United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-012

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.4100:

Seedling Emergence 

and Seedling Growth
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 30

OCSPP 850.4100: Seedling emergence and seedling growth.

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

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

Vigor (Tier II) Testing (Pesticide Assessment Guidelines Subdivision J—Hazard 

Evaluation: Nontarget Plants); OPP Standard Evaluation Procedure Nontarget Plants: 

Seed Germination/Seedling Emergence - Tier 1 and 2, and ASTM E 1963-02, Standard 

Guide for Conducting Terrestrial Plant Toxicity Tests. This guideline incorporates what 

were formerly Public Drafts OCSPP 850.4100 and OCSPP 850.4225 (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 seedling emergence test is to measure the effect of test 

substances upon plants during early critical stages of their development. Procedures for testing a 

single exposure concentration (Tier I testing for pesticides) as well as procedures for testing 

multiple exposure concentrations (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 seedling emergence tests in assessing the hazard and risk a 

test substance may present in the terrestrial environment. This guideline incorporates earlier 

guidelines that addressed seed germination and root elongation separately.

(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).

Emerged refers to a plant that has successfully germinated but may be dead or alive at the 

time of observation (i.e. if the plant died after emerging, remains of the plant should be 

observable on the surface and the plant is counted as successfully emerging).

Mortality refers to a plant that has successfully emerged (i.e. germinated) but is dead at 

the time of observation.

Survival refers to a plant that has successfully emerged (i.e. germinated) and is still alive 

at the time of final observation.
Page 2 of 30

(d) General considerations—

(1) Summary of the test. Seeds of a terrestrial plant species are exposed to a test 

substance through the periods of germination, emergence, and early stages of seedling 

growth. Effects measured include percent emergence, number of emerged plants, 

seedling survival, seedling length, and seedling 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%) reduction (EC25 or IC25 as 

appropriate) in a number of seedling emergence and growth effect measures 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 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 soil surface 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., emergence)) 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. The protocol to examine seedling growth 

under TSCA requirements is the OCSPP 850.4230 guideline for early seedling growth.

(3) Range-finding test. A range-finding test is usually conducted to establish the 

appropriate test substance concentrations to be used for the definitive test. In the rangefinding 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 rangefinding test do not have to be the same as for definitive testing in that the number of 

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

than that used 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 (seedling emergence, survival, shoot length, and shoot biomass) its concentrationresponse curve and NOEC (and lowest observed effect concentration (LOEC)); the EC25

value (with 95% confidence interval and standard error) for seedling emergence, and for 

survival; and the IC25 value (with 95% confidence interval and standard error) for shoot 

length, and for shoot biomass. The slope of the concentration-response curve, its 

associated standard error, and 95% confidence interval should be determined for each 

measure of effect, if possible. However, at a minimum, the full concentration-response 

curve (e.g., 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 
Page 3 of 30

the most sensitive NOEC (or EC05 or IC05) and most sensitive EC25 or IC25 value. For a 

satisfactory test, the lowest treatment concentration is below the EC25 (seedling 

emergence and survival) and IC25 (shoot length and biomass) values for all measures of 

effect. A summary of test conditions is provided in Table 3 and validity elements for an 

acceptable definitive test are listed in Table 4.

(5) Limit test. In some situations, it is only necessary to ascertain that for a given plant 

species the seedling emergence and survival EC25 values and the shoot length and shoot 

biomass IC25 values occur above a certain limit concentration, and that at this limit 

concentration there is no observable adverse effect. In a seedling emergence and 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 the following two conditions are met for seedling emergence, 

survival, shoot length, and shoot biomass. First, the “limit” treatment response is both 

statistically less than a 25% decrease from the 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 at least 3 times the estimated environmental 

concentration 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, mixture, or 

end-use product. OCSPP 850.4000 lists the type of information that should be known 

about the test substance before testing.
Page 4 of 30

(2) Test duration. The limit and definitive tests last for 14 days after 50% of the control 

plants have emerged. If phytotoxic symptoms start to appear between day 7 and 14 postemergence, the test should be extended to 21 days post-emergence of 50% of the control 

plants.

(3) Test organism―

(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 listing of crop and non-crop plant taxa 

that have been used in toxicity tests can be found in Table1 and Table 2, 

respectively, 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. Seeds of plant species 

with low or variable germination potential should be avoided for this study.

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

specific plant(s) used are selected on a case-by-case basis. For pesticide 

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)(3) of this 

guideline 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 root crop species (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 that are acceptable 

test species are listed in Table 1.
Page 5 of 30

Table 1.—Crop species and families for use in the seedling emergence and 

seedling growth 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 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, the use of sensitive non-crop 

plant species is recommended. Table 2 provides a list of recommended 

non-crop species. The information provided for each species is a 

compilation from several sources. References are provided in brackets to 

each entry. The references appearing in brackets in Table 2 appear in

paragraph (k) of this guideline. The table is based on a table from the 

reference in paragraph (j)(5) of this guideline.
Page 6 of 30

Table 2.—Table of non-crop species for use in the seedling emergence and seedling growth test

FAMILY Lifespan Seed Photoperiod Planting Time to Special Toxicity

Species Botanical Name and Habitat Weight for germination Depth Germinate Treatments(d) Test(e)

(English Common Name) mg or growth(a) mm(b) days

(c)

