United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-011

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.4150:

Vegetative Vigor
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 20

OCSPP 850.4150: Vegetative vigor.

(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 OPP 122-1 Seed Germination/Seedling Emergence and Vegetative 

Vigor (Tier I) and 123-1 Seed Germination/Seedling Emergence and Vegetative Vigor 

(Tier II) guidelines (Pesticide Assessment Guidelines Subdivision J—Hazard Evaluation: 

Nontarget Plants); OPP Standard Evaluation Procedure Nontarget Plants Vegetative 

Vigor (Tier I and 2); OECD 208 Terrestrial Plants, Growth Test; OECD 227 Terrestrial 

Plant Test: Vegetative Vigor Test, and ASTM E 1963-02, Standard Guide for Conducting 

Terrestrial Plant Toxicity Tests. This guideline incorporates what were formerly Public 

Drafts OCSPP 850.4150 and OCSPP 850.4250 (April, 1996).

(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. The objective of the vegetative vigor test is to measure the effect of a foliar 

applied test substance upon terrestrial plants during the vegetative growth period of their 

development. Procedures for testing a single exposure concentration (i.e. Tier I testing for 

pesticides) as well as procedures for testing multiple exposure concentrations (i.e. Tier II testing 

for pesticides) are described. This guideline should be used in conjunction with OCSPP

850.4000, which provides general information and overall guidance for the guidelines in OCSPP

Series 850, Group D. The Environmental Protection Agency will use data from vegetative vigor 

tests in assessing the hazard a test substance may present in the terrestrial environment. 

(c) Definitions. The definitions in OCSPP 850.4000 apply to this guideline. In addition, the 

more specific definitions in this paragraph also apply:

Biomass is defined as all portions of the plant above the soil surface (i.e., does not 

include roots).

Shoot height is defined as the length of the above-ground vegetation from the soil surface 

to the apical tip or highest aerial part of the plant.

(d) General considerations―

(1) Summary of the test. The vegetative vigor test is a test that evaluates the affect of a 

test substance upon the growth of a number of plant species. Effects measured include 

survival, plant height, and plant biomass. In addition, qualitative phytotoxic effects are 

observed and evaluated. The test is designed to determine the quantity of test substance

required to cause a 25 percent (25%) inhibition in plant height and plant biomass (IC25

values and to determine the no observed effect concentration (NOEC) for these effect 

measures. A number of crop and non-crop plant species may be used. It may be 
Page 2 of 20

performed in a growth chamber, greenhouse, or in small field plots. A natural or 

synthetic soil serves as the substrate and the test substance is sprayed on the foliage at test 

initiation. The results are used to establish toxicity levels, evaluate hazards or risks to 

terrestrial plants, and to indicate if further testing at a higher tier is necessary. 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% of 

the population exhibit the effect (e.g., survival)) and ICx (concentration resulting in an x% 

decrease or inhibition effect on an attribute of the population (e.g., plant yield)).

(2) General test guidance. The general guidance in OCSPP 850.4000 applies to this 

guideline, except as specifically noted herein.

(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 (e.g., 1, 10, 100 pounds per acre (lbs/acre)). The details of the range-finding 

test do not have to be the same as for definitive testing in that there are no replicates, and 

the number of test organisms used, and the duration of exposure may be less than in 

definitive testing. In addition, the types 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 (shoot height and shoot biomass) its concentration-response curve and NOEC (and 

lowest observed effect concentration (LOEC)); the shoot height and weight IC25 values 

(with 95% confidence intervals and standard errors). If possible the slopes of the 

concentration-response curves, associated standard errors, and the 95% confidence 

intervals of the slopes should also be determined. However, at a minimum, the full 

concentration-response curve (concentration range covers IC05 to IC90) is determined for 

the most sensitive measure of effect using a minimum of five concentrations of the test 

chemical, plus appropriate controls. Recommend adding one or two additional test 

concentrations in the lower tail of the concentration-response curve for the most sensitive 

endpoint to ensure bracketing of both the most sensitive NOEC (or IC05) and IC25 values. 

For a satisfactory test, the lowest treatment concentration is below both the shoot height 

and biomass IC25 values. A summary of test conditions is provided in Table 2 and 

validity elements for an acceptable definitive test are listed in Table 3.

