&EPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention
(7101)
EPA712-C-010
January 2012
Ecological Effects
Test Guidelines
OCSPP 850.4230:
Early Seedling Growth
Toxicity Test
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NOTICE
This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention
(OCSPP) for use in testing pesticides and chemical substances to develop data for
submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601,
et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et
seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic
Substances (OPPTS). To distinguish these guidelines from guidelines issued by other
organizations, the numbering convention adopted in 1994 specifically included OPPTS as
part of the guideline's number. Any test guidelines developed after April 22, 2010 will use
the new acronym (OCSPP) in their title.
The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the
test, and is also used by EPA, the public, and the companies that are subject to data
submission requirements under TSCA, FIFRA, and/or the FFDCA. As a guidance
document, these guidelines are not binding on either EPA or any outside parties, and the
EPA may depart from the guidelines where circumstances warrant and without prior notice.
At places in this guidance, the Agency uses the word "should." In this guidance, the use of
"should" with regard to an action means that the action is recommended rather than
mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline, but EPA recognizes that departures
may be appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the Agency will assess them for
appropriateness on a case-by-case basis
For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.gov/ocspp and select "Test Methods &
Guidelines" on the left side navigation menu. You may also access the guidelines in
http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-
0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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OCSPP 850.4230: Early seedling growth toxicity test.
(a) Scope—
(1) Applicability. This guideline is intended to be used to help develop data to submit to
EPA under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and
the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).
(2) Background. The source materials used in developing this harmonized OCSPP test
guideline include the OPPT guideline under 40 CFR 797.2800 Early Seedling Growth
Toxicity Test, and OECD guideline 227, Terrestrial Plant Test: Vegetative Vigour Test.
(b) Purpose. This guideline is intended for use in developing data on the toxicity of chemical
substances and mixtures ("test chemicals" or "test substances") subject to environmental effects
test regulations. This guideline describes tests mainly using commercially important terrestrial
plants (crop species) to develop data on the phytotoxicity of test substances. Either a foliar
exposure or a root exposure scenario is employed. If the anticipated fate of the test substance is
occurrence in soil or pore water, and the mechanism of concern is root uptake, the test substance
should be applied in nutrient solution to the root support media (or by coating onto sand or glass
beads). With a test substance whose anticipated mode of exposure to plants is surface deposition
by atmospheric transport, or irrigation water, the appropriate testing method is foliar application
allowing subsequent movement into the rooting zone with watering. This guideline should be
used in conjunction with the OCSPP 850.4000 guideline, which provides general information
and overall guidance for plant test guidelines. The Environmental Protection Agency will use
data from these tests to assess the hazard a test substance may present in the terrestrial
environment.
(c) Definitions. The definitions in the OCSPP 850.4000 guideline apply to this guideline. In
addition definitions in this paragraph also apply:
Shoot length is defined as the above-ground vegetation from the support media surface
(or water surface for hydroponic solutions) to the apical tip or highest aerial part of the
shoot.
Root length (tap root system) is defined as the below-ground vegetation from the support
media surface (or water surface for hydroponic solutions) to the apical tip of the primary
root (tap root) and from the primary root to the apical tip of a first order lateral root.
Root length (fibrous root system) is defined as the below-ground vegetation from the
support media surface (or water surface for hydroponic solutions) to the apical tip of a
fibrous or first order lateral root.
(d) General considerations—
(1) Summary of the test. This test evaluates the effect of a test substance applied to the
roots or the leaves of a number of terrestrial plant species. In preparation for the test,
seeds are planted in the potting containers (or in cotton or glass-wool plugs supported in
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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 substance. Seedlings emerging
after this time are also pinched off at the surface. Potting mixtures of sand or glass beads
are sub-irrigated with nutrient solution. The test substance is applied to the plants via
nutrient solution or sorbed to the support media (to produce a root exposure scenario) or
applied to the plants by either spraying or dusting the foliage or by exposing the plants to
gas in a fumigation chamber (to produce a foliar exposure scenario). Plants are harvested
after 14 days and analyzed for growth. Parameters that may be measured include
seedling survival, length and weight of whole plants, shoot length, shoot dry weight, root
length, and root dry weight, and observed phytotoxicity. Results are reported as ECio and
ECso values for seedling survival and ICio and ICso values for weight and length of
shoots, roots and entire plants for a multiple-concentration test. Also, for industrial
chemicals, the no observed effect concentration (NOEC) and lowest observed effect
concentration (LOEC) for the measures of effect (survival; lengths of roots, shoots, and
entire plant; weights of roots, shoots, and entire plant) may be determined. For a limit
test, results are reported simply as percent effect at the test treatment relative to the
control(s). At a minimum, these endpoints should be calculated for survival, shoot
length, and shoot dry weight. Note historically in OCSPP pesticide and industrial
chemical guidelines the term ECX was used to cover both the current OCSPP 850.4000
definition of ECX (concentration where x percent (x%) of the population exhibit the effect
(e.g., immobility)) and ICX (concentration resulting in an x% decrease or inhibition effect
on an attribute of the population (e.g., growth rate)).
(2) General test guidance. The general guidance in OCSPP 850.4000 and the references
in paragraphs (j)(2), G)(7) and 0)00) of this guideline applies to this guideline, except as
specifically noted herein. The protocol to examine seedling emergence and growth under
FIFRA requirements is the OCSPP 850.4100 guideline.
(3) Range-finding test. A range-finding test is usually conducted to establish the
appropriate test concentrations to be used for the definitive test. In the range-finding
test, the test organisms are exposed to a series of widely-spaced concentrations of the test
substance (i.e., 0.01, 0.1, 1.0, 10, 100, 1000 milligrams per liter (mg/L)). The details of
the range-finding test do not have to be the same as the definitive testing in that there are
not replicates, and the number of test organisms used, and duration of exposure may be
less than in definitive testing. In addition, the types and frequency of observations made
on test organisms are not as detailed or as frequently observed as that of a definitive test
and results are analyzed using nominal concentrations. However, the range-finding test
will be more useful the greater the similarity between the range-finding and the definitive
test.