APIACEAE

Torilis japonica Annuals, Biennials 1.7 - 1.9 L = D 0 5 (50%) cold stratification [7, 14, 18, 19] POST

(Japanese Hedge-parsley) disturbed areas, [14, 19] [14] [1, 19] [19] maturation may be necessary [19] [5]

hedgerows, pastures germination inhibited 

[16, 19] by darkness [1, 19]

no special treatments [5]

ASTERACEAE

Bellis perennis Perennials 0.09 - 0.17 L = D 0 3 (50%) [19] germination not affected by irradiance POST

(English Daisy) grassland, arable [4, 19] [14] [4] 11 (100%) [18] [18, 19] [4]

fields, turf [16, 19] no special treatments [4, 14]

Centaurea cyanus Annuals 4.1 - 4.9 L = D 0 - 3 14 - 21 (100%) no special treatments [2, 4] POST 

(Cornflower) fields, roadsides, [4, 14] [14] [2, 4, 14] [14] [2, 4]

open habitats [16]

Centaurea nigra Perennials 2.4 - 2.6 L = D 0 3 (50%) [19] maturation may be necessary [18, 19] POST 

(Black Knapweed) fields, roadsides, [14, 19] [14] [19] 4 (97%) [18] germination inhibited by darkness [19] [5, 22, 26]

open habitats [16, 19] no special treatments [5, 14, 26]

Inula helenium Perennials 1 - 1.3 0 no special treatments [4] POST 

(Elecampane) moist, disturbed sites 

[16]

[4, 14, 29] [4, 29] [4]

Leontodon hispidus Perennials 0.85 - 1.2 L = D 0 4 (50%) [19] germination inhibited by darkness POST

(Big Hawkbit) fields, roadsides, [14, 19] [14] [19] 7 (80%) [18] [17, 18, 19] [5, 22, 23]

disturbed areas [16, 19]

no special treatments [5, 23]

Rudbeckia hirta Biennials, Perennials 0.3 L = D 0 < 10 (100%) no special treatments POST

(Black-eyed Susan) Disturbed [16] [4, 14] [14] [4, 33] [33] [4, 14, 33] [4, 33]

Solidago canadensis Perennials 0.06 - 0.08 L = D 0 14 – 21 mix with equal part sand and POST 

(Canada Goldenrod) pasture, open areas [4, 14] [11] [4] [11] soak in 500ppm GA for 24 hrs [11] [4]

[16] no special treatments [4]
Page 7 of 30

FAMILY Lifespan Seed Photoperiod Planting Time to Special Toxicity

Species Botanical Name and Habitat Weight for germination Depth Germinate Treatments(d) Test(e)

(English Common Name) mg or growth(a) mm(b) days

(c)

ASTERACEAE continued

Xanthium pensylvanicum Annuals 25 - 61 0 [1] germination may be inhibited by PRE & 

(Common Cocklebur) fields, open habitats [16] [14, 29] 5 [29] darkness [1] POST [31]

soak in warm water for 12 hrs [29]

Xanthium spinosum Annuals 200 L = D [14] 10 scarification [14] PRE &

(Spiny Cocklebur) open habitats [16] [14] L > D [6] [6] no special treatments [6] POST [6]

Xanthium strumarium Annuals 67.4 L = D 10 - 20 no special treatments PRE &

(Italian Cocklebur) fields, open habitats [16] [14] [14] [6, 21] [6, 14, 21] POST [6, 21, 

28, 31]

BRASSICACEAE

Cardamine pratensis Perennials 0.6 L = D 0 5 (50%) [19] germination inhibited by darkness POST 

(Cuckoo Flower) fields, roadsides, turf [14, 19] [14] [19] 15 (98%) [18] [18, 19] [5, 22]

[16, 19] no special treatments [5, 14, 22]

CARYOPHYLLACEAE

Lychnis flos-cuculi Perennials 0.21 L = D < 14 (100%) maturation may be necessary [18] POST

(Ragged Robin) [16] [14] [14] [14, 25] no special treatments [5, 14, 15, 22-

26]

[5, 15, 22-

26]

CHENOPODIACEAE

Chenopodium album Annuals 0.7 - 1.5 L = D 0 2 (50%) treatment differs depending on PRE &

(Lamb's Quarters) field margins, [14, 19, [14] [1, 19] [19] seed color [19] POST [28, 

disturbed areas [16, 19] 34] dry storage dormancy [19] 31, 34]

germination inhibited by darkness

[1, 18, 19]

cold stratification [18]

no special treatments [14, 34]

CLUSIACEAE

Hypericum perforatum Perennials 0.1 - 0.23 L = D 0 3 [19] germination inhibited POST 

(Common St. John's Wort) fields, arable land, [14, 19] [14] [1, 19] 11 (90%) [18] by darkness [1, 18, 19] [5, 15, 25, 

open habitats [16, 19] no special treatments [5, 14, 15, 25, 

27]

27]

CONVOLVULACEAE

Ipomoea hederacea Annuals 28.2 L > D 10 - 20 4 (100%) germination not affected by PRE & 

(Purple Morning Glory) roadsides, open [14] [6, 10] [6, 10, 21] [10] irradiance [1] POST [6,

habitats, corn fields [16] no special treatments [6, 21] 12, 21, 28]
Page 8 of 30

FAMILY Lifespan Seed Photoperiod Planting Time to Special Toxicity

Species Botanical Name and Habitat Weight for germination Depth Germinate Treatments(d) Test(e)