(5) Limit test. In some situations, it is only necessary to ascertain that the EC25 value

and the shoot height and shoot biomass IC25 values for a given plant species occur above 

a certain limit concentration, and that at this limit concentration there is no observable 

adverse effect. In a vegetative vigor limit test, at least 40 seedlings (divided into at least 

4 replicates of 10 plants each) are exposed to a single “limit concentration,” with the 

same number of replicates and organisms in appropriate controls. The multipleconcentration definitive test may be waived for a given test species if the following two 

conditions are met for survival, shoot height, shoot weight, and phytotoxic ranking. First, 

the “limit” treatment response is both statistically less than a 25% decrease from the 
Page 3 of 20

control response (i.e., EC25 and IC25 values > limit concentration), and second, the limit 

treatment responses are not significantly reduced (or inhibited) as compared to the control 

responses (i.e., NOEC > limit concentration). The limit concentration is 3 times the 

estimated environmental concentration for industrial chemicals or for pesticides the limit 

concentration is based upon the maximum label rate, e.g. the maximum recommended 

amount of active ingredient (a.i.), in the recommended minimum quantity of carrier (such 

as water), to be used per land area. Results are reported in grams or pounds of a.i. per 

acre. Except for the number of treatment groups, an acceptable limit test follows the 

same test procedures, is the same duration, and has the same number of controls as the 

definitive test. Acceptable limit tests like definitive tests include analytical confirmation 

of the applied dose.

(e) Test standards―

(1) Test substance. For pesticides the substance to be tested is the typical end-use 

product (TEP). If there is more than one TEP with the same inert substances, the one 

with the highest percent a.i. and/or the one most commonly used should be tested. If

there is more than one TEP with different inert substances, a TEP representative of each 

different inert substance should be tested in the range-finding test and at a minimum the 

most sensitive one tested in the definitive or limit test. In addition, if an adjuvant is 

recommended for use on a TEP label, the adjuvant is added with the TEP at the label rate 

to constitute the test substance. If a TEP is not available (i.e. new pesticide), the 

technical grade a.i. is used with a representative formulation, which should include any

adjuvant that will be recommended for use on the label. If products are applied in a tank 

mixture, dosages of each a.i. should be reported with identification and formulation for 

each product in the tank mix. For industrial chemicals the substance to be tested should 

be technical grade, unless the test is designed to test a specific formulation or mixture. 

OCSPP 850.4000 lists the type of information that should be known about the test 

substance before testing.

(2) Test duration. The limit and definitive tests last for 21 days after test substance

application. If phytotoxic symptoms first start to appear between day 14 and 21 postapplication, the test is extended to 28 days post-application.

(3) Test organisms―

(i) Species. The test is performed using crop and/or non-crop terrestrial plant 

species selected from a cross-section of the terrestrial plant species that have been 

historically used for this type of testing. A list of crop and non-crop plant taxa 

that have been used in toxicity tests can be found in Table 1 of this guideline and 

Table 2 of the OCSPP 850.4100 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. 

(A) Number of species tested. For testing industrial chemicals, the 

specific plant(s) used are selected on a case-by-case basis. For pesticide 
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testing, at a minimum ten plant species from the plant groups and families 

identified in paragraphs (e)(3)(i)(A)(1) through (e)(3)(i)(A)(2) are tested:

(1) Dicotyledoneae: Six species of at least four families, one 

species of which is soybean (Glycine max).

(2) Monocotyledoneae: Four species of at least two families, one 

species of which is corn (Zea mays).

(3) At least one test species should be a root crop (either a monocot 

such as onion or dicot such as carrot, table beet or sugar beet).

(B) Crop species. A representative list of crop species which are 

acceptable test species are listed in Table 1.

Table 1.—Crop species and families acceptable for use in the vegetative vigor 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

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 Turnip, Rape

Fabaceae Phaseolus vulgaris Garden bean

Malvaceae Gossypium spp. Cotton

Polygonaceae Fagopyrum esculentum Buckwheat

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.

(C) Non-crop plant species. When selecting plant species other than the 

three crop species (soybean, corn, and a root crop (onion, carrot, table beet 

or sugar beet)), which are tested at a minimum for pesticide testing, the 

use of sensitive non-crop plant species is recommended. Table 2 of 

OCSPP 850.4100, which is based on a table from the reference in 

paragraph (j)(5) of this guideline, provides a list of recommended noncrop species.
Page 5 of 20

(ii) Source. Within a given test, all seedlings of a given species, including the 

controls, should be from the same source and lot number, and same cultivation 

group.