(4) Definitive test. The goal of the definitive test is to determine for each measure of
effect (seedling survival; length, and weight of the shoots, roots and entire plant) its
concentration-response curve; the ECso and ECio values (with 95% confidence intervals
and standard errors) for seedling survival; and the ICso and ICio values (with 95%
confidence intervals and standard errors) for length and weight of shoots, roots and entire
plant. The slope of the concentration-response curve, its associated standard error, and
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95% confidence interval should be determined for each measure of effect, if possible.
However, at a minimum, the full concentration-response curve (curve between the ECio
and ECgo (or ICio and ICgo)) is determined for the most sensitive measure of effect using
at a minimum five treatment levels, plus appropriate controls. For a satisfactory test, the
lowest treatment concentration is below the ECio (seedling survival) and ICio (length and
weight of shoots, roots and entire plant) values for all measures of effect. The test
substance should be added to the hydroponic or nutrient solution or coated on the support
medium for the root exposure test, or sprayed, dusted, or gassed directly on the foliage in
the foliage exposure tests. Analytical confirmation of the test concentrations is
performed as described in OCSPP 850.4000. A summary of test conditions is provided in
Table 3 and validity elements for an acceptable definitive test are given in Table 4.
(5) Limit test. In some situations, it is only necessary to ascertain that the 14 day post-
germination ECio, ECso, ICio and ICso values are above a certain limit concentration. In
an early seedling growth limit test, at least 40 plants (divided into at least 4 replicates of
10 plants each) are exposed to a single "limit concentration," with the same number of
organisms in appropriate controls. The multiple-concentration definitive test may be
waived for a given test species if for all measures of effect (seedling survival, length, and
weight of the shoots, roots and entire plant), the "limit" concentration has not caused an
effect greater than 10% on a plant species (e.g. ICio >limit concentration). For most
industrial chemicals, 1,000 mg/L is considered appropriate as the "limit" concentration.
Alternatively, the "limit" concentration, for water-soluble compounds, should be the
saturation concentration. Except for the number of test concentrations, number of
organisms used, an acceptable limit test follows the same test procedures, is the same
duration, and has the same number of controls as the multi-concentration definitive tests
(see Table 3). Acceptable limit tests like definitive tests include analytical confirmation
of the test concentration.
(e) Test standards—
(1) Test substance. The substance to be tested should be technical grade, unless the test
is designed to test a specific formulation, mixture, or end-use product. OCSPP 850.4000
lists the type of information that should be known about the test substance before testing,
and discusses methods for preparation on test substances.
(2) Test duration. The test lasts for at least 14 days from the time that 50% of the
control seeds have germinated.
(3) Test organisms—
(i) Species.
(A) The test is performed on species from a cross-section of the terrestrial
plant population that have been historically used for this type of testing,
and, therefore, have known types of responses (see reference in paragraph
(j)(l) of this guideline). At a minimum ten plant species are tested, and
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include the six dicot species and the four monocot species listed in Table
1.
Table 1.—Species and families tested at a minimum for the early seedling growth toxicity
test
Dicots
Monocots
Family
Solanaceae
Cucurbitaceae
Asteraceae
Fabaceae1
Brassicaceae
Apiaceae
Poaceae
Poaceae
Poaceae
Liliaceae
Species
Lycopersicon esculentum or
Solarium lycopersicum
Cucumis sativus
Lactuca sativa
Glycine max
Brassica oleracea
Caucus carota
Avena sativa
Lolium perenne
Zea mays
Allium cepa
Common name
Tomato
Cucumber
Lettuce
Soybean
Cabbage
Carrot
Oat
Perennial ryegrass
Corn
Onion
Inoculation with Rhizobium japonicum is unnecessary.
(B) Eight additional dicot species are listed in Table 2. Some or all of
these may be tested in addition to the minimal data set of species (see
paragraph (e)(3)(i) of this guideline). For example, if cabbage is initially
found to be sensitive to the test substance, it may be important to then test
turnips and canola plants, which are additional species in the same plant
family (Brassicaceae). Other economically or ecologically important
species (e.g., see Table 2 species list in the OCSPP 850.4100 guideline
(see reference in paragraph (j)(8) of this guideline) at a specific site or
region of impact (from production, use or disposal) may also be
appropriate and can be selected for testing on a case-by-case basis, in
addition to those listed in Tables 1 and 2 of this guideline. Endangered or
threatened species as determined by the Endangered Species Act of 1973
(Public Law 93-205) may not be used without permission from the Fish
and Wildlife Service.
Table 2.—Dicot species for additional consideration in the early seedling growth
toxicity test
Family
Chenopodiaceae
Asteraceae
Fabaceae
Brassicaceae
Brassicaceae
Fabaceae
Malvaceae
Polygonaceae
Species
Beta vulgaris
Helianthus annuus
Pisum sativum
Brassica rapa
Brassica napus
Phaseolus vulgaris
Gossypium spp.
Fagopyrum esculentum
Common name
Sugar beet or table beet
Sunflower
Pea
Field mustard, Canola
Rape, Turnip
Garden bean
Cotton
Buckwheat
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(ii) Source. Within a given test, all test organisms of a given species, including
the controls, should be from the same source and lot number and should be
selected to have uniform size.
(iii) Growth stage. Newly germinated plants are used. Seedlings used in testing
are produced by planting seeds of the desired species in the selected support
medium and allowing them to germinate, at which time they are thinned to 10
plants of similar size and condition.
(iv) Condition. For a satisfactory test, healthy seeds should be used with reported
germination rate for a lot number which met or exceed minimum seed
germination standards (see paragraph (e)(5)(i) of this guideline). Within a given
test, all test organisms of a given species, including the controls, should be from
the same source and lot number and should be selected to have uniform size.
Seeds should be selected from a single class size within each species. The seeds
of most test species are sorted by the supplier according to size. However, if
necessary, the seeds should be further sorted prior to use to provide a more
uniform size class that reduces the potential for bias from testing different sizes of
seed. In addition broken or damaged seeds should be separated and removed
from those used in the test.
(v) Care and handling.
(A) Seeds should be stored in a dessicator and refrigerated until used.
Pesticide treated seeds should be avoided.