(English Common Name) mg or growth(a) mm(b) days

(c)

CYPERACEAE

Cyperus rotundus Perennials 0.2 L = D 0 [1] 12 (91%) germination inhibited by darkness [1] PRE & 

(Purple Nutsedge) arable land, pastures, [14] [14] 10-20 [6, 10] [10] no special treatments [6, 10, 14] POST [6, 28, 

roadsides [16, 30] 31]

FABACEAE

Lotus corniculatus Perennials 1 - 1.67 L = D 1 (50%) scarification [14, 19] POST

(Bird's-foot Trefoil) Grassy areas, [14, 19] [14] [19] germination not affected by irradiance [5, 23, 25]

roadsides, open [18, 19]

habitats [16, 19] no special treatments [23, 25]

Senna obtusifolia Annuals 23 - 28 L = D [14] 10 - 20 soak seeds in water for 24 hours [9] POST

(Cassia, Sicklepod) moist woods [16] [9] L > D [9] [6, 9] scarification [14] [6, 9]

seed viability differs depending on 

color [1]

no special treatments [6]

Sesbania exaltata Annuals 11 - 13 L > D 10 - 20 soak seeds in water for 24 hours [9] PRE & 

(Hemp) alluvial soil [16] [9, 14] [9] [9, 21] germination not affected POST [9, 21, 

by irradiance [1] 28, 31]

no special treatments [21]

Trifolium pratense Perennials 1.4 - 1.7 L = D 1 (50%) scarification [14, 18] POST

(Red Clover) fields, roadsides, [14, 19] [14] [19] may need maturation [19] [5]

arable land [16, 19] germination not affected by 

irradiance [1, 19]

no special treatments [5]

LAMIACEAE

Leonurus cardiaca Perennials 0.75 - 1.0 L = D 0 [4] no special treatments [4, 14] POST

(Motherwort) open areas [16] [4, 14] [14] [4]

Mentha spicata Perennials 2.21 0 [4] no special treatments [4] POST

(Spearmint) moist areas [16] [4] [4]

Nepeta cataria Perennials 0.54 L = D 0 [4] no special treatments [2, 4, 14] POST

(Catnip) disturbed areas [16] [4, 14] [14] [2, 4]
Page 9 of 30

FAMILY Lifespan Seed Photoperiod Planting Time to Special Toxicity

Species Botanical Name and Habitat Weight for germination Depth Germinate Treatments(d) Test(e)

(English Common Name) mg or growth(a) mm(b) days

(c)

LAMIACEAE continued

Prunella vulgaris Perennials 0.58 - 1.2 L = D 0 5 (50%) [19] germination inhibited POST

(Self-heal) arable fields, grassy [4, 14, 19] [14] [4, 19] 7 (91%) [18] by darkness [18, 19]

areas, disturbed sites greater germination with larger [4, 22]

[16, 19] seeds [1]

no special treatments [4, 14, 22]

Stachys officinalis Perennials 14 - 18 L = D 7 (50%) no special treatments POST

(Hedge-nettle) grasslands, field 

margins [19]

[14, 19] [14] [19] [5, 14, 22] [5, 22]

MALVACEAE

Abutilon theophrasti Annuals 8.8 L = D 10 - 20 4 (84%) scarification [14] PRE & 

(Velvetleaf) fields, open habitats [16] [14] [14] [6, 10, 21] [10] no special treatments [5, 10, 21] POST [6, 22, 

28, 31]

Sida spinosa Annuals 3.8 L = D 10 - 20 scarification [14] PRE & 

(Prickly Sida) fields, roadsides [16] [14] [14] [6, 21] germination not affected by POST

irradiance [1] [6, 21, 28,

no special treatments [6, 21] 31]

PAPAVERACEAE

Papaver rhoeas Annuals 0.1 - 0.3 L = D 0 4 (50%) cold stratification & scarification POST [4]

(Poppy) fields, arable land, [4, 14, 19, [14] [4, 29] [19] [1, 19, 32]

disturbed sites [16, 19] 29] no special treatments [4, 14, 29]

POACEAE

Agrostis tenuis lawns, pastures [16] 0.07 L > D 20 10 (62%) germination inhibited by POST 

(Common Bentgrass) [14] [10] [10] [10] darkness [1, 17-19] [10]

no special treatments [10]

Alopecurus myosuroides Annuals 0.9 - 1.6 L = D 2 < 24 (30%) scarification [14] PRE & 

(Foxtail) Fields, open habitats [29, 34] [14] [29] [34] treat with 101 mg/L KNO3 [14] POST [28, 

[16] warm stratification [1] 34]

germination inhibited by darkness [1]

no special treatments [34]

Avena fatua Annuals 7 - 37.5 L = D [14] 10 - 20 3 (70%) scarification [7, 32] PRE & 

(Wild Oats) cultivated areas, [14, 30] L > D [6] [6, 10] [18] darkness inhibits germination [1] POST [6, 10, 

open habitats [16] cold stratification [1, 18] 28, 31]

no special treatments [6, 10, 14]
Page 10 of 30

FAMILY Lifespan Seed Photoperiod Planting Time to Special Toxicity

Species Botanical Name and Habitat Weight for germination Depth Germinate Treatments(d) Test(e)

(English Common Name) mg or growth(a) mm(b) days

(c)