(iii) Age or growth stage and size. Seedlings used in testing are selected from 

groups of plants grown from seed to the 2 to 4 true leaf stage. Selected seedlings 

should also be of uniform size.

(iv) Condition. Healthy plants should be used. For cultivation of seedlings from 

seed, healthy seeds should be used with reported germination rate for a seed lot of 

70% or better. Seeds should be examined before use and sorted to remove broken 

or damaged seeds. 

(v) Care and handling. Seeds, used to cultivate seedlings for this test, should be

stored in a desiccator and refrigerated until needed. Pesticide treated seeds should 

be avoided except for approved seed treatments. The Agency should be consulted 

prior to test initiation if seed treatments other than a weak hypochlorite solution 

(recommended by Environment Canada), captan, or thiram are used. Captan and 

thiram seed treatments (nonsystemic mode of action; see paragraph (j)(4) of this 

guideline) are the only Agency-approved pesticide seed treatments. When 

unapproved pesticide seed treatments are used in a study, the test should be 

designed to demonstrate no synergistic or antagonistic interactions occur between 

or among the seed treatment and test substance. Steam sterilization of soil is 

recommended as a non-pesticide alternative for killing pathogens, fungi, and 

insects in soil media.

(4) Administration of test substance. Within 48 hours prior to the application of the 

test substance, seedlings of similar size and condition (morphological symptoms) are 

selected for use in the test, and the height and condition of each plant is determined and 

recorded. At test initiation, test substance is applied to the seedling foliage as a spray. 

(i) Preparation of spray solutions. The amount of water used in the spray as a 

carrier is equivalent to the recommendation on the pesticide label. For example, 

vegetation within a given pot (test container) is sprayed with x milligrams of TEP 

mixed into 10 milliliters of water, where the 10 milliliters of water per the pot 

area is equivalent to the minimum number of gallons per acre specified on the 

label. For a satisfactory test, all pots in all treatment levels and controls have the 

same equivalent volume of water applied (e.g., in the example just given this is 10 

milliliters).

(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., IC25, 

IC05), test substance concentrations should immediately bracket the point 
Page 6 of 20

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 lb/acre). While a 

twofold progression is preferred, threefold and fourfold progressions are 

acceptable. If a fourfold series progression is used, the rationale for using 

this large an interval between concentrations and the effect on the 

accuracy and reproducibility of the point estimate or NOEC should be 

provided. For an acceptable study for a given plant species, the lowest test 

treatment level should be lower than the IC25 values for shoot height and 

biomass 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 5% inhibition concentration 

(IC05). It is recommended 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 both the most sensitive IC25

value and the most sensitive NOEC (or IC05) value.

(B) For pesticides dosages should be expressed as mass of test substance

per unit of soil surface area in the pot or container (i.e. lb/acre). 

Additionally for pesticides, dosages should be expressed in unit mass of 

a.i. or acid equivalent per unit of land area treated, as appropriate.

(C) 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).

(5) Controls. 

(i) Every test includes controls consisting of the same support medium, 

conditions, procedures, seedling source, seed source and lot, except that no test 

substance is added. In addition, vehicle (solvent) controls are also included if a 

solvent is used. However for pesticides, because TEP is typically tested, solvents 

are generally not necessary.

(ii) If the negative control is contaminated with the test substance, the study is not 

acceptable and should be repeated.

(iii) A test is not acceptable if at test termination less than 90% of control plants 

survive.

(6) Number of test organisms and replicates. 

(i) For each species, the minimum number of test organisms is 30 seedlings per 

dose level (a minimum of six replicates, each with a minimum of 5 seedlings). 
Page 7 of 20

Alternatively, the number of test organisms is 40 seedlings per dose level (a 

minimum of four replicates, each replicate with a minimum of 10 seeds). 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 

seedlings; the loading for rape, onion, wheat, or other small seed species is three 

to four seedlings. To prevent bias, seedlings are impartially or randomly assigned 

to the containers (pots, flats) or plots. Within a given test, all test organisms of a 

given species, including the controls, should be from the same source and 

cultivation group. 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.

(ii) Randomization. 

(A) Placement of the flats or pots within the greenhouse or arrangement of 

the field plots should follow a random or randomized block design. 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 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) Reliability. All equipment used in conducting the test, including equipment 

used to prepare and administer the test substance, and equipment to maintain and 

record environmental conditions, should be of such design and capacity that tests 

involving this equipment can be conducted in a reliable and scientific manner. 

