or environmental
chambers should provide adequate environmental controls to meet the car-
bon dioxide, humidity, irradiation, photoperiod, and temperature specifica-
tions. Chambers should be designed to prevent escape of internal air into
the external environment other than through appropriate filtering material
or media to prevent contamination of the external environment with the
test chemical.
(B) Laboratory facilities for chemical determinations should include
nonporous floor covering, absorbent bench covering with nonporous back-
ing, and adequate disposal facilities to accommodate plant nutrient, test
and wash solutions containing test chemicals at the end of each run, and
any bench covering, lab clothing, or other contaminated materials.
(ii)	Containers and support media. For each run, 18 polyethylene
pots sufficiently large to grow at least 10 plants up to 14 days, are required
for each species. It is equally acceptable to use small, individual containers
if plants are grown in hydroponic solution. An additional three pots will
be needed if a carrier control is needed. Potting containers used in each
experiment should be of equal size and volume and possess the same con-
figuration. When sand or glass beads are used, the potting containers
should be filled to within 2.5 cm of their tops. Perlite, vermiculite, native
soils, etc., should not be used for root support.
(iii)	Cleaning and sterilization. (A) Potting and receiving containers,
nutrient storage containers, and root support medium should be cleaned
before use. All equipment should be washed according to good standard
laboratory procedures to remove any residues remaining from manufactur-
ing or prior use. Dichromate solution should not be used for cleaning beads
or pots.

(B) Rooting media other than glass beads should be discarded at the
end of the experiment. Disposal should conform to existing regulations.
(iv) Nutrient media. Half-strength modified Hoagland nutrient solu-
tion should be utilized as nutrient medium for this test. When sand or
glass beads are used as support media, the potting containers should be
filled with nutrient solution and drained periodically. An automated system
design is recommended.
(3) Test parameters. Environmental conditions should be maintained
as specified in this paragraph.
(i)	Carbon dioxide concentration at 350 ±50 ppm.
(ii)	Relative humidity should approach 70 ±5 percent during light pe-
riods and 90 percent during dark periods.
(iii)	The level of irradiation, measured at 1 m from the source, at
350 ±50 (iE/m2 sec at 400 to 700 nrn.
(iv)	Photoperiods of 16 h light/8 h darkness.
(v)	Day/night temperatures at 25°/20°± 3°C.
(f) Reporting. Reporting requirements of 40 CFR Part 792—Good
Laboratory Practice Standards apply to this guideline. The following data
should be reported for each of the species tested in tabular form:
(1)	Concentration of chemical in nutrient solution and in the root sup-
port material when the chemical is soluble in water or solubilized with
a carrier compound, or the concentration of carrier compound in nutrient
solution when carrier is used, or the quantity of chemical per unit weight
of root support material when it is coated on the material.
(2)	The quantity of chemical, the concentration at which it was ap-
plied, and the number of applications for those chemicals applied to the
(3)	Environmental conditions (day/night temperatures, relative humid-
ity, light intensity, carbon dioxide concentration, and photoperiod).
(4)	Mass of above ground (shoot) and below ground (root) portion
of each plant and mass of each whole plant (dry weight at 70 °C).
(5)	Length of shoot, root, and entire plant.
(6)	Visible effects of chemical, if any, on the intact plants.
(7)	Means and standard deviations for mass and length of roots,
shoots, and entire plants in each treatment and control. In addition,
concentration- response curves with 95 percent confidence limits delin-
eated, goodness-of-fit determination, and EC 10s and EC50s identified.