&EPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention
(7101)
EPA712-C-012
January 2012
Ecological Effects
Test Guidelines
OCSPP 850.4100:
Seedling Emergence
and Seedling Growth
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NOTICE
This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention
(OCSPP) for use in testing pesticides and chemical substances to develop data for
submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601,
et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et
seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic
Substances (OPPTS). To distinguish these guidelines from guidelines issued by other
organizations, the numbering convention adopted in 1994 specifically included OPPTS as
part of the guideline's number. Any test guidelines developed after April 22, 2010 will use
the new acronym (OCSPP) in their title.
The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the
test, and is also used by EPA, the public, and the companies that are subject to data
submission requirements under TSCA, FIFRA, and/or the FFDCA. As a guidance
document, these guidelines are not binding on either EPA or any outside parties, and the
EPA may depart from the guidelines where circumstances warrant and without prior notice.
At places in this guidance, the Agency uses the word "should." In this guidance, the use of
"should" with regard to an action means that the action is recommended rather than
mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline, but EPA recognizes that departures
may be appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the Agency will assess them for
appropriateness on a case-by-case basis.
For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.qov/ocspp and select "Test Methods &
Guidelines" on the left side navigation menu. You may also access the guidelines in
http://www.requlations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-
0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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OCSPP 850.4100: 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.
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(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 range-
finding test, the test organisms are exposed to a series of widely spaced concentrations of
the test substance (e.g., 1, 10, 100 pounds per acre (Ibs/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 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 concentration-
response 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., ICos to ICgo) 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 2 of30
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the most sensitive NOEC (or ECos or ICos) and most sensitive £€25 or 1C25 value. For a
satisfactory test, the lowest treatment concentration is below the £€25 (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 £€25 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.., £€25 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.
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(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 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 type of testing. A listing of crop and non-crop plant taxa
that have been used in toxicity tests can be found in Tablel 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)(7j through (e)(3)(i)(A)(%) 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.
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Table 1.—Crop species and families for use in the seedling emergence and
seedling growth test
Family
Species
Common name
Dicots
Solanaceae
Cucurbitaceae
Asteraceae
Fabaceae1
Brassicaceae
Apiaceae
Chenopodiaceae
Asteraceae
Fabaceae
Brassicaceae
Brassicaceae
Fabaceae
Malvaceae
Polygonaceae
Monocots Poaceae
Poaceae
Poaceae
Liliaceae
Lycopersicon esculentum or Solarium
lycopersicum
Cucumis sativus
Lactuca sativa
Glycine max
Brassica oleracea
Caucus carota
Beta vulgaris
Helianthus annuus
Pisum sativum
Brassica rapa
Brassica napus
Phaseolus vulgaris
Gossypium spp.
Fagopyrum esculentum
Avena sativa
Lolium perenne
Zea mays
Allium cepa
Tomato
Cucumber
Lettuce
Soybean
Cabbage
Carrot
Sugar beet or table beet
Sunflower
Pea
Field mustard, Canola
Turnip, Rape
Garden bean
Cotton
Buckwheat
Oat
Perennial ryegrass
Corn
Onion