(B) 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 centimeters (cm) of the top
and to sow seeds directly on the support medium. A sufficient number of
seeds (e.g., 30) should be planted to provide an excess number of
seedlings. After 50% of the seeds have germinated, the seedlings should
be thinned to the 10 most uniform per pot. The condition and length of
each of the 10 seedlings used in the test is recorded. Any plants
subsequently emerging are also pinched off.
(C) Alternative planting methods are used when the test substance is
highly volatile. An impervious barrier of polyethylene film, a
modification 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 degrees Celsius (°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. Control pots should be handled
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identically to the test pots except there is no exposure to the test substance.
This transplanting procedure, without the volatilization barrier, is also
recommended when the test substance is adsorbed to the support medium.
(4) Administration of test substance—
(i) Methods. The choice of method for test substance application to newly
germinated seedlings is dependent upon the expected route of exposure of plants
in the environment and the properties of the test substance.
(A) Root exposure. Root exposure is accomplished by adding the test
substance to the nutrient medium used to subirrigate the plants, as
described in paragraphs (e)(4)(i)(A)(7y) and (e)(4)(i)(A)f2y) of this guideline
for soluble and insoluble test substances, respectively, or by sorbing the
test substance to the sand or glass beads used as support medium, as
described in paragraph (e)(4)(i)(A)(%) of this guideline. For hydroponic
solutions the entire test solution should be replaced weekly or earlier if the
concentration of test substance in the test or nutrient solution varies by
more than 20% of that specified. The volume of solution added should be
recorded. An automated system design is recommended. Sand or glass
filled potting containers should be periodically (dependent on the rate of
loss of the nutrient solution) filled with nutrient solution and drained to
provide aeration.
(1) Test substances that are soluble in water should be dissolved in
the nutrient solution just prior to the beginning of the test. Reagent
water should be used in making stock solutions of the test
substance. Sufficient quantities of each concentration should be
made up as needed to minimize storage time and disposal volume.
The OCSPP 850.4000 guideline contains additional information on
preparation of stock solutions.
(2) Test substances that are insoluble in water, but which can be
suspended in an aqueous solution by a vehicle, should be added to
the nutrient solution. The vehicle should be soluble in water,
relatively nontoxic to plants, and should be used in the minimum
amount needed to dissolve or suspend the test substance. There are
no preferred vehicles; however, acetone, gum arable, polyethylene
glycol, ethanol, and others have been used extensively in testing
herbicides, plant growth regulators, fungicides, and other
chemicals that affect plants. Vehicle controls should be included
in the experimental design of the test and tested simultaneously.
OCSPP 850.4000 contains additional information on preparation of
stock solutions using a vehicle.
(3) Water-insoluble test substances for which no nontoxic, water-
soluble vehicle is available should be dissolved in an appropriate
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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 test substance on the
sand or beads. A weighed portion of sand or beads should be
extracted with the same organic solvent and the concentration of
test substance determined before the potting containers are filled.
Solvent controls are included in the experimental design and tested
simultaneously.
(B) Foliar exposure. Foliar exposure is accomplished by spraying the
plants, as described in paragraph (e)(4)(i)(B)(7) of this guideline, by
dusting the plants, as described in paragraph (e)(4)(i)(B)0 of this
guideline or by exposing the plants to gas in a fumigation chamber, as
described in paragraph (e)(4)(i)(B)(3y) of this guideline.
(1) Water-soluble test substances should be dissolved in reagent
water just prior to use. Test substances that can be suspended in an
aqueous solution by a vehicle should be prepared as described in
paragraph (e)(4)(i)(A)f2y) of this guideline except using reagent
water not nutrient solution. Sufficient quantities of each test
concentration should be made up as needed. These solutions
should be applied daily (during a normal 5-day work week) to the
foliage. Plants should be placed in an exhaust hood and the test
substance applied to the foliage. 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 test substance to plant and pot, facilitates
expression of test substance dosage to quantity per pot area (i.e.,
micrograms per square meter). Shoots of control plants are also
sprayed, using reagent water. In addition, a vehicle control group
is included if a vehicle was used to prepare the test substance.
Alternatively, a miniature compressed-air sprayer may be mounted
on a pendulum and equipped to automatically spray a plant
positioned directly beneath the center of its arc of swing. Track or
belt sprayers may also be used (see the OCSPP 850.4000 and
OCSPP 850.4100 guidelines for additional information). When
radioisotope-labeled test substances are applied, health and safety
considerations prohibit spray application. Instead, specific
quantities of labeled chemical should be applied directly to plants
in single drops.
(2) Water-insoluble test substances, existing as solids, may be
prepared for testing by grinding or other reduction methods to
particles smaller than 200 microns (|im) in diameter. 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 quantity of the test substance sprinkled uniformly over the
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potted seedlings. Prior to test substance application, plants should
be misted with water to promote foliar retention of the test
substance. Control plants are also misted with reagent water at
each treatment date and dusted with an inert material of the same
particle size as the test substance. Applications are expressed as
quantity per unit pot area (i.e., micrograms per square meter).
(3) Test substances existing in gaseous form at normal ambient
temperatures and pressures can be generated as needed or stored
under pressure. The bottled gas may be 100% test substance or
may be mixed with an inert carrier, such as nitrogen, to known
concentrations. Controlled or measured concentrations of test
substance should be metered into the exposure chamber, uniformly
mixed about the plants, and exhausted through the outlet port
where the flow rate and concentration are again measured. Use of
this system design provides an alternate method of analysis if the
quantity of test substance sorbed by plants is less than the
sensitivity of the chemical analysis method.
(ii) Treatment levels.
(A) For a given plant species, five treatment levels are tested at a
minimum. A range-finding test can be used to establish the appropriate
test doses for the definitive test (see paragraph (d)(3) of this guideline).
For scientifically sound estimates of a given point estimate (e.g., ECso,
ECio), test substance concentrations should immediately bracket the point
estimate(s). The concentrations should be a geometric progression of
twofold at a minimum (e.g., 0.1, 0.2, 0.4, 0.8, and 1.6 pounds per acre
(lb/acre)). For an acceptable study for a given plant species, the lowest
test treatment level should be lower than the ECio value for seedling
survival and the ICio values for weight and length of shoots, roots and
entire plant for that plant species. The NOEC should be determined by
hypothesis testing for each effect measure. The lack of a NOEC for an
effect measure is not critical as long as the response-curve for the effect
measure is acceptable for calculation of the 10% effect or inhibition
concentrations (ECio or ICio). It is recommend that one or two additional
test concentrations in the lower tail of the concentration-response curve of
the most sensitive endpoint be added to insure bracketing of the most
sensitive ECio or ICio value.