POACEAE continued

Bromus tectorum Annuals 0.45 - 2.28 L = D 3 maturation period [1, 7, 32] PRE & 

(Downy Brome) fields, roadsides, [14, 29] [14] [29] germination inhibited by light [1] POST [28, 

arable land [16] no special treatments [14] 31]

Cynosurus cristatus Perennials 0.5 - 0.7 L = D 0 3 (50%) germination not affected by POST

(Dog's-tail Grass) fields, roadsides, [14, 19, [14] [29] [19] irradiance [19] [5]

open habitats [16, 19] 29] no special treatments [14, 29]

Digitaria sanguinalis Annuals 0.52 - 0.6 L = D 10 - 20 7 (75%) scarification, cold stratification, & PRE & 

(Crabgrass) fields, turf, [14, 30] [14] [21] 14 (94%) maturation [1, 7, 14, 32] POST [18, 

open habitats [16] [7] treat with 101 mg/L KNO3 [14] 25, 31]

germination inhibited by darkness [1]

no special treatments [21]

Echinochloa crusgalli Annuals 1.5 L = D [14] 10 - 20 scarification [7, 32] PRE & 

(Barnyard Grass) [16] [14] L > D [3] [7, 21] germination not affected by POST [3, 21, 

irradiance [1] 28, 31]

no special treatments [3, 14, 21]

Elymus canadensis Perennials 4 - 5 L = D 1 14 - 28 no special treatments POST

(Canada Wild Rye) riparian, disturbed sites 

[16]

[14, 30] [11] [11] [11] [2, 11] [2]

Festuca pratensis Perennials 1.53 - 2.2 L = D [14] 20 9 (74%) [10] no special treatments POST

(Fescue) fields, moist areas [16, 

19]

[16, 19] L > D [10] [10] 2 (50%) [19] [10, 19] [10]

Hordeum pusillum Annuals 3.28 warm stratification [1] PRE

(Little Barley) pastures, roadsides, [14] germination not affected by [31]

open habitats [16] irradiance [1]

Phleum pratense Perennials 0.45 L > D 0 - 10 2 (74%) [10] germination inhibited by darkness [19] POST

(Timothy) pastures, arable fields, [14, 19] [10, 14] [10, 19] 8 (50%) [19] germination not affected [10]

disturbed sites [16, 19] by irradiance [17]

no special treatments [10, 14, 17, 19]
Page 11 of 30

FAMILY Lifespan Seed Photoperiod Planting Time to Special Toxicity

Species Botanical Name and Habitat Weight for germination Depth Germinate Treatments(d) Test(e)

(English Common Name) mg or growth(a) mm(b) days

(c)

POLYGONACEAE

Polygonum convolvulus Annuals 5 - 8 L = D 0 - 2 cold stratification for 4 - 8 weeks PRE & 

(Black Bindweed) open habitats, [4, 14, 29] [20] [4, 29] [1, 2, 4, 20, 29] POST [1, 2, 

roadsides [16] germination not affected 

by irradiance [1]

20, 28, 31]

Polygonum lapathifolium Annuals 1.8 - 2.5 L > D 5 (94%) germination not affected PRE & 

(Pale Persicaria) moist soil [16] [14] [6] [18] by irradiance [1] POST [6]

germination inhibited by darkness [18]

cold stratification [1]

no special treatments [5]

Polygonum Annuals 3.6 - 7 2 cold stratification for 4 wks at 0 – 5 o

C PRE

pennsylvanicum Fields, open habitats [14, 29] [29] [1, 29]

(Pennsylvania [16] germination inhibited by darkness [1] [31]

Smartweed)

Polygonum periscaria Annuals 2.1 - 2.3 L > D 0 < 14 [13] scarification, cold stratification, GA POST 

(Smartweed) disturbed areas, [14, 19] [13] [19] 2 (50%) [19] treatment [14] [13]

arable land [16, 19] cold stratification, maturation [17-19]

germination inhibited by darkness [19]

no special treatments [13]

Rumex crispus Perennials 1.3 - 1.5 L = D 0 3 (50%) [19] germination inhibited by POST

(Curly Dock) arable fields, roadsides [4, 14, 19] [14, 33] [4, 19, 33] 6 (100%) [33] darkness [18, 19] [4, 33]

open areas [16, 19] maturation may be necessary [18]

no special treatments [4, 14, 33]

PRIMULACEAE

Anagallis arvensis Annuals 0.4 - 0.5 L = D 1 (50%) cold stratification, GA treatment POST

(Scarlett Pimpernel) arable fields, open [4, 14, 19] [14] [19] [1,14, 18, 19, 32] [2, 4]

areas, disturbed sites light required for germination [1]

[16, 19] no special treatments [2, 4]

RANUNCULACEAE

Ranunculus acris Perennials 1.5 - 2 L = D 1 41 - 56 no special treatments POST 

(Common Buttercup) arable fields, roadsides, [14, 19, [14] [29] [19, 29] [5, 14, 22, 24 - 26] [5, 22, 24-26]

Open areas [16, 19] 29]
Page 12 of 30

FAMILY Lifespan Seed Photoperiod Planting Time to Special Toxicity

Species Botanical Name and Habitat Weight for germination Depth Germinate Treatments(d) Test(e)

(English Common Name) mg or growth(a) mm(b) days

(c)

ROSACEAE

Geum urbanum Perennials 0.8 - 1.5 L = D 0 5 (50%) [19] germination inhibited by POST