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.
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Table 2.—Table of non-crop species for use in the seedling emergence and seedling growth test
FAMILY
Species Botanical Name
(English Common Name)
APIACEAE
Torilisjaponica
(Japanese Hedge-parsley)
ASTERACEAE
Bellis perennis
(English Daisy)
Centaurea cyanus
(Cornflower)
Centaurea nigra
(Black Knapweed)
Inula helenium
(Elecampane)
Leontodon hispidus
(Big Hawkbit)
Rudbeckia hirta
(Black-eyed Susan)
Solidago canadensis
(Canada Goldenrod)
Lifespan
and Habitat
Annuals, Biennials
disturbed areas,
hedgerows, pastures
[16, 19]
Perennials
grassland, arable
fields, turf [16, 19]
Annuals
fields, roadsides,
open habitats [16]
Perennials
fields, roadsides,
open habitats [16, 19]
Perennials
moist, disturbed sites
[16]
Perennials
fields, roadsides,
disturbed areas [16, 19]
Biennials, Perennials
Disturbed [16]
Perennials
pasture, open areas
[16]
Seed Photoperiod Planting
Weight for germination Depth
mg or growth'3' mm(b)
1.7
[14,
0.09
[4,
4.1
[4,
2.4
[14,
1 -
-1.9
19]
-0.17
19]
-4.9
14]
-2.6
19]
1.3
L=D
[14]
L=D
[14]
L=D
[14]
L=D
[14]
[4, 14, 29]
0.85
[14,
0
[4,
0.06
[4,
-1.2
19]
3
14]
-0.08
14]
L=D
[14]
L=D
[14]
L=D
[11]
0
[1,19]
0
[4]
0-3
[2, 4, 14]
0
[19]
0
[4, 29]
0
[19]
0
[4, 33]
0
[4]
Time to
Germinate
days(c)
5 (50%)
[19]
3 (50%) [19]
11 (100%) [18]
14-21 (100%)
[14]
3 (50%) [19]
4 (97%) [18]
4 (50%) [19]
7 (80%) [18]
< 10 (100%)
[33]
14-21
[11]
Special
Treatments'111
cold stratification [7, 14, 18, 19]
maturation may be necessary [19]
germination inhibited
by darkness [1, 19]
no special treatments [5]
germination not affected by irradiance
[18, 19]
no special treatments [4, 14]
no special treatments [2, 4]
maturation may be necessary [18, 19]
germination inhibited by darkness [19]
no special treatments [5, 14, 26]
no special treatments [4]
germination inhibited by darkness
[17, 18, 19]
no special treatments [5, 23]
no special treatments
[4, 14, 33]
mix with equal part sand and
soak in SOOppm GA for 24 hrs [1 1 ]
no special treatments [4]
Toxicity
Test'6'
POST
[5]
POST
[4]
POST
[2,4]
POST
[5, 22, 26]
POST
[4]
POST
[5, 22, 23]
POST
[4, 33]
POST
[4]
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FAMILY
Species Botanical Name
(English Common Name)
ASTERACEAE continued
Xanthium pensylvanicum
(Common Cocklebur)
Xanthium spinosum
(Spiny Cocklebur)
Xanthium strumarium
(Italian Cocklebur)
BRASSICACEAE
Cardamine pratensis
(Cuckoo Flower)
CARYOPHYLLACEAE
Lychnis flos-cuculi
(Ragged Robin)
CHENOPODIACEAE
Chenopodium album
(Lamb's Quarters)
CLUSIACEAE
Hypericum perforatum
(Common St. John's Wort)
CONVOLVULACEAE
Ipomoea hederacea
(Purple Morning Glory)
Lifespan
and Habitat
Annuals
fields, open habitats [16]
Annuals
open habitats [16]
Annuals
fields, open habitats [16]
Perennials
fields, roadsides, turf
[16, 19]
Perennials
[16]
Annuals
field margins,
disturbed areas [16, 19]
Perennials
fields, arable land,
open habitats [16, 19]
Annuals
roadsides, open
habitats, corn fields [16]
Seed
Weight
mg
25-61
[14, 29]
200
[14]
67.4
[14]
0.6
[14, 19]
0.21
[14]
0.7-1.5
[14, 19,
34]
0.1 -0.23
[14, 19]
28.2
[14]
Photoperiod
for germination
or growth(a)
L = D[14]
L > D [6]
L=D
[14]
L=D
[14]
L=D
[14]
L=D
[14]
L=D
[14]
L>D
[6, 10]
Planting
Depth
mm(b)
0[1]
5 [29]
10
[6]
10-20
[6,21]
0
[19]
0
[1,19]
0
[1,19]
10-20
[6, 10,21]
Time to
Germinate
days(c)
5 (50%) [19]
15 (98%) [18]
< 14 (100%)
[14, 25]
2 (50%)
[19]
3 [19]
11 (90%) [18]
4(100%)
[10]
Special
Treatments™
germination may be inhibited by
darkness [1]
soak in warm water for 12 hrs [29]
scarification [14]
no special treatments [6]
no special treatments
[6, 14,21]
germination inhibited by darkness
[18, 19]
no special treatments [5, 14, 22]
maturation may be necessary [18]
no special treatments [5, 14, 15, 22-
26]
treatment differs depending on
seed color [19]
dry storage dormancy [19]
germination inhibited by darkness
[1, 18, 19]
cold stratification [18]
no special treatments [14, 34]
germination inhibited
by darkness [1, 18, 19]
no special treatments [5, 14, 15, 25,
27]
germination not affected by
irradiance [1]
no special treatments [6, 21]
Toxicity
Test16'
PRE&
POST [31]
PRE&
POST [6]
PRE&
POST [6, 21,
28, 31]
POST
[5, 22]
POST
[5, 15,22-
26]
PRE&
POST [28,
31, 34]
POST
[5, 15,25,
27]
PRE&
POST [6,
12,21,28]