Equipment should be inspected, cleaned, and maintained regularly, and be 

properly calibrated.

(ii) Application equipment. The application equipment used in testing products 

in small field plot studies should be designed to simulate conventional farm 

equipment using the basic components of commercial application equipment in 

the design of the small-plot equipment. For example, nozzle types, sizes, and 
Page 8 of 20

arrangements on small plot sprayers should be identical to those used by growers 

on commercial ground sprayers. The application equipment used in greenhouse 

or growth chamber tests (such as a spray chamber or computerized belt sprayer) 

should provide uniform coverage and should be calibrated at spray volumes (to 

the extent practical) that are representative of field conditions. Acceptable foliar 

spray methods are found in the reference in paragraph (j)(8) of this guideline. 

The amount of water used in spray as a carrier is to be equivalent to the 

recommendation on the label.

(iii) Facilities. Vegetative vigor tests should be conducted under controlled 

conditions in greenhouses and growth chambers, or under ambient conditions in 

small field plots.

(iv) 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 plastic pots or flats that have not been previously used are 

acceptable test containers, provided they are free of toxic materials. Do not use 

clay or peat containers. Containers should be thoroughly cleaned prior to use. A

dichromate solution should not be used to clean containers. The volume of the 

pot should be large enough so that seedling growth is not restricted during the 

test, see paragraph (e)(6)(i) of this guideline. Containers should be consistent 

within species.

(v) Support medium. Plants may be grown in a natural soil (free of pesticide 

contamination) or synthetic soil. Growth chamber and greenhouse tests are 

performed using a sterilized standardized soil that consists primarily of sandy 

loam, loamy sand, loamy clay, or clay loam soil that contains up to 3% organic 

matter (up to 1.5% organic carbon). Commercial potting soil or synthetic soil 

mixes may be used as the soil medium provided that the organic matter does not 

exceed 3%. Clay soils should not be used if the test substance is known to have a 

high affinity for clay. Field soils should be sieved to remove coarse (greater than 

2 millimeters) particles and recommend pasteurizing or heat treating the soil to 

reduce the effect of soil pathogens. The soil pH may be adjusted to the optimum 

growing range of 6.0 to 7.5 by the addition of a basic substance (calcium 

carbonate) or an acidic substance (gypsum, ammonium sulfate, or sulfuric acid). 

A slow-release fertilizer may be added to the soil to provide nutrients for plant 

growth. 

(vi) Plant supply. Seeds are planted and cultivated ahead of time to provide 

sufficient seedlings at the 2- to 4-true leaf stage of growth (typically one to four 

weeks post-emergence) and of uniform size to initiate the test for a given test 

species on the same day. Thinning the seedlings to uniform size and condition is 

performed before use. For non-crop species guidance on the time to germination 

may be found in Table 2 of OCSPP 850.4100.
Page 9 of 20

(8) Environmental conditions. For greenhouse and growth chamber studies, general 

conditions in paragraphs (e)(8)(i) through (e)(8)(v) of this guideline are recommended; 

however, excursions outside these recommendations do not invalidate the study if other 

acceptance criteria are met. Additional guidance for non-crop species is provided in 

Table 2 of OCSPP 850.4100.

(i) Temperature. Air temperature should be uniform throughout the greenhouse 

or growth chamber. Air temperature during the day should be 25 ± 6 degrees 

Celsius (oC) while temperature during the night should be 20 ± 6 oC. 

(ii) Humidity. Humidity should be uniform throughout the greenhouse or growth 

chamber. Relative humidity should approach 70 ± 15% during light periods.

(iii) Lighting and photoperiod. Luminance of 350 ± 50 μmol/m2/sec, measured 

at the top of the canopy, is desirable, on a photoperiod of 16 hours light and 8 

hours darkness. 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. For greenhouse and growth chambers, top watering below the 

canopy is used at the first watering after the test substance has been applied to 

initiate the capillary movement of water for bottom watering. Bottom watering of 

test containers is used for the duration of the study in order to prevent washing the 

chemical off the foliage and through the soil during watering. The watering 

method should prevent the test substance from washing off the foliage or leaching 

out of the soil or the pots/flats.

(v) Nutrients. Nutrients may be supplied during the study by using a nutrient 

solution of defined chemical composition, such as half-strength modified 

Hoagland nutrient solution, to water the plants. Alternatively, nutrients may be 

supplied by amending the test soil at the start of the test with fertilizer including 

standard nutrients for the species tested.