(Yellow Avens) hedgerows, moist areas [14, 19] [14] [19] 16 (79%) [18] darkness [18, 19] [5, 22, 25, 

[16, 19] warm stratification [1] 26]

no special treatments [5, 14, 22, 25, 

26]

RUBIACEAE

Galium aparine Annuals 7 - 9 L = D 5 (50%) [19] cold stratification [1, 18, 19] PRE & 

(Cleavers) arable fields, moist [14, 19] [14] 6 (100%) [18] germination not affected by POST [6, 28]

areas disturbed sites irradiance [18, 19]

[16, 19] light inhibits germination [1]

no special treatments [6, 14]

Galium mollugo Perennials 7 L = D 2 no special treatments POST 

(Hedge Bedstraw) hedge banks, open [29] [14] [29] [5, 14, 22, 24, 26, 29] [5, 22, 24, 

areas [8] 26]

SCROPHULARIACEAE

Digitalis purpurea Biennials, Perennials 0.1 - 0.6 L = D 0 6 (50%) [19] germination inhibited by darkness POST 

(Foxglove) Hedge rows, open [4, 14, 19] [14] [4, 19] 8 (99%) [18] [1, 17-19] [4, 22 - 26]

areas [16, 19] no special treatments [4, 22-26]

Veronica persica Annuals 0.5 - 0.6 L = D 0 3 [19] germination inhibited by PRE & 

(Speedwell) arable fields, open [14, 19] [14] [19] 5 (96%) [18] darkness [18, 19] POST [28]

areas, disturbed cold stratification [18]

sites [16, 19] no special treatments [14]

(a) Proportion of light (L) and darkness (D) required to induce seed germination

(b) 0 millimeters (mm) indicates seeds sown on the soil surface or that seeds need light to germinate.

(c) The numbers provided represent the number of days in which a percentage of seeds germinated according to the provided reference in brackets, e.g., 3 days 

(50% germination) in reference [19]; the references appearing in brackets appear in paragraph (k) of this guideline.

(d) Duration of maturation and or stratification not always available. Except for cold treatment requirements, temperature conditions are not specified since in 

greenhouse testing there is limited temperature control. Most seeds will germinate under normal fluctuation of temperatures found in greenhouses.

(e) Indicates species was utilized in either a pre-emergence (PRE) and/or post-emergence (POST) plant toxicity test involving herbicides.
Page 13 of 30

(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) Condition. For a satisfactory test, healthy seeds should be used with 

reported germination rate for a lot number of 70% or better. Seeds should be 

examined and sorted to remove broken or damaged seeds.

(iv) Care and handling. Seeds should be stored in a dessicator 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 nonpesticide alternative for killing pathogens, fungi, and insects in soil media.

(4) Administration of test substance. The seeds are planted in the substrate and then 

the test substance is immediately (i.e. within the day) applied to the substrate surface. To 

prevent bias, seeds are impartially or randomly assigned to the pots (or flats or plots).

(i) Preparation of spray mixtures. 

(A) At test initiation, test substance is applied to surface soil immediately

after the seeds have been planted using a properly calibrated sprayer. 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 of water 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).

(B) For tests with industrial chemicals under TSCA, the preferred 

application methods are discussed in OCSPP 850.4230.

(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., EC25, 

EC05), test substance concentrations should immediately bracket the point 

estimate(s). The concentrations should be a geometric progression of 
Page 14 of 30

twofold (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 higher 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 EC25 values for seedling 

emergence and survival and the IC25 values for shoot length and biomass 

for that plant species. 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% effect or inhibition concentration (EC05, IC05, 

respectively). It is recommended that one or two additional test 

concentrations in the lower tail of the concentration-response curve of the 

most sensitive endpoint to insure bracketing of both the most sensitive 

EC25 or IC25 value and the most sensitive NOEC (or EC05 or IC05) value.

(B) Dosages should be expressed as mass of test substance per unit of land 

area treated. For pesticides, dosages should be expressed in units 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. Every test includes controls consisting of the same support medium, test

conditions, procedures, and seed source and lot, except that no test substance is added. 

Vehicle (solvent) controls are also included if a solvent is used. However for pesticides, 

because a TEP is typically tested, solvents are generally not necessary. 

(i) Contamination. If the negative control is contaminated with the test 

substance, the study is not acceptable and should be repeated.

(ii) Seed germination standards. A test is not acceptable if less than 70% of 

control plants emerged.

(iii) Control survival. A test is not acceptable if at test termination less than 90% 

of those control plants that emerged survived.

(6) Number of test organisms and replicates. 

(i) For each species, the minimum number of test organisms is 40 seeds per dose 

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

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 
Page 15 of 30

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.

(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 

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

on commercial ground sprayers. The application equipment used in greenhouse 

tests (such as a spray chamber) should provide uniform coverage and should be 
Page 16 of 30

calibrated at spray volumes (to the extent practical) that are representative of field 

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

the recommendation on the label.

(iii) Facilities. Seedling emergence tests should be conducted under controlled 

conditions in growth chambers, greenhouses, 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 or chamber 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. 

(A) For tests with pesticides, seeds should be planted in a natural soil 

(free of pesticide contamination) or synthetic soil. Growth chamber or 

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 they do not exceed 3% organic matter. 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 (mm)) 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. The use of 100% 

acid washed sand or hydroponic methods (glass beads, rockwool, etc.) is 

not recommended for testing with pesticides, and test methods and 

protocols for hydroponic tests with pesticides should be submitted to the 

OPP for review prior to test initiation. Support media should be consistent 

within test species.