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FAMILY
Species Botanical Name
(English Common Name)
CYPERACEAE
Cyperus rotundus
(Purple Nutsedge)
FABACEAE
Lotus corniculatus
(Bird's-foot Trefoil)
Senna obtusifolia
(Cassia, Sicklepod)
Sesbania exaltata
(Hemp)
Trifolium pratense
(Red Clover)
LAMIACEAE
Leonurus cardiaca
(Motherwort)
Mentha spicata
(Spearmint)
Nepeta cataria
(Catnip)
Lifespan
and Habitat
Perennials
arable land, pastures,
roadsides [16, 30]
Perennials
Grassy areas,
roadsides, open
habitats [16, 19]
Annuals
moist woods [16]
Annuals
alluvial soil [16]
Perennials
fields, roadsides,
arable land [16, 19]
Perennials
open areas [16]
Perennials
moist areas [16]
Perennials
disturbed areas [16]
Seed
Weight
mg
0.2
[14]
1 -1.67
[14, 19]
23-28
[9]
11 -13
[9, 14]
1.4-1.7
[14, 19]
0.75-1.0
[4, 14]
2.21
[4]
0.54
[4, 14]
Photoperiod
for germination
or growth(a)
L=D
[14]
L=D
[14]
L = D[14]
L > D [9]
L>D
[9]
L=D
[14]
L=D
[14]
L=D
[14]
Planting
Depth
mm(b)
0[1]
10-20 [6, 10]
10-20
[6,9]
10-20
[9,21]
0[4]
0[4]
0[4]
Time to Special
Germinate Treatments(d)
days(c)
12 (91%) germination inhibited by darkness [1]
[10] no special treatments [6, 10, 14]
1(50%) scarification [14, 19]
[19] germination not affected by irradiance
[18, 19]
no special treatments [23, 25]
soak seeds in water for 24 hours [9]
scarification [14]
seed viability differs depending on
color [1]
no special treatments [6]
soak seeds in water for 24 hours [9]
germination not affected
by irradiance [1]
no special treatments [21]
1 (50%) scarification [14, 18]
[19] may need maturation [19]
germination not affected by
irradiance [1, 19]
no special treatments [5]
no special treatments [4, 14]
no special treatments [4]
no special treatments [2, 4, 14]
Toxicity
Test16'
PRE&
POST [6, 28,
31]
POST
[5, 23, 25]
POST
[6,9]
PRE&
POST [9, 21,
28, 31]
POST
[5]
POST
[4]
POST
[4]
POST
[2,4]
Page 8 of30
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FAMILY
Species Botanical Name
(English Common Name)
LAMIACEAE continued
Prunella vulgaris
(Self-heal)
Stachys officinalis
(Hedge-nettle)
MALVACEAE
Abutilon theophrasti
(Velvetleaf)
Sida spinosa
(Prickly Sida)
PAPAVERACEAE
Papaver rhoeas
(Poppy)
POACEAE
Agrostis tenuis
(Common Bentgrass)
Alopecurus myosuroides
(Foxtail)
Avena fatua
(Wild Oats)
Lifespan
and Habitat
Perennials
arable fields, grassy
areas, disturbed sites
[16, 19]
Perennials
grasslands, field
margins [19]
Annuals
fields, open habitats [16]
Annuals
fields, roadsides [16]
Annuals
fields, arable land,
disturbed sites [16, 19]
lawns, pastures [16]
Annuals
Fields, open habitats
[16]
Annuals
cultivated areas,
open habitats [16]
Seed
Weight
mg
0.58-1.2
[4, 14, 19]
14-18
[14, 19]
8.8
[14]
3Q
.0
[14]
0.1 -0.3
[4, 14, 19,
29]
0.07
[14]
0.9-1.6
[29, 34]
7-37.5
[14, 30]
Photoperiod
for germination
or growth(a)
L=D
[14]
L=D
[14]
L=D
[14]
L=D
[14]
L=D
[14]
L>D
[10]
L=D
[14]
L = D[14]
L > D [6]
Planting
Depth
mm(b)
0
[4, 19]
10-20
[6, 10,21]
10-20
[6,21]
0
[4, 29]
20
[10]
2
[29]
10-20
[6, 10]
Time to
Germinate
days(c)
5 (50%) [19]
7 (91%) [18]
7 (50%)
[19]
4 (84%)
[10]
4 (50%)
[19]
10(62%)
[10]
< 24 (30%)
[34]
3 (70%)
[18]
Special
Treatments™
germination inhibited
by darkness [18, 19]
greater germination with larger
seeds [1]
no special treatments [4, 14, 22]
no special treatments
[5, 14, 22]
scarification [14]
no special treatments [5, 10, 21]
scarification [14]
germination not affected by
irradiance [1]
no special treatments [6, 21]
cold stratification & scarification
[1, 19, 32]
no special treatments [4, 14, 29]
germination inhibited by
darkness [1, 17-19]
no special treatments [10]
scarification [14]
treat with 101 mg/L KNOs [14]
warm stratification [1]
germination inhibited by darkness [1]
no special treatments [34]
scarification [7, 32]
darkness inhibits germination [1]
cold stratification [1, 18]
no special treatments [6, 10, 14]
Toxicity
Test16'
POST
[4, 22]
POST
[5, 22]
PRE&
POST [6, 22,
28, 31]
PRE&
POST
[6,21,28,
31]
POST [4]
POST
[10]
PRE&
POST [28,
34]
PRE&
POST [6, 10,
28, 31]
Page 9 of30
-------
FAMILY
Species Botanical Name
(English Common Name)
POACEAE continued
Bromus tectorum
(Downy Brome)
Cynosurus cristatus
(Dog's-tail Grass)
Digitaria sanguinalis
(Crabgrass)
Echinochloa crusgalli
(Barnyard Grass)
Elymus canadensis
(Canada Wild Rye)
Festuca pratensis
(Fescue)
Hordeum pusillum
(Little Barley)
Phleum pratