United States Environmental Protection Agency Office of Chemical Safety and Pollution Prevention (7101) EPA 712

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-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 situati ons. 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 e lectronically, 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**

**O CSPP**  **850. 41**  **0 0**  **: Seedling emergence and seedling grow** **th**

**.** (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 o f chemical substances and mixtures (“test chemicals” or “test substance s”) subject to environmental effects test regulations. The objective of the seedling emergence test is to measure the effect of test substance s 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) **Defini tions.**  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 phy totoxic effects are observed and evaluated. The test is designed to determine the quantity of test substance required to cause a 25 percent (25%) reduction (EC 25 or IC 25 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 b e used. It may

b e performed in a growth chamber, greenhouse, or in small field plots. Anatural or sy nthetic 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 range finding 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 b e less than that used in definitive testing. In addition, the ty pes 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 concentration

response curve and NOEC (and lowest observed effect concentration (LOEC)); the EC 25 value (with 95% confidence interval and standard error) for seedling emergence, and for survival; and the IC 25 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., IC 05 to IC 90) 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 f or the most sensitive endpoint to ensure bracketing of bothPage **3** of **30**

the most sensitive NOEC (or EC 05 or IC 05 ) and most sensitive EC25 or IC 25 value. For a satisfactory

test, the lowest treatment concentration is below the EC 25 (seedling emergence and survival) and IC 25 (shoot length and biomass) values for all measures of effect. Asummary

o f test conditions is provided in Table 3 and validity elements for an acceptable definitive test are listed in Table 4. (5) **Lim it**  **test.** In some situations, it is only necessary

to ascertain that for a given plant species the seedling emergence and survival EC 25 values and the shoot length and shoot biomass IC 25 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 b e 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. , EC 25 and IC 25 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 o f 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 s ame 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** 

. F or pesticides the substance to be tested is the ty pical end

-use prod uct (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 substance s, 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 adj uvant 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 ty pe 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 phy totoxic sy mptoms start to appear between day 7and 14 post

emergence, 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 ty

pe 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. S eeds 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 soy

bean ( Glycine max). (2 ) Monocotyl edoneae: 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 fo**  **r use in the seedling emergence and**  **seedling gro wth** **test**

**Family Species**  **Common name** Dicots Solanaceae Lycopersicon esculentum or Solanum lycopersicum To mato
Cucurbitaceae Cucumis sativus Cucumber Asteraceae Lactuca sativa Lettuce Fabaceae 1 Glycine max So ybean 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 (soyb ean, 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 gro** **wth 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) day** **s (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) day** **s (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 Wo rt) 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) day** **s (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) day** **s (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 KNO 3 [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 & (W ild 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) day** **s (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 KNO 3 [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 Wi ld 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) day** **s (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) day** **s (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 green houses. (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

s eeds should be used with reported germination rate for a lot number of 70% or better. S eeds 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. P esticide 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 hy pochlorite 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 sy nergistic 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 subs** **tance .**  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**  s**pray 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 leve ls.**  (A) For a given plant species, five treatment levels are tested at a minimum. Arange- 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. , EC 25 , EC 05 ), test substance concentrations should immediately bracket the point estimate(s). The concentrations should be a geometric progression ofPage **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

o f 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 EC 25 values for seedling emergence and survival and the IC 25 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 (EC 05 , IC 05 , 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 EC 25 or IC 25 value and the most sensitive NOEC (or EC 05 or IC 05 ) value. (B) Dosages should be expressed as mass of test substance per unit of land area treated. F or 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 sy nergistic 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 ty

pically 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 thePage **15** of **30** number of plants per pot is left to the discretion of the laboratory conducting the test, avoid overcrowding. Atest 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, soy bean, 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 substance s 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 ty pes, 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 bePage **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. Polyethyl ene plastic pots or flats that have not been previously used are acceptable test containers, provided they are free of toxic materials. D o not use clay or peat containers. C ontainers 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. C ontainers 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 sy nthetic soil. Growth chamber or greenhouse tests are performed using a sterilized standardized soil that consists primarily

o f sandy loam, loamy

s and, loamy

clay, or clay

loam s oil that contains up to 3% organic matter (up to 1.5% organic carbon). Commercial potting soil or sy nthetic soil mixes may

b e used as the soil medium provided they

do not exceed 3% organic matter. Clay s oils 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 b e 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 (gy psum, ammonium sulfate, or sulfuric acid). Aslow