(B) For tests with industrial chemicals under TSCA, seeds should be 

planted in an artificial matrix consisting of quartz sand or glass beads (see 

OCSPP 850.4230).
Page 17 of 30

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

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

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

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

Table 2 of this guideline).

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

or growth chamber. Air temperature during the day should be 25 ± 6 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 soil surface, is desirable, on a photoperiod of 16 hour light and 8 hour 

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 is used at the 

first watering after the test substance has been applied to stimulate seed 

germination, and 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 through the soil during watering.

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

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

Hoagland’s 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). If one or more 

dosing solutions batches are prepared, chemical analysis should be conducted at a 

minimum at the start and end of application of each batch. The total volume of 

dosing solution used, and total area treated for a given treatment level, and the 

dosing batch used 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) should be 

determined, including soil type and texture, pH, particle size distribution, and 

organic matter content.
Page 18 of 30

(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, 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 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 daily 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) Controls are observed daily for number of seedlings emerged until 

50% of seedlings have emerged. Number of seedlings emerged, number 

of surviving and dead seedlings, and visual symptoms are observed and 

recorded for treatment and controls at day 0, 7 and 14 post-emergence 

(and day 21 post-emergence if test is extended (see paragraph (e)(2) of this 

guideline)) of 50% of control plants. At test termination, shoot length and 

shoot biomass of surviving seedlings is determined and recorded. 

Seedling condition is determined using a standard visual phytotoxicity 

rating scale. Shoot lengths are recorded for each individual surviving 
Page 19 of 30

plant. After shoot lengths are measured, plants are dried (constant weight 

at 70 oC) and the total plant biomass (i.e. dry weight) in a replicate is 

measured and recorded. Biomass measurements should be made for each 

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 signs (e.g., chlorosis, necrosis, pigmentation, leaf curling, and 

wilting), and formative (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 recorded.

(f) Treatment of results―

(1) Summary statistics―

(i) Environmental conditions. Descriptive statistics (mean, standard deviation, 

and range) are calculated by treatment level for air temperature, humidity, and 

light intensity data. 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 length. Calculate and plot the average shoot length and standard 

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

length and standard deviation. 

(iv) Shoot weight. 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) Emergence and survival. Calculate and plot the percent seedling emergence 

(cumulative number emerged divided by number of seeds planted) and percent 

survival (number of surviving seedlings divided by the cumulative number of 

emerged seedlings) for each replicate and the treatment level mean and standard 
Page 20 of 30

deviation for percent emergence and percent survival. For each treatment level 

calculate mean percent survival as a percent of the control mean percent survival 

and mean percent emergence as a percentage of the control mean percent 

emergence.

(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 length and shoot weight 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 length or shoot weight); and

X = the treatment mean response value (shoot length or shoot weight, 

respectively). Stimulation is reported as negative percent inhibition.

(3) Limit test―

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

length, shoot weight, and emergence IC25 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 response 

measure. For a comparison of sample means, the difference defining the EC25 or 

IC25, compared to controls, is operationally defined as a 25% 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 25% 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., 

emergence, survival shoot length, and shoot weight) 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 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).
Page 21 of 30

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

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., emergence, shoot length, 

shoot weight, survival); 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 (emergence, survival, 

shoot length, and shoot weight), 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 if either the effect or 

inhibition level for one or more response measures (i.e., emergence, 

survival shoot length, and shoot weight) 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) and/or the NOEC is

less than the limit concentration.

(B) Multiple-dose testing may be waived for a 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 

(seedling emergence, survival, shoot length, and shoot biomass).

(4) Multiple-dose definitive test―

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

(A) Shoot length and shoot weight. For dose-response tests the IC25

value (standard error and 95% confidence interval) is calculated for each

of shoot length and shoot weight (see OCSPP 850.4000 and references in 

paragraphs (j)(2) and (j)(8) 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 
Page 22 of 30

result in the determination of a definitive IC25 value for a response 

measure, test and document that the IC25 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 is captured too many additional 

treatments would be needed to capture the full response relationship for 

the other less sensitive response measure(s). Methods, assumptions, and 

results of the statistical approaches used should be recorded.

(B) Percent emergence and percent survival. The EC25 value (standard 

error and 95% confidence interval) is calculated for each of percent 

emergence and 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 paragraph (f)(3) of this guideline and 

OCSPP 850.4000). 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 for the other less 

sensitive response measure(s). Methods, assumptions, and results of the 

statistical approaches used should be recorded. 

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

survival, shoot length, 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 length or shoot weight) or the dose 

at which 5% of the population is effected (i.e., the EC05 for emergence and 

survival) is estimated and used in place of the NOEC. The standard error and 

95% confidence interval should also be calculated for IC05 and EC05 values. 

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

recorded.

(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 conditions will greatly 

increase the likelihood that the completed test will be acceptable or valid.
Page 23 of 30

Table 3.—Summary of Test Conditions for Seedling Emergence and Seedling 

Growth Test

Test type Seedling emergence and seedling growth

Test duration 14 days post-emergence of 50% of control plants (extended 

to 21 days post-emergence if phytotoxic symptoms initially 

show between day 7 and 14 post-emergence)

Substrate Natural or synthetic soil with at most 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 soil surface

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

2 of this guideline)

Watering For greenhouse and growth chamber, the initial watering 

event is by top watering and bottom watering is used 

throughout the remainder of the study. In field plots top 

watering is used.