(9) Observations―

(i) Measurement of test substance. The dosing solution (i.e. spray tank) is 

sampled at the start of and end of application and analyzed for the test substance

concentration (in a.i. or acid equivalent units for pesticides). The total volume of 

dosing solution used, and the total soil surface area per pot or container in a 

replicate and treatment level should be recorded. The analytical methods used to 

measure the amount of test substance in a sample are validated before beginning 

the test, as described in OCSPP 850.4000.

(ii) Support medium. Characteristics of a batch of soil representative of that 

used in the study or the native soil (for tests done in field plots) are determined, 

including soil type and texture, pH, particle size distribution, and organic matter 

content.
Page 10 of 20

(iii) Environmental conditions. Environmental conditions to be monitored in 

greenhouses and growth chambers include air temperature, humidity, and light 

intensity. For field plots, environmental conditions should be monitored at the 

field site during and after test substance application and daily throughout the 

duration of the study. Environmental information to be collected in the field 

should include air temperature, precipitation, relative humidity, wind speed, light 

intensity at the canopy, and cloud cover. Source of environmental field condition 

data can be taken from a weather station that is within 5 miles of the field.

(A) Air temperature and humidity. The air temperature and humidity 

during the study should be recorded at representative locations throughout 

the area in which the test plants are growing. Measurements are made 

preferably continuously, but alternatively as maximum and minimum 

values over each 24-hour period.

(B) Light intensity. Light intensity at the canopy should be determined 

every 3 to 5 days 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. For field plots, ideally

PAR measurements should be taken between eleven in the morning and 

two in the afternoon. Additional information on the use of lighting in 

plant toxicity tests can be found in the references given in OCSPP

850.4000.

(C) Watering and precipitation. Frequency of watering should be 

recorded for greenhouse and growth chamber studies, and observations of 

moisture stress should be made and recorded daily. Frequency of watering 

and frequency and amount of precipitation should be recorded for field 

studies, and observations of moisture stress should be recorded daily.

(D) 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.

(iv) Phytotoxic Effects. 

(A) Observations on the number of alive and dead plants, and visual 

morphological symptoms of phytotoxicity are made and recorded at least 

on days 7, 14 and 21 post-application, and if the test is extended (see 

paragraph (e)(2) of this guideline) on day 28. Shoot height for each 

individual surviving plant is recorded at a minimum on days 14 and 21 

post-application, and if the test is extended on day 28. At test termination 

after shoot heights are measured, plants are dried (constant weight at 70 

oC) and the total plant biomass (i.e. dry weight) in each replicate is 

measured and recorded. Biomass measurements should be made for each 
Page 11 of 20

replicate not each individual plant.

(B) Seedling condition is determined using a standard morphological 

phytotoxicity rating scale. Observations on morphological symptoms of 

phytotoxicity should include all variations, either inhibitory or stimulatory, 

between the treated and the untreated organisms. Such variations may be 

phytotoxic symptoms (e.g., chlorosis, necrosis, pigmentation, leaf curling 

and wilting) and formative (e.g., leaf and stem deformation) effects. 

Observations should include the 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 reported.

(f) Treatment of results―

(1) Summary statistics―

(i) Environmental conditions. Air temperature, humidity, and light intensity

data should be summarized in tabular form, showing the mean, standard 

deviation, and range during the test at each measurement location. For field 

studies precipitation events and the amount of precipitation should be summarized 

in tabular form by date of occurrence, and total precipitation calculated. 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) Shoot height. Calculate and plot the average shoot height and standard 

deviation of shoot height for each replicate and the treatment level mean shoot 

height and standard deviation at each observation period (including test 

initiation).

(iv) Shoot biomass. Calculate and plot the average plant dry weight (total dry 

weight biomass divided by number of surviving plants) for each replicate and the 

treatment level mean shoot weight and standard deviation.

(v) Survival. Calculate and plot the percent survival (number of surviving 

seedlings divided by the number of seedlings at test initiation) for each replicate 

and the treatment level mean and standard deviation percent survival at each 

observation time. For each treatment level calculate mean percent survival as a 

percentage of the control mean percent survival at test termination.
Page 12 of 20

(vi) Phytotoxic effects. 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 shoot height and shoot biomass calculate the percent 

inhibition (%I) at each treatment level using Equation 1.