-release fertilizer may b e 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) **T emperature.**  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) **Light ing**  **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 b e used to lengthen short

- day p eriods 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 b e 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 b e 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 analy tical methods used to measure the amount of test substance in a sample are validated before beginning the test, as described in O CSPP 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 ty pe 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. S ource 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. Aphotosy nthetically 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 o f 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. P est 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 f or 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 phy totoxicity rating scale. Shoot lengths are recorded for each individual survivingPage **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. B iomass measurements should be made for each replicate not each individual plant. (B) Seedling condition is determined using a standard morphological phy totoxicity rating scale. Observations on morphological sy mptoms of phy totoxicity should include all variations, either inhibitory or stimulatory, between the treated and the untreated organisms. Such variations may b e phy totoxic 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 biomas s 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 s eeds 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 standardPage **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 sy mptoms of plant injury

should be summarized in tabular form by time of observation, treatment, and replicate. Definition of any index values used for morphological sy mptoms, 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 X C 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) **EC 25**  **and IC**  **25 values.**  To ascertain that the survival EC 25 value and the shoot length, shoot weight, and emergence IC 25 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 EC 25 or IC 25 , compared to controls, is operationally defined as a 25% reduction from the control sample mean (Equation 2). The null hy pothesis (H0) stated in terms of true population parameters is that the difference of the ‘limit’ treatment mean response (μ limit) from the control mean response (μ control) is greater than or equal to a 25% reduction, compared to the control (i.e ., H0 : μ control - μ limit > δ 0). The alternative hy

pothesis (HA) is that this difference is less than a 25% reduction, compared to the control ( i.e ., HA : μ control - μ limit < δ 0). An example of a parametric two

- sample comparison test of this is the Student’s t

-test. If the null hy pothesis 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., IC 25 > limit dose). If the null hy

pothesis i s 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., IC 25 < limit dose).Page **21** of **30** 

##    

100 0 

### p x control 

###    **Equation 2**
where: δ 0 = 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 EC 25 and IC 25 . (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 O CSPP 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 hy pothesis is not rejected) and/or the NOEC is less than the limit concentration. (B) Multiple

- dose testing may b e 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**  **IC 25**  **and EC 25**  **values**

― (A) **Shoot length**  **and shoot**  **weight.** For dose

-response tests the IC 25 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 IC 25 the model parameters (e.g. , slope) and their uncertainty e stimates (e.g. , standard error) should be recorded. Where the dose

-response range tested does notPage **22** of **30** result in the determination of a definitive IC 25 value for a response measure, test and document that the IC 25 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 EC 25 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 EC 25 the model parameters (e.g. , slope) and their uncertainty e stimates (e.g., standard error) should be recorded. Where the dose

-response range tested does not result in the determination of a definitive EC 25 value, test and document that the EC 25 value is above the highest treatment level tested (see statistical guidance in paragraph (f)(3) o f 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 IC 05 value for shoot length or shoot weight) or the dose at which 5% of the population is effected (i.e ., the EC 05 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 IC 05 and EC 05 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**  **Gro wth** **Test** Test type Seedling emergence and seedling growth Test duration 14 days post