(B) The use of pesticide treatments to control pests during the test should
be avoided. Mechanical, cultural, and biological pest control methods are
suggested. If other pesticides are used in the test for pest control, for a
satisfactory test a demonstration should be performed (i.e., additional test
data) documenting that the pesticide is not toxic to the test species and that
no synergistic or antagonistic interactions with the test substance exist
(additional test data).
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(5) Controls. Every test includes controls consisting of the same support medium,
conditions, procedures, and test population, except that no test substance is added. In
addition, vehicle (solvent) controls are also included if a solvent is used.
(i) Seed germination standards.
(A) For a satisfactory test, seed germination percentage in each of the
controls should be at least 65% or the percentages in minimum acceptable
United States Department of Agriculture (USDA) seed germination
standards listed in paragraph (e)(5)(i)(B) of this guideline. For native
species that do not have germination standards, sufficient numbers of
seeds should be used to insure the identified number of seedlings per pot
for test initiation.
(B) The minimum acceptable USDA seed germination (control) standards
in this paragraph for vegetable crops (as described in the Federal Seed Act
Regulation, 7 CFR parts 201-202) and other available standards for
agronomic crops will be used: Field corn (85%), pop corn (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%), sugar
beet (55%), small grains—wheat, oats, barley, rice (80%), ryegrass (75%),
vetch (75%), alfalfa-clover (70%), rape (75%). Refer to regulation for
additional vegetable crops.
(ii) Control survival. A test not acceptable if at test termination the mean control
seedling survival is less than 90%.
(6) Number of test organisms and replicates.
(i) For each species, the minimum number of test organisms is 40 plants per
concentration level (a minimum of four replicates each with 10 plants or five
replicates each with 8 plants). Larger populations (i.e. more seeds) and more
replicates are recommended for plants with lower germination rates to increase
the power of the test. While the number of plants per pot is left to the discretion
of the laboratory conducting the test, avoid overcrowding. A test is not
acceptable if a container is too small, resulting in overcrowding and competition
among plants in the container. The recommended loading in a 6 inch container
for corn, soybean, tomato, cucumber or sugar beets is one to two seeds; the
loading for rape or pea is three seeds; and the loading for onion, wheat, or other
small grain is a maximum of six seeds. Note that a replicate in this case will
consist of several pots and/or flats and the integrity of the replicate should be
maintained throughout the duration of the study (i.e. pots/flats from one replicate
are not moved to or mixed with another replicate and if the replicate is moved all
pots/flats composing the replicate are moved as a unit). Do not mix species
within a replicate.
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(ii) Randomization.
(A) For a satisfactory test, a system of impartial or random assignment of
the seeds to the pots and the pots to test and control groups is used to
prevent bias. For this test a randomized complete block design with
blocks delineated within the growth chambers or over greenhouse benches
and randomization of treatment occurring within the blocks is
recommended for placement of replicates. If, because of the use of very
large pots, there is inadequate space within chambers for blocking, total
randomization of replicates within chambers is acceptable. To minimize
spatial differences, which can have a significant impact upon plant growth,
the placement of replicates should be randomized during the test (at least
every three to five days).
(B) Alternative placement should be used with volatile test substances to
prevent cross contamination. For example, in a greenhouse setting use
positive air flow throughout the duration of the study with placement of
controls and treatment levels such that air flows first across controls and
then from the lowest treatment to the highest treatment. In addition,
include another set of replicate controls which are placed in a separate
greenhouse for comparison with control results from the greenhouse
containing the treatments. Where tests are conducted using growth
chambers where positive air flow can not be achieved, the controls and
each treatment level should be placed in separate growth chambers.
(7) Facilities, apparatus and supplies—
(i) Environmental chamber, growth room or greenhouse. Environmental
chambers and some greenhouses or growth rooms should provide adequate
environmental controls to meet the carbon dioxide, humidity, luminance,
photoperiod, and temperature specifications. 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 radioactivity and/or test substances. Growth chambers and
growth rooms are preferred over greenhouses, because in the first two,
environmental conditions are more easily controlled.
(ii) Test containers. Test containers should be nonporous so that the test
substance is not absorbed or does not react in any way with the container. Glass
or stainless steel containers with drainage holes can be used as plant pots.
Polyethylene pots that have not been previously used are acceptable test
containers, provided they are free of toxic materials. Do not use clay or peat
containers. For a satisfactory test, pots sufficiently large to grow (e.g., large
enough to effectively not result in competition among the plants for space,
lighting, or nutrition) at least 10 plants up to 14 days should be used for each
species. It is equally acceptable to use small, individual containers if plants are
grown in hydroponic solution. Potting containers used in each experiment should
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be of equal size and volume and possess the same configuration. When sand,
glass beads, or soil are used, the potting containers should be filled to within 2.5
cm of their tops.
(iii) Cleaning and disposal.
(A) Potting and receiving containers, nutrient storage containers, and root
support medium should be cleaned before use. All equipment should be
washed to remove any residues remaining from manufacturing or prior
use. Bichromate 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. Glass
beads can be re-used provided they are not contaminated with previous
test substances.
(iv) Support medium. An artificial support medium, consisting of quartz sand or
glass beads, is used. Alternatively, a hydroponic system may be used, in which
the seeds are planted in plugs of cotton or glass wool supported at the top of the
solution. Perlite, vermiculite, natural (native) soils, or any formulated soils
should not be used as a root support medium in this test.
(v) Nutrient media. A nutrient solution of defined chemical composition, such
as half-strength modified Hoagland nutrient solution (see the reference in
paragraph (j)(5) of this guideline), should be utilized as nutrient medium for this
test.