  

C

C X

I

100 %

 Equation 1

where:

C = the control mean response value (shoot height or shoot biomass); and

X = the treatment mean response value (shoot height or shoot biomass, 

respectively). Stimulation is reported as negative %I.

(3) Limit test―

(i) EC25 and IC25 values. To ascertain that the survival EC25 value and shoot 

height and shoot biomass IC25 values for a given plant species occur above the 

“limit” concentration, 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 EC25 and IC25 compared to controls is operationally defined as a 25% 

reduction or inhibition 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 25% inhibition or reduction, compared to the 

control (i.e., H0: μcontrol - μlimit > δ0). The alternative hypothesis (HA) is that this 

difference is less than a 25% reduction, compared to the control (i.e., HA: μcontrol -

μlimit < δ0). An example of a parametric two-sample comparison test of this is the 

Student’s t-test. If the null hypothesis is rejected, the effect level or inhibition 

level for the given response measure (i.e., survival, shoot height and biomass) in 

the limit treatment as compared to the control is declared to be less than 25% 

(e.g., IC25 > limit dose). If the null hypothesis is not rejected, the effect level or 

inhibition level in the limit treatment as compared to the control response is 

declared to be 25% or greater (e.g., IC25 < limit dose).

   100  0  xcontrol  p Equation 2

where:

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

reduction from the control sample mean;
Page 13 of 20

control x = control sample mean response (e.g., survival, shoot height and 

biomass); and

p = percent reduction from the control sample mean, which is 25 in the 

case of the EC25 and IC25.

(ii) NOEC. To ascertain that there is no observable effect at the limit treatment 

(i.e., NOEC > limit dose) for a given response measure (survival, shoot height and 

biomass), the limit treatment response is compared to the control treatment 

response using a one-sided two-sample parametric or nonparametric test, as 

appropriate (see OCSPP 850.4000). The minimum significant difference 

detectable by the test or a similar estimate of the sensitivity of the test should be 

determined and reported.

(iii) Multiple-dose definitive testing. 

(A) A multiple-dose definitive test is performed for a given test species if 

either the effect or inhibition level for one or more response measures (i.e., 

survival, shoot height and biomass) in the limit treatment as compared to 

the control response at test termination are declared to be 25% or greater 

effect (i.e., the null hypothesis is not rejected) or the NOEC is less than the 

limit concentration.

(B) Multiple-dose definitive testing may be waived for a given test species 

if at test termination the “limit” treatment response is both statistically less 

than a 25% decrease from the control response and there is no observable 

adverse effect from the control response for all measures of effect 

(survival, shoot height and biomass).

(4) Multiple-dose definitive test―

(i) Dose-response curves, slopes and IC25 and EC25 values―

(A) Shoot height and shoot biomass. For dose-response-response tests 

the IC25 value (standard error and 95% confidence interval) is calculated 

for each of shoot height and shoot biomass (see OCSPP 850.4000 and 

references in paragraphs (j)(2) and (j)(9) of this guideline for statistical 

guidance). If a dose-response curve was fit to the data to determine the 

IC25, the model parameters (e.g., slope) and their uncertainty estimates 

(e.g., standard error) should be recorded. Where the dose-response range 

tested does not result in the determination of a definitive IC25 value for a 

given response measure, test and document that the IC25 value is above the 

highest treatment level tested (see the statistical guidance in OCSPP

850.4000 and in paragraph (f)(3) of this guideline). Such an event may 

arise if one of the other response measures is much more sensitive, and 

while the full response curve for that response measure is captured too 

many additional treatments would be needed to capture the full response 
Page 14 of 20

relationship for the other less sensitive response measure(s). Methods, 

assumptions, and results of the statistical approaches used should be 

recorded. 

(B) Percent survival. The EC25 value (standard error and 95% 

confidence interval) is calculated for percent survival (see OCSPP

850.4000 for statistical guidance). If a dose-response curve was fit to the 

data to determine the EC25 the model parameters (e.g., slope) and their 

uncertainty estimates (e.g., standard error) should be recorded. Where the 

dose-response range tested does not result in the determination of a 

definitive EC25 value, test and document that the EC25 value is above the 

highest treatment level tested (see statistical guidance in OCSPP 850.4000

and in paragraph (f)(3) of this guideline). Such an event may arise if one 

of the other response measures is much more sensitive, and while the full 

response curve for that response measure is captured too many additional 

treatments would be needed to capture the full response relationship or a 

definitive EC25 for survival. Methods, assumptions, and results of the 

statistical approaches used should be recorded. 

