United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-010

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.4230:

Early Seedling Growth 

Toxicity Test
i

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.
Page 1 of 23

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 EC10 and 

EC50 values for seedling survival and IC10 and IC50 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), (j)(7) and (j)(10) 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 EC50 and EC10 values (with 95% confidence intervals 

and standard errors) for seedling survival; and the IC50 and IC10 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 
Page 3 of 23

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 EC10

and EC90 (or IC10 and IC90)) 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 EC10 (seedling survival) and IC10 (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 postgermination EC10, EC50, IC10 and IC50 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. IC10 >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)(1) 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

Family Species Common name

Dicots Solanaceae Lycopersicon esculentum or 

Solanum lycopersicum

Tomato

Cucurbitaceae Cucumis sativus Cucumber

Asteraceae Lactuca sativa Lettuce

Fabaceae1 Glycine max Soybean

Brassicaceae Brassica oleracea Cabbage

Apiaceae Daucus carota Carrot

Monocots Poaceae Avena sativa Oat

Poaceae Lolium perenne Perennial ryegrass

Poaceae Zea mays Corn

Liliaceae Allium cepa Onion

1

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 Species Common name

Chenopodiaceae Beta vulgaris Sugar beet or table beet

Asteraceae Helianthus annuus Sunflower

Fabaceae Pisum sativum Pea

Brassicaceae Brassica rapa Field mustard, Canola

Brassicaceae Brassica napus Rape, Turnip

Fabaceae Phaseolus vulgaris Garden bean

Malvaceae Gossypium spp. Cotton

Polygonaceae Fagopyrum esculentum Buckwheat
Page 5 of 23

(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 (

oC) 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 
Page 6 of 23

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)(1) and (e)(4)(i)(A)(2) 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)(3) 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 arabic, 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, watersoluble vehicle is available should be dissolved in an appropriate 
Page 7 of 23

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)(1) of this guideline, by

dusting the plants, as described in paragraph (e)(4)(i)(B)(2) of this 

guideline or by exposing the plants to gas in a fumigation chamber, as 

described in paragraph (e)(4)(i)(B)(3) 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)(2) 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 (μm) 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 
Page 8 of 23

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., EC50, 

EC10), 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 EC10 value for seedling 

survival and the IC10 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 (EC10 or IC10). 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 EC10 or IC10 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).
Page 9 of 23

(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.
Page 10 of 23

(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 
Page 11 of 23

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. 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. 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 oC while temperature during the night should be 20 ± 3 oC. 

Temperature in greenhouses is more difficult to control. Under these 

circumstances, temperature during the day should be 25 ± 6 oC while temperature 

during the night should be 20 ± 6 oC. 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 
Page 12 of 23

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 (μmol/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. 
Page 13 of 23

(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.
Page 14 of 23

(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 
Page 15 of 23

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 oC) 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. 
Page 16 of 23

(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.

  

C

C X

I

100 %

 Equation 1

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 EC10 and the length and weight IC10 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 IC10 or EC10, compared to 

the controls, is operationally defined as a 10% reduction from the control sample mean 

(Equation 2). The null hypothesis (H0) stated in terms of true population parameters is 

that the difference of the “limit” treatment mean response (μlimit) from the control mean 

response (μcontrol) is greater than or equal to a 10 reduction, compared to the control (i.e., 

H0: μcontrol - μlimit > δ0). The alternative hypothesis (HA) is that this difference is less than 

a 10% reduction, compared to the control (HA: μcontrol - μlimit < δ0). 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).

   100  0  xcontrol  p Equation 2
Page 17 of 23

where:

δ0 = difference between two parameters, defined in this case as a p% reduction 

from the control sample mean;

control x = 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 

EC10 and IC10.

(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 EC10 and EC50 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 IC10 and IC50 values for 

each of shoot, root and entire plant length and weight at test termination (see 

references in paragraph (j)(4) and (j)(11) of this guideline). Standard error and 

95% confidence interval (CI) values for the calculated IC 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.
Page 18 of 23

Table 3.—Summary of Test Conditions for Early Seedling Growth Toxicity Test1

Test duration At least 14 days after 50% of the control plants have germinated

Substrate Quartz sand or glass beads. Hydroponic system may also be used.

Nutrients Watered with nutrient solution (e.g., half-strength modified Hoagland’s 

medium)

Temperature 25/20 oC (day/night) ± 3 oC

Relative humidity 70/90% (day/night) ± 5%

Carbon dioxide 350 ± 50 ppm at the top of the canopy

Light quality Fluorescent or representative of natural sunlight

Light intensity 350 ± 50 μmol/m2/sec at the top of the canopy

Photoperiod 16 hours light: 8 hours dark

Watering Bottom watering as needed, using nutrient solution

Test chamber (pot) size Varies with plant species selected. Six-inch diameter plastic pots are 

typical. 

Number of organisms per test 

chamber

8 - 10 seedlings of one species per pot

Number of replicate chambers 

per test treatment

4 – 5

Number of organisms per test 

treatment

40 (minimum) 

Test treatment levels Unless performing limit test, minimum of 5 treatment levels plus 

appropriate controls

Test substance application 

method

Root exposure (via watering with nutrient solution or sorbed onto the 

substrate) or foliar exposure (via spray, dust or gas)

Measures of effect 

(measurement endpoints)

EC10, EC50, NOEC and LOEC based upon survival

IC10, IC50, NOEC and LOEC for each of roots, shoots and entire plant 

length

IC10, IC50, NOEC and LOEC for each of roots, shoots and entire plant 

weight

Visual phytotoxicity reported 

1 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).
Page 19 of 23

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)(1) 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.
Page 20 of 23

(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.
Page 21 of 23

(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.
Page 22 of 23

(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 EC10 and EC50 values for survival and 

IC10 and IC50 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 D.J. Versteeg, 1992. A statistical procedure for modeling continuous 

toxicity data. Environmental Toxicology and Chemistry 11:1485-1494.

(5) Downs, R.J. and Helmers, H., 1975. Environment and Environmental Control of 

Plant Growth. Academic Press, NY. 

(6) Gulley, D.D. et al, 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.
Page 23 of 23

(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.