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

pots are typical. Flats are also encouraged

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

Number of replicates per test treatment 4 (minimum) 

Number of seeds per test treatment 40 (minimum)

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

plus appropriate controls

Test substance application method Applied to soil surface immediately after planting seeds (i.e.

within the day)

Measures of effect or measurement 

endpoints

EC25 and NOEC (or EC05) for each of percent emergence 

and percent survival

IC25 and NOEC (or IC05) for each of shoot length and shoot 

weight

(h) Test validity elements. This test would be considered to be unacceptable or invalid if one or 

more of the conditions in Table 4 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 4 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 

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

resulting effects on test endpoints noted and discussed. 
Page 24 of 30

Table 4.—Test Validity Elements for the Seedling Emergence and Seedling Growth 

Test

1. Mean control seedling 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 test species, all seeds used in the test are from the same source and lot number.

4. All test chambers (including soil medium) used for a particular species are identical.

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

the test.

6. The lowest test concentration level was lower than the most sensitive effect EC25 and IC25.

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.

(iv) If a vehicle (e.g., solvent) is used to prepare stock or test substance provide: 

the name and source of the vehicle, the nominal concentration(s) of the vehicle in 

stock solutions or mixtures, and the vehicle dose(s) used in the treatments.
Page 25 of 30

(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 before use in 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 seeds in each plot.

(iii) For greenhouse or growth chambers the description of pots/flats: type, 

material, and dimensions.

(iv) Number of seeds 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, pH, percent 

organic matter, type and amounts of soil amendments.

(vii) Volume of soil per pot or test container.

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

seeds to test plots, pots, and/or flats.

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

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

solution applied. 

(x) Date of test substance application and test duration.

(xi) Culture practices during the test such as cultivation, pest control, and 

irrigation practices (type and watering schedule or regime).

(xii) The photoperiod and light source.
Page 26 of 30

(xiii) 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.

(xiv) 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 emerged 

seedlings, survival, and shoot length and weight, 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 seeds exposed at test initiation, the number of emerged 

seedlings, the number of surviving and number of dead emerged seedlings at each 

observation time, and the shoot length for each individual plant and biomass at 

test termination (provide the raw data).

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

emerged seedlings, number of surviving and number of dead emerged seedlings, 

plant length for each individual plant and total plant biomass (provide the raw 

data).

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

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

test termination.

(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 percent emerged, percent survival, 

shoot length 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.
Page 27 of 30

(x) For the definitive study and for those effect measures (emerged, survival, 

shoot length 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, provide table of EC25 values for percent emergence and 

percent survival and IC25 values for plant length and plant biomass.

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

of effect (percent emergence, percent survival, plant length, and plant biomass). 

The EC05 or IC05 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 EC25 and IC25 values, fitting the dose-response 

model, and the basis for the choice of method. Provide results of any goodnessof-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 EC05 or 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 EC05 and 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.

(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. 2006. New Test 

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

Vigour Test, 21pp.; 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 28 of 30

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

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

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

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

Evaluation Procedure, Nontarget Plants: Seed Germination/Seedling Emergence - Tiers 1 

and 2. Office of Pesticides Programs, Washington, D.C. EPA 540/9-86-132, June 1986.

(8) 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.

(k) References to Table 2. The references in this section are cited in Table 2 in paragraph 

(e)(3)(iii) of this guideline. (Table 2 and the references are from Annex 3 in references given in 

paragraph (j)(5) of this guideline.)

(1) Baskin, C.C. & Baskin, J.M. 1998. Seeds. Academic Press, Toronto

(2) Blackburn, L.G. & Boutin, C. 2003. Subtle effects of herbicide use in the context of 

genetically modified crops: a case study with glyphosate (Round-Up). Ecotoxicology, 

12:271-285.

(3) Boutin, C., Lee, H-B., Peart, T., Batchelor, P.S., & Maguire, R.J. 2000. Effects of 

the sulfonylurea herbicide metsulfuron methyl on growth and reproduction of five 

wetland and terrestrial plant species. Environmental Toxicology & Chemistry, 

19(10):2532-2541.

(4) Boutin, C., Elmegaard, N., & Kjaer, C. 2004. Toxicity testing of fifteen non-crop 

plant species with six herbicides in a greenhouse experiment: implications for risk 

assessment. Ecotoxicology, 13:349-369.

(5) Breeze, V., Thomas, G., & Butler, R. 1992. Use of a model and toxicity data to 

predict the risks to some wild plant species from drift of four herbicides. Annals of 

Applied Biology, 121:669-677.

(6) Brown, R.A., & Farmer, D. 1991. Track-sprayer and glasshouse techniques for 

terrestrial plant bioassays with pesticides. In: Plants for toxicity assessment: 2nd 

volume. ASTM STP 1115, J.W. Gorsuch, W.R. Lower, W.Wang, & M.A. Lewis, eds. 

American Society for Testing & Materials, Philadelphia. pp 197 – 208.

(7) Buhler, D.D. & Hoffman, M.L. 1999. Anderson’s guide to practical methods of 

propagating weeds and other plants. Weed Science Society of America, Lawrence, K.