(8) Environmental conditions. Controlled environmental conditions are used to
maintain uniform growth and ensure reliable data. Standardization of environmental
conditions is essential because it has been demonstrated that differences in environmental
conditions influence the response of plants exposed to chemicals. For growth chamber
studies, the general conditions listed in paragraphs (e)(8)(i) through (e)(8)(v) of this
guideline should be maintained. If greenhouses or large growth rooms are used,
comparability of the environment between small groups of plants is more difficult to
achieve and therefore not as critical. Environmental variability specifications may be
relaxed on a case-by-case basis.
(i) Temperature. Air temperature should be uniform throughout the greenhouse
or growth chamber or greenhouse. In growth chambers air temperature during the
day should be 25 ± 3 °C while temperature during the night should be 20 ± 3 °C.
Temperature in greenhouses is more difficult to control. Under these
circumstances, temperature during the day should be 25 ± 6 °C while temperature
during the night should be 20 ± 6 °C. For other very warm or cool crop species
the test conditions should approximate those optimal conditions for the species
and varieties tested.
(ii) Humidity. Humidity should be uniform throughout the greenhouse or growth
chamber or greenhouse. In growth chambers relative humidity should approach
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70 ± 5% during light periods and 90 ± 5% during dark periods. Humidity in
greenhouses is more difficult to control. Under these circumstances, relative
humidity should approach 70 ± 15% during light periods.
(iii) Lighting and photoperiod. Luminance of 350 ± 50 micromoles per square
meter per second (|imol/m2/s), measured at the top of the canopy, is desirable, on
a photoperiod of 16 hours light and 8 hours darkness assuming an average
wavelength of 400 to 700 nanometers (nm). For greenhouses, artificial lighting
may be used to lengthen short-day periods or to supplement natural sunlight on
overcast days. Care should be taken to ensure that plants are not affected from the
heat generated from supplemental lighting.
(iv) Watering and nutrients. Nutrient media (containing test substance, if this is
the selected application route) is used to subirrigate the plants as needed, i.e.,
frequent enough to maintain good health (should not see nutrition deficiency in
controls) and moisture. Pots should be irrigated regularly (for example, 30
minutes per hour (min/h)) with nutrient solution, preferably using a system in
which water flows from the bottom of the pot upward (level should reach at least
half way up the pot). An automatic system design is recommended such as that
described in OCSPP 850.4600.
(v) Carbon dioxide. Carbon dioxide concentration at 350 ± 50 parts per million
(ppm) (678 ±97 milligrams per cubic meter (mg/m3)) of air.
(9) Observations—
(i) Measurement of test substance. For a satisfactory test, analytical
confirmation of the concentration of test substance in the vehicle used to expose
the plants (nutrient medium, glass beads or sand, spray, dust or gas) should be
performed. The analytical methods used to measure the amount of test substance
in a sample are validated before beginning the test, as described in the OCSPP
850.4000 guideline.
(A) Nutrient solution. Where test substance was introduced by addition
to the nutrient medium, chemical analysis should be conducted at a
minimum for each treatment level and controls at test initiation (0-hour) in
the bulk solutions and test termination in at least three replicates per
treatment and controls at test termination when nutrient solutions are not
renewed. Where nutrient solution is renewed during the study, chemical
analysis should be conducted at a minimum for each treatment level and
controls at test initiation (0-hour) in the bulk solutions and at the end of
this first cycle in the used nutrient solution, at the beginning (fresh
solution) and end (used solution) of the longest renewal cycle and the last
renewal cycle. It is also recommended that measurements be made at the
beginning of each renewal cycle.
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(B) Quartz sand or glass bead support medium. Where test substance
was introduced by addition to the quartz sand or glass bead support
medium, chemical analysis should be conducted at a minimum in the bulk
support medium for each treatment level and control at test initiation and
from at least three pots for each treatment level and control at test
termination.
(C) Foliar spray. Where test substance is applied by foliar spray and a
single batch of test solution for each treatment level and control is used
throughout the study, chemical analysis in the spray solutions should be
conducted at a minimum at test initiation and termination for each
treatment level and control batch. If one or more spray solution batches
are prepared during the study, chemical analysis of spray solutions should
be conducted at a minimum for each treatment level and controls at test
initiation (0-hour) and at the end of the use of this nutrient solution batch,
at the beginning (fresh solution) and end (used solution) of the longest
time between batches and the last batch. It is also recommended that
measurements be made at the beginning of each batch.
(D) Foliar dusting. Where test substance is applied as a dust and a single
batch of test substance for each treatment level and control is used
throughout the study, chemical analysis of the dust (to determine test
substance exposure) should be conducted at a minimum at test initiation
(0- hour) and at test termination for each treatment level and control batch.
If one or more batches of dust are prepared and used during the study,
chemical analysis of each dust batch should be conducted at a minimum
for each treatment level and control at test initiation (0- hour) and at the
end of the use of a particular dust batch, at the beginning (using fresh dust)
and end (used dust) of the longest time between batches and the final
batch. It is also recommended that measurements be made at the
beginning of each batch. When dusting the plants, care should be taken to
uniformly dust all plant surfaces, to reduce exposure variability of the
different areas of the plant.
(E) Gas application. Where test substance is applied as a gas in a growth
chamber, and a single source of the gas is used for each treatment level
and control throughout the study (recommended), chemical analysis of the
gas should be conducted at a minimum at test initiation and test
termination for each treatment level and control. Use of the gas only in a
growth chamber will help minimize variability in exposure concentrations
over the test period. For the gas exposure system, at the time of fumigation
the air temperature, dew point temperature or water vapor pressure of
incoming and outgoing air streams, air speed within chamber, carbon
dioxide concentration at inflow and outflow ports, and gas flow rate into
and out of exposure system are measured and recorded.
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(ii) Environmental conditions—
(A) Carbon dioxide. In environmental chambers, carbon dioxide
measurements should be made at the top of the plant canopy, preferably
on a continuous basis.
(B) Air temperature and humidity. The air temperature and humidity
during the study should be recorded continuously in growth chambers, but
alternatively as maximum and minimum values over each 24-hour period,
especially in greenhouses at representative locations throughout the area in
which the test plants are growing.
(C) Light intensity. Light intensity should be determined daily at
representative locations throughout the area in which the test plants are
growing. A photosynthetically active radiation (PAR) sensor should be
used to measure light quality. Additional information on the use of
lighting in plant toxicity tests can be found in the reference in paragraph
(j)(2) of this guideline and the references given in the OCSPP 850.4000
guideline.