- em ergence of 50% of control plants (extended to 21 days post

- em ergence if phytotoxic symptoms initially show between day 7 and 14 post

- em ergence) 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 Te mp erature 25/20 o C (daytime/nighttime) ± 6 o C Relative humidity 70% (daytime)± 15% Light intensity 350 ± 50 μmol/m 2/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 us ed. 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 EC 25 and NOEC (or EC 05 ) for each of percent emergence and percent survival IC 25 and NOEC (or IC 05 ) 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. B efore 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 seedl ing 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 te st concentration level was lower than the most sensitive effect EC 25 and IC 25 . 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 o f 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

o f the seed: source, name of supplier, seed ye ar 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 ty pe 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: ty pe, 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 ty pe designation, pH, percent organic matter, typ e 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 ty pe, equipment design, method for calibrating the application equipment, dose levels, volume of dosing solution applied. (x) Date of test sub stance 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 o f environmental monitoring performed during the definitive or limit study f or 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

o f 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). Adescription and count of plants with visual phy totoxic 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 phy totoxicity 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 stati stical 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 IC 25 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 EC 05 or IC 05 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 EC 25 and IC 25 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 EC 05 or IC 05 value is used in place of a NOEC provide a description of statistical method(s) used for point estimates, including software package, for determining EC 05 and IC 05 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) Br uce, 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. 2 006. 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. B oth guidelines adopted on 19 July 2 006 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. P esticide Assessment Guidelines, Subdivision J Hazard Evaluation: Nontarget Plants. O ffice 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. 2 003. 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. 2 000. 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. 2 004. 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. 1 992. 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. 1 991. Track

- sprayer and glasshouse techniques for terrestrial plant bioassays with pesticides. In: Plants for toxicity assessment: 2nd volume. A STM 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. 1 999. 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. 1 981. Excursion flora of the British Isles, 3rd ed. Cambridge University Press, Cambridge (9) Clay, P.A. & Griffin, J.L

. 2 000. Weed seed production and seedling emergence response to late

- season gly phosate applications. Weed Science, 48:481

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(10) Cole, J.F.H. & Canning, L. 1 993. Rationale for the choice of species in the regulatory

testing of the effects of pesticides on terrestrial non-target plants. B CPC

– Weeds. pp. 151

– 156. (11) Fiely, M. (Ernst Conservation Seeds). 2 004. P ersonal communication. (www.ernstseed.com) (12) Fletcher, J.S., Johnson, F.L., & McFarlane, J.C. 1 990. 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. 1 996. P otential 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. 1 991. Manual of vascular plants of northeastern United States and adjacent Canada, 2nd ed. New York Botanical Garden, Bronx, NY (17) Grime, J.P. 1 981. The role of seed dormancy in vegetation dy

namics. Annals of Applied Biology, 9 8:555

- 558. (18) Grime, J.P., Mason, G., Curtis, A.V., Rodman, J., Band, S.R., Mowforth, M.A.G., Neal, A.M., & Shaw, S. 1 981. Acomparative study o f germination characteristics in a local flora. Journal of Ecology, 69:1017

- 1059. (19) Grime, J.P., Hodgson, J.G., & Hunt, R. 1 988. C omparative plant ecology : a functional approach to common British species. Unwin Hy man Ltd., London (20) Kjaer, C. 1 994. 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. 1 989. 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. 1 991. 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

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(24) Marrs, R.H., Frost, A.J., & Plant, R.A. 1 991. 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. 1 993. D etermination of buffer zones to protect seedlings of non-target plants from the eff ects of glyphosate spray

drift. Agriculture, Ecosystems, & Environment, 45:283

-293. (26) Marrs, R.H. & Frost, A.J. 1 997. Amicrocosm 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. 1 985. Herbicide effects on field margin flora. BCPC

– Weeds. pp. 1021

- 1028. (28) McKelvey, R.A., Wright, J.P., & Honegger, J.L

. 2 002. Acomparison of crop and non- crop plants as sensitive species for regulatory testing. P est 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. 1 999. One-Liner Database. [U.S. E.P.A./Office of Pesticides Programs, Environmental Fate and Effects Division, Environmental Epidemiology Branch]. (32) Webster, R.H. 1 979. 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. 2 000. Testing the phy totoxic effects of herbicides on higher terrestrial non

-target plants using a plant life

- cycle test. Z. P flKrankh. PflSchutz, Sonderh., 17:711

- 718.