(8) Clapham, A.R., Tutin, T.G., & Warburg, E.F. 1981. Excursion flora of the British 

Isles, 3rd ed. Cambridge University Press, Cambridge

(9) Clay, P.A. & Griffin, J.L. 2000. Weed seed production and seedling emergence 

response to late-season glyphosate applications. Weed Science, 48:481-486.
Page 29 of 30

(10) Cole, J.F.H. & Canning, L. 1993. Rationale for the choice of species in the 

regulatory testing of the effects of pesticides on terrestrial non-target plants. BCPC –

Weeds. pp. 151 – 156.

(11) Fiely, M. (Ernst Conservation Seeds). 2004. Personal communication. 

(www.ernstseed.com)

(12) Fletcher, J.S., Johnson, F.L., & McFarlane, J.C. 1990. Influence of greenhouse 

versus filed testing and taxonomic differences on plant sensitivity to chemical treatment. 

Environmental Toxicology & Chemistry, 9:769-776.

(13) Fletcher, J.S., Pfleeger, T.G., Ratsch, H.C., & Hayes, R. 1996. Potential impact of 

low levels of chlorsulfuron and other herbicides on growth and yield of nontarget plants. 

Environmental Toxicology & Chemistry, 15(7):1189-1196.

(14) Flynn, S., Turner, R.M., and Dickie, J.B. 2004. Seed Information Database (release 

6.0, Oct 2004) Royal Botanic Gardens, Kew http://www.rbgkew.org.uk/data/sid

(15) Franzaring, J., Kempenaar, C., & van der Eerden, L.J.M. 2001. Effects of vapors of 

chlorpropham and ethofumesate on wild plant species. Environmental Pollution, 114:21-

28.

(16) Gleason, H.A. & Cronquist, A. 1991. Manual of vascular plants of northeastern 

United States and adjacent Canada, 2nd ed. New York Botanical Garden, Bronx, NY

(17) Grime, J.P. 1981. The role of seed dormancy in vegetation dynamics. Annals of 

Applied Biology, 98:555-558.

(18) Grime, J.P., Mason, G., Curtis, A.V., Rodman, J., Band, S.R., Mowforth, M.A.G., 

Neal, A.M., & Shaw, S. 1981. A comparative study of germination characteristics in a 

local flora. Journal of Ecology, 69:1017-1059.

(19) Grime, J.P., Hodgson, J.G., & Hunt, R. 1988. Comparative plant ecology: a 

functional approach to common British species. Unwin Hyman Ltd., London

(20) Kjaer, C. 1994. Sublethal effects of chlorsulfuron on black bindweed (Polygonum 

convolvulus L.). Weed Research, 34:453-459.

(21) Klingaman, T.E., King, C.A., & Oliver, L.R. 1992. Effect of application rate, weed 

species, and weed stage of growth on imazethapyr activity. Weed Science, 40:227-232.

(22) Marrs, R.H., Williams, C.T., Frost, A.J., & Plant, R.A. 1989. Assessment of the 

effects of herbicide spray drift on a range of plant species of conservation interest. 

Environmental Pollution, 59:71-86.

(23) Marrs, R.H., Frost, A.J., & Plant, R.A. 1991. Effects of herbicide spray drift on 

selected species of nature conservation interest: the effects of plant age and surrounding 

vegetation structure. Environmental Pollution, 69:223-235.
Page 30 of 30

(24) Marrs, R.H., Frost, A.J., & Plant, R.A. 1991. Effects of mecoprop drift on some 

plant species of conservation interest when grown in standardized mixtures in 

microcosms. Environmental Pollution, 73:25-42.

(25) Marrs, R.H., Frost, A.J., Plant, R.A., & Lunnis, P. 1993. Determination of buffer 

zones to protect seedlings of non-target plants from the effects of glyphosate spray drift. 

Agriculture, Ecosystems, & Environment, 45:283-293.

(26) Marrs, R.H. & Frost, A.J. 1997. A microcosm approach to detection of the effects 

of herbicide spray drift in plant communities. Journal of Environmental Management, 

50:369-388.

(27) Marshall, E.J.P. & Bernie, J.E. 1985. Herbicide effects on field margin flora. 

BCPC – Weeds. pp. 1021-1028.

(28) McKelvey, R.A., Wright, J.P., & Honegger, J.L. 2002. A comparison of crop and 

non-crop plants as sensitive species for regulatory testing. Pest Management Science, 

58:1161-1174.

(29) Morton, S. (Herbiseed). 2004. Personal communication. (www.herbiseed.com)

(30) USDA, NRCS. 2004. The Plants Database, version 3.5. (http://plants.usda.gov). 

National Plant Data Centre, Baton Rouge, LA 70874-4490 USA

(31) USEPA. 1999. One-Liner Database. [U.S. E.P.A./Office of Pesticides Programs, 

Environmental Fate and Effects Division, Environmental Epidemiology Branch].

(32) Webster, R.H. 1979. Technical Report No. 56: Growing weeds from seeds and 

other propagules for experimental purposes. Agricultural Research Council Weed 

Research Organization, Oxford.

(33) White, A. & Boutin, C. (National Wildlife Research Centre, Environment Canada). 

2004. Personal communication. 

(34) Zwerger, P. & Pestemer, W. 2000. Testing the phytotoxic effects of herbicides on 

higher terrestrial non-target plants using a plant life-cycle test. Z. PflKrankh. PflSchutz, 

Sonderh., 17:711-718.