(D) Watering. Records should be kept noting the days upon which the
plants are watered and observations of possible moisture stress should be
made and recorded daily.
(E) Pests. Daily observations should be made on pest pressure using an
index of the extent of infestation. Pest infestation may affect the
interpretation of study results and therefore should be adequately
described. Frequency, methods, and rates used for treating an insect or
disease should be recorded.
(F) Nutrient media pH. The pH of nutrient solutions are measured prior
to use in bulk solutions and in used solutions at test termination in each
replicate. If more than one batch of nutrient solution is used throughout
the study the pH of each nutrient solution treatment concentration and
control(s) are measured in the nutrient solution batch prior to use, and at
replacement in the used solution.
(iii) Phytotoxic effects—
(A) Survival and morphology. Observations of plants should be made
approximately every 3 to 4 days (e.g., day 3 or 4, 7, 10, and 14), or more
often if necessary, after treatment for mortality and visual symptoms of
phytotoxicity. All abnormalities (visible effects of the test substance on
plant growth and morphology including stunting of growth, discoloration,
chlorosis and/or necrosis of the leaves, or morphological abnormalities)
should be recorded. Observations on root symptomology may also be
useful. Seedling and root condition should be determined using a standard
visual phytotoxicty rating scale. Observations should include the
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treatment level and replicate, stage of development and dates when
adverse results occurred, subsided or recovered, and counts for each plant
affected. 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. Any lack of effects by the
test substance should also be recorded.
(B) Length of roots, shoots and entire plant. Length of roots, shoots,
and entire plants (root and shoot) are measured at test termination for each
individual surviving plant, to the nearest millimeter (mm). For fibrous
root systems with a high root density where it is not practical to measure
all fibrous or first order lateral roots, take representative samples of the
root system and measure and record the length of each root in the samples
and calculate the mean and median lengths. Where fibrous root density is
low and where it is practical all fibrous or first order lateral roots are
measured and the mean and median lengths calculated. For a tap root
system measure and record the length of the tap root and each first order
lateral root and calculate the mean and median lengths of the lateral roots.
(C) Weight of roots, shoots and entire plant After lengths are
determined at test termination, the mass of roots, shoots, and entire plants
(root and shoot) are measured. Dry weight (to constant weight at 70 °C) is
preferred because it is less variable than wet weight. Biomass
measurements should be made for each replicate not each individual plant.
(f) Treatment of results—
(1) Summary statistics—
(i) Environmental conditions. Air carbon dioxide, temperature, humidity, and
light intensity data should be summarized in tabular form, showing the mean,
standard deviation, and range during the test. Watering frequency and duration
should be summarized in tabular form by date of occurrence.
(ii) Test substance concentration. Compare the initial test substance dosing
solution concentration with test substance concentration in the dosing solution at
end of application. If the substance was not stable calculate a rate of decline of
the test substance. The total volume of dosing solution used, total area treated,
and the time application started and ended for a given treatment level summarized
in tabular form.
(iii) Seedling survival. Means and standard deviations are calculated and plotted
for each treatment and control. For each treatment level calculate mean, relative
to control mean survival.
(iv) Length of roots, shoots and entire plant. Means and standard deviations
and medians are calculated and plotted for each treatment and control.
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(v) Weight of roots, shoots and entire plant. Means and standard deviations are
calculated and plotted for each treatment and control.
(vi) Morphological conditions. Morphological symptoms of plant injury should
be summarized in tabular form by time of observation, treatment, and replicate.
Definition of any index values used for morphological symptoms, indicating the
severity of the symptom(s), should be provided.
(2) Percent inhibition. For length of roots, shoots, and entire plant and weight of roots,
shoots and entire plant calculate the % inhibition (%I) at each treatment level using
Equation 1.
Equation1
where:
C = the control mean response value (length of roots, shoots, or entire plant;
weight of roots, shoots, or entire plant); and
X = the treatment mean response value (length of roots, shoots, or entire plant;
weight of roots, shoots, or entire plant, respectively). Stimulation is reported as
negative %I.
(3) Limit test. For a limit test, calculate and report the percent effect at the limit
treatment, relative to the control, for each response measure. To ascertain that the
survival ECio and the length and weight ICio values for a given plant species occur above
the "limit" dose, a one-sided test which compares the difference between two sample
groups to a fixed value (or difference) is performed for each of these response measures.
For a comparison of sample means, the difference defining the ICio or ECio, compared to
the controls, is operationally defined as a 10% reduction from the control sample mean
(Equation 2). The null hypothesis (Ho) stated in terms of true population parameters is
that the difference of the "limit" treatment mean response (|iiimit) from the control mean
response (|icontroi) is greater than or equal to a 10 reduction, compared to the control (i.e.,
HQ: ^control - Hiimit > So). The alternative hypothesis (HA) is that this difference is less than
a 10% reduction, compared to the control (HA: ^control - Hiimit < 5o). An example of a
parametric two-sample comparison test of this is the Student's t-test. If the effect level or
inhibition level for all response measures (i.e., survival and length and weight of roots,
shoots and entire plant) in the limit treatment as compared to the control response at test
termination are declared to be less than 10% (i.e., the null hypothesis is rejected), the
multiple-dose definitive test may be waived. A multiple-dose definitive test is performed
if the effect or inhibition level for one or more response measures (i.e., survival and
length and weight of roots, shoots, and entire plant) in the limit treatment as compared to
the control response at test termination are declared to be not distinguishable from a 10%
or greater effect (i.e., the null hypothesis is not rejected).
80=(xcontml)x(p/lOO) Equation 2
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where:
5o = difference between two parameters, defined in this case as a p% reduction
from the control sample mean;
^control = control sample mean response (e.g., survival and length and weight of
roots, shoots, and entire plant); and
p = percent reduction from the control sample mean, which is 10 in the case of the
and ICio.