(ii) NOEC. The NOEC (and LOEC) for each response measure (survival, shoot 

height and shoot weight) is determined (see OCSPP 850.4000 and the reference in 

paragraph (j)(3) of this guideline). If a NOEC value can not be determined for a 

given response measure, as appropriate, the dose at which there is a 5% inhibition 

(i.e., an IC05 value for shoot height and shoot weight) or the dose at which there is 

a 5% reduction in survival of the exposed population (i.e., an EC05 value) is 

estimated and used in place of the given NOEC. The standard error and 95% 

confidence interval should also be calculated for the IC05 and EC05 values. 

Methods, assumptions, and results of the statistical approaches used should be 

recorded.

(g) Tabular summary of test conditions. Table 2 lists the important conditions that should 

prevail during the definitive test. Except for the number of test treatments, Table 2 also lists the 

important conditions that should prevail during a limit test. Meeting these conditions will greatly 

increase the likelihood that the completed test will be acceptable or valid.
Page 15 of 20

Table 2.—Summary of Test Conditions for Vegetative Vigor Test

Test type Vegetative vigor test

Test duration Minimum of 21 days after test substance application 

(extended to 28 days post-application if phytotoxic 

symptoms initially show between days 14 and 21 postapplication).

Substrate Natural or synthetic soil with 3% organic matter and pH 

6.0 - 7.5

Nutrients As naturally available, or supplemented with either a 

soil fertilizer or watered with nutrient solution 

Temperature 25/20 oC (daytime/nighttime) ± 6 oC

Relative humidity 70% (daytime) ± 15%

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

Photoperiod 16 hours light: 8 hours dark (for non-crop species see 

Appendix 1 of this guideline)

Watering For greenhouse and growth chamber, the initial 

watering event is by top watering below the canopy and 

bottom watering is used throughout the remainder of 

the study. In field plots top watering below the canopy 

is used.

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

plastic pots are typical. Flats are also encouraged.

Number of plants per test chamber Varies with species and test chamber (pot) size.

Number of replicates per test treatment 6 (a minimum of 4 if increase number of seedlings per 

replicate to 10)

Number of plants per test treatment 30 plants (a minimum of 40 seedlings if there is only 4 

replicates)

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

levels plus appropriate controls

Test substance application method Applied to foliage

Measures of effect or measurement endpoints IC25 and NOEC (or IC05) for each of shoot height and 

shoot weight

EC25 and NOEC (or EC05) for survival

(h) Test validity elements. This test would 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 3 

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 3 and in 

OCSPP 850.4000, it is unlikely that 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 
Page 16 of 20

departures from the guideline should be identified, reasons for these changes given, and any 

resulting effects on test endpoints noted and discussed.

Table 3.—Test Validity Elements for the Vegetative Vigor Test

1. Mean control plant survival is at least 90% at test termination.

2. Control seedlings do not contain any visible phytotoxic symptoms during the test that are the same as 

due to the test substance.

3. For a given species, all seedlings in a test are from the same cultivation group and source.

4. All test chambers used for a particular species should be identical and should contain the same 

amount of soil from the same source.

5. A negative (untreated) control [and solvent (or vehicle) control, when a solvent was used] is included in 

the test.

6. For a given plant species, the lowest test concentration level was below both the shoot height and 

biomass IC25 values for the plant species.

7. If pesticides are used for pest control during the test, additional test data was submitted to document 

that the pesticide used was not toxic to the test species and that there were no synergistic or antagonistic 

interactions with the test substance.

8. The water carrier used at the time of test substance application did not excessively exceed the amount 

of water on the label (gallon per acre).

(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 

OCSPP 850.4000.

(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.
Page 17 of 20

(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.

(4) Plant test species. 

(i) Scientific and common name, plant family, and variety.

(ii) History of the young test plants including source, name of supplier, batch or 

lot number of the seed.

(iii) Test date of germination rating and germination percentage.

(iv) Date of planting.

(v) Pre-test planting and seedling cultivation conditions, grow out conditions and 

handling including pest and disease treatments, for the young plants before use in 

the test.

(vi) Height and morphological condition (i.e., any abnormalities, pigmentation, 

wilting, etc.) 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) Location of testing, and if not a field plot study the type of growth chamber or 

greenhouse.