(4) Multiple-dose definitive test —
(i) Seedling survival. For dose-response tests, statistical procedures (see
references in paragraphs (j)(3) and (j)(9) of this guideline) are employed to
calculate the ECio and ECso values based upon number of surviving plants at test
termination. Standard error and 95% confidence interval (CI) values for the
calculated EC values are to be included. The slope of the concentration-response
curve, its standard error, and 95% CI should also be recorded. Appropriate
statistical analyses should provide a goodness-of-fit determination for the
concentration-response curves. Hypothesis testing procedures can be used to
determine NOEC (and LOEC) values.
(ii) Length and weight of roots, shoots and entire plant. For dose-response
tests, statistical procedures are employed to calculate the ICio and ICso values for
each of shoot, root and entire plant length and weight at test termination (see
references in paragraph (j)(4) and G)(H) of this guideline). Standard error and
95% confidence interval (CI) values for the calculated 1C values are to be
included. The slope of the concentration-response curve, its standard error, and
95% CI should also be recorded. Appropriate statistical analyses should provide a
goodness-of-fit determination for the concentration-response curves. Hypothesis
testing procedures can be used to determine NOEC (and LOEC) values (see
reference in paragraph (j)(6) of this guideline).
(g) Tabular summary of test conditions. Table 3 lists the important conditions that should
prevail during the definitive test. Except for the number of test treatments, Table 3 also lists the
important conditions that should prevail during a limit test. Meeting these test conditions will
greatly increase the likelihood that the completed test will be acceptable or valid. These test
conditions apply mainly to growth chambers and growth rooms; they are preferred to
greenhouses where for the latter it may be more difficult to meet or control these conditions.
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Table 3.—Summary of Test Conditions for Early Seedling Growth Toxicity Test1
Test duration
Substrate
Nutrients
Temperature
Relative humidity
Carbon dioxide
Light quality
Light intensity
Photoperiod
Watering
Test chamber (pot) size
Number of organisms per test
chamber
Number of replicate chambers
per test treatment
Number of organisms per test
treatment
Test treatment levels
Test substance application
method
Measures of effect
(measurement endpoints)
At least 14 days after 50% of the control plants have germinated
Quartz sand or glass beads. Hydroponic system may also be used.
Watered with nutrient solution (e.g., half-strength modified Hoagland's
medium)
25/20 °C (day/night) ± 3 °C
70/90% (day/night) ± 5%
350 ± 50 ppm at the top of the canopy
Fluorescent or representative of natural sunlight
350 ± 50 umol/m^/sec at the top of the canopy
16 hours light: 8 hours dark
Bottom watering as needed, using nutrient solution
Varies with plant species selected. Six-inch diameter plastic pots are
typical.
8-10 seedlings of one species per pot
4-5
40 (minimum)
Unless performing limit test, minimum of 5 treatment levels plus
appropriate controls
Root exposure (via watering with nutrient solution or sorbed onto the
substrate) or foliar exposure (via spray, dust or gas)
EC-io, EC50, NOEC and LOEC based upon survival
IC-io, IC50, NOEC and LOEC for each of roots, shoots and entire plant
length
IC-io, IC50, NOEC and LOEC for each of roots, shoots and entire plant
weight
Visual phytotoxicity reported
These environmental conditions are mainly applicable to growth chamber or growth room studies.
(h) Test validity elements. This test would likely be considered to be unacceptable or invalid if
one or more of the conditions in Table 3 did not occur or one or more performance objectives in
Table 4 were not met. This list should not be misconstrued as limiting the reason(s) that a test
could be found unacceptable or invalid. However, except for the conditions listed in Table 4 and
in the OCSPP 850.4000 guideline, it is unlikely a study will be rejected when there are slight
variations from guideline environmental conditions and study design unless the control
organisms are significantly affected, the precision of the test is reduced, the power of a test to
detect differences is reduced, and/or significant biases are introduced in defining the magnitude
of effect on measurement endpoints as compared to guideline conditions. Before departing
significantly from this guideline, the investigator should contact the Agency to discuss the reason
for the departure and the effect the change(s) will have on test acceptability. In the test report, all
departures from the guideline should be identified, reasons for these changes given, and any
resulting effects on test endpoints noted and discussed. It is preferred that this test be conducted
in growth chambers or growth rooms, instead of in greenhouses (see paragraphs (e)(7)(i) and (g)
of this guideline).
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Table 4.—Test validity elements for the early seedling growth toxicity test
1. Seed germination percentage in each of the controls is at least 65% for the recommended test species;
or the minimum acceptable USDA seed germination standards listed in paragraph (e)(5)(i)(B) of this
guideline. For native species that do not have germination standards, sufficient numbers of seeds, are
used to provide statistically valid measures of effect.
2. Mean control seedling survival is at least 90% for the duration of the study.
3. Control seedling condition should not indicate any visible phytotoxic or developmental symptoms
during the test.
4. For a given test species, all seeds used in the test are from the same source and lot number.
5. All test chambers used for a particular species are identical and contain the same amount of substrate
from the same source.
6. An untreated (or negative) control [and solvent (or vehicle) control, when a solvent was used] is
included in the test.
(i) Reporting—
(1) Background information. Background information to be supplied in the report
consists at a minimum of those background information items listed in paragraph (j)0) of
the OCSPP 850.4000 guideline.
(2) Guideline deviations. Provide a statement of the guideline or protocol followed.
Include a description of any deviations from the test guideline or any occurrences which
may have influenced the results of the test.
(3) Test substance.
(i) Identification of the test substance: common name, IUPAC and CAS names,
CAS number, structural formula, source, lot or batch number, chemical state or
form of the test substance, and its purity (i.e. for pesticides, the identity and
concentration of active ingredient(s)).
(ii) Storage conditions of the test chemical or test substance and stability of the
test chemical or test substance under storage conditions if stored prior to use.
(iii) Methods of preparation of the test substance and the treatment doses used in
the range-finding and definitive test, or limit test.
(iv) If a vehicle (e.g., solvent, dust) is used to prepare stock or test substance
provide: the name and source of the vehicle, the nominal concentration(s) of the
test substance in the vehicle in stock solutions or mixtures, and the vehicle dose(s)
used in the treatments.
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(4) Plant test species.
(i) Scientific and common name, plant family, and variety.
(ii) Test date of germination rating and germination percentage.
(iii) History of the seed: source, name of supplier, seed year or growing season
collected, batch or lot number, seed treatment(s), and storage conditions.
(iv) Seed size class.
(v) Description of handling and processing of seeds, pre-test planting, production,
and handling of test seedlings before use in test.
(vi) Length and condition of seedlings selected for the test.
(5) Test system and conditions. Description of the test system and conditions used in
the definitive or limit test, and any preliminary range-finding tests.
(i) A description of the test system, including type of growth chamber, growth
room, or greenhouse.
(ii) The description of pots or test containers: type, material, dimensions.
(iii) Number of seedling plants per pot or test container.
(iv) Number of pots per replicate, and number of replicates per treatment level.
(v) Description of the support medium or hydroponic system: source, type,
material.
(vi) Volume of support medium per pot or test container.
(vii) Methods used for treatment randomization.
(viii) Methods for preparing the test treatments: exposure route (root or foliar
exposure), application methods (including equipment type and method for
calibrating the application equipment), information about any solvent used to
dissolve and apply the test substance, and the concentrations (concentration of the
test substance in nutrient solution or in the foliar application vehicle, or the
quantity of test substance per unit weight of root support material when it is
coated on the support material, the concentration of test substance applied to the
soil surface; the concentration of test substance in the dust or spray; or the
concentration of gaseous test substances at inflow and outflow ports) and the
amounts used.
(ix) Number of applications and dates applied.
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(x) Culture practices during the test such as pest control, and irrigation practices
(type and watering schedule or regime).
(xi) The photoperiod and light source.
(xii) Methods and frequency of environmental monitoring performed during the
definitive or limit study for air temperature, humidity, and light intensity.
(xiii) For the definitive, or limit test, all analytical procedures should be
described. The accuracy of the method, method detection limit, and limit of
quantification should be given.
(6) Results.
(i) Environmental monitoring data results (carbon dioxide concentrations, air
temperature, humidity and light intensity) in tabular form (provide raw data for
measurements not made on a continuous basis), and descriptive statistics (mean,
standard deviation, minimum, maximum).
(ii) For preliminary range-finding tests, if conducted, the number of initial
seedlings, number of surviving seedlings, and length and dry weight of roots,
shoots, and entire plants, if measured, at each dose level and in the control(s). A
description and count of plants with visual phytotoxic effects, if recorded, at each
dose level and in the control(s).
(iii) For a limit test, tabulate for the limit concentration and the control by
replicate, the number of seedlings exposed at test initiation, the number of
surviving and number of dead seedlings at each observation time, and the length
and dry weight of roots, shoots and entire plants at test termination (provide the
raw data).
(iv) For the definitive test, tabulation by treatment and replicate of the number of
seedlings exposed at test initiation, the number of surviving and number of dead
seedlings at each observation time, and the length and dry weight of roots, shoots
and entire plants (provide the raw data).
(v) For the limit and definitive test, means and standard deviations for survival,
length and weight of roots, shoots, and entire plants in each treatment and control.
(vi) For the limit and definitive test, tabulation by treatment of the percent
reduction in mean length and dry weight of roots, shoots and entire plants relative
to control mean length and dry weight of roots, shoots and entire plants.
(vii) For the limit and definitive test, a description of visual (morphological) signs
of phytotoxicity including: time of onset, duration, severity (e.g. rank), and
number affected at each dose level and control(s) (provide the raw data). A
description of the phytotoxicity rating system used should be included.
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(viii) Graphs of the dose-response data for survival, and length and dry weight of
roots, shoots, and entire plant at test termination.
(ix) For the definitive study and for those effect measures (seedling survival,
length and dry weight of roots, shoots and entire plant) with sufficient data
coverage to fit a concentration-response relationship, tabulation of the slope of the
dose-response curve and its standard error and 95% confidence limits and any
goodness of fit results.
(x) For the definitive test, provide table of ECio and ECso values for survival and
ICio and ICso values for length and dry weight of roots, shoots and entire plants.
Also provide 95% confidence intervals and standard errors for these point
estimates.
(j) References. The references in this paragraph should be consulted for additional background
material on this test guideline.
(1) American Society for Testing and Materials. ASTM E 1963-02. Standard guide for
conducting terrestrial plant toxicity tests. In Annual Book of ASTM Standards, Vol.
11.06, ASTM, West Conshohocken, PA. Current edition approved December 10, 2002.
(2) American Society for Testing and Materials. ASTM E 1733-95. Standard guide for
the use of lighting in laboratory testing. In Annual Book of ASTM Standards, Vol.
11.05, ASTM, West Conshohocken, PA. Current edition approved September 10, 1995.
(3) Boutin, C. et al, 1993. 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.
(4) Bruce, R.D. and DJ. Versteeg, 1992. A statistical procedure for modeling continuous
toxicity data. Environmental Toxicology and Chemistry 11:1485-1494.
(5) Downs, RJ. and Helmers, H., 1975. Environment and Environmental Control of
Plant Growth. Academic Press, NY.
(6) Gulley, D.D. etal, 1989. Toxstat Release 3.0. University of Wyoming, Laramie, WY.
(7) Nyholm, N., P.S. Sorenson, K.O. Kusk, and E.R. Christensen, 1992. Statistical
treatment of data from microbial toxicity tests. Environmental Toxicology and Chemistry
11:157-167.
(8) Organization for Economic Cooperation and Development. 2006. New Test
Guideline (Section 2- Effects On Biotic Systems), 227, Terrestrial Plant Test: Vegetative
Vigour Test, 21 pp.; Revised Test Guideline (Section 2), 208, Terrestrial Plant Test:
Seedling Emergence and Seedling Growth Test, 21pp. Both guidelines adopted on 19
July 2006 and published as part of the 17th Addendum to the OECD Guidelines for the
Testing of Chemicals, 972006131, ISBN 92-64-01553-1.
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(9) Stephan, C.E., 1977. 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.
(10) Truelove, B., (ed)., 1977. Research Methods in Weed Science. Southern Weed
Science Society, Auburn Printing Inc., Auburn, AL 36830.
(11) VanEwijk, P.H. and J.A. Hoekstra, 1993. Calculation of the EC50 and its confidence
interval when a subtoxic stimulus is present. Ecotoxicology and Environmental Safety
25:25-32.
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