(ii) For small field plot testing describe the plot design: size of field plots, number 

of control and experiment plots, the number of plots per treatment and control, the 

plot lay-out, the number of plants in each plot.

(iii) For greenhouse or growth chambers the description of the containers, pots 

and flats: type, material, dimensions, and the soil surface area per pot or test 

container.

(iv) Number of seedlings per pot/flat.

(v) Number of pots or flats per replicate, and number of replicates per treatment 

level.

(vi) Description of the support medium: source, soil type designation, soil 

composition, pH, percent organic matter, type and amounts of soil amendments.

(vii) Methods used for treatment randomization and impartial assignment of 

plants to test plots, pots, and/or flats.
Page 18 of 20

(viii) Method of test substance application: equipment type, equipment design, 

method for calibrating the application equipment, dose levels, volume of dosing 

solution applied. 

(ix) Date of test substance application and test duration (date plants were 

harvested at test termination).

(x) Culture practices during the test such as cultivation, 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 and 

additionally for field plot tests rainfall and cloud cover.

(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 (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 surviving 

plants, and shoot and biomass, if measured, at each dose level and in the 

control(s). A description and count of 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 and their shoot height at test initiation, 

the number of surviving and number of dead seedlings at each observation time, 

and the shoot height and biomass at test termination (provide the raw data).

(iv) For the definitive test, tabulation by treatment and replicate the number of 

seedlings exposed and their shoot height at test initiation, the number of surviving 

and number of dead seedlings at each observation time, and the shoot height for 

each individual plant and total plant biomass at test termination (provide the raw 

data).

(v) For the limit and definitive tests, tabulation by treatment of the percent 

reduction in mean height, biomass, and survival as compared to control plants at 

test termination.
Page 19 of 20

(vi) 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.

(vii) Graphs of the dose-response data for shoot height and weight at test 

termination.

(viii) For a limit test, provide the results of hypothesis tests.

(ix) For the limit test, provide a description of the statistical methods used 

including software package, and the basis for the choice of method.

(x) For the definitive study and for those effect measures (shoot height and 

weight) with data sufficient 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.

(xi) For the definitive test, tabulation of IC25 values for plant height and plant 

biomass.

(xii) For the definitive test, a tabulation of the NOEC and LOEC for each measure 

of effect (plant height, and plant biomass). The EC05 or IC05, as applicable, 

should be reported for effect measure data where an NOEC could not be 

determined.

(xiii) Description of statistical method(s) used for point estimates, including 

software package, for determining IC25 values, fitting the dose-response model, 

and the basis for the choice of method. Provide results of any goodness-of-fit 

tests.

(xiv) Description of statistical method(s) used for NOEC and LOEC 

determination, including software package, and the basis for the choice of 

method. If an IC05 value is used in place of a NOEC provide a description of 

statistical method(s) used for point estimates, including software package, for 

determining IC05 values, fitting the dose-response model, and the basis for the 

choice of method. Provide results of any goodness-of-fit tests.

(j) References. The following references 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) Bruce, R.D. and D.J. Versteeg, 1992. A statistical procedure for modeling continuous 

toxicity data. Environmental Toxicology and Chemistry 11:1485-1494.
Page 20 of 20

(3) Gulley, D.D. et al., 1989. Toxstat Release 3.0. University of Wyoming, Laramie, WY.

(4) Hatzios, K.K. and D. Penner, (1985). Interactions of herbicides with other 

agrochemicals in higher plants. Rev. Weed Sci. 1:1-63.

(5) Organization for Economic Cooperation and Development (OECD). 2006. New Test 

Guideline (Section 2- Effects On Biotic Systems), 227, Terrestrial Plant Test: Vegetative 

Vigour Test, 21pp. 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.

(6) OECD. 2006. Revised Test Guideline (Section 2), 208, Terrestrial Plant Test: 

Seedling Emergence and Seedling Growth Test, 21pp. 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.

(7) U.S. Environmental Protection Agency, 1982. Pesticide Assessment Guidelines 

Subdivision J, Hazard Evaluation: Nontarget Plants. Office of Pesticides and Toxics, 

Washington, D.C. EPA 540/9-82-020, October 1982.

(8) U.S. Environmental Protection Agency, 1986. Hazard Evaluation Division Standard 

Evaluation Procedure, Nontarget Plants: Vegetative Vigor -Tiers 1 and 2. Office of 

Pesticides Programs, Washington, D.C. EPA 540/9-86-133, June 1986.

(9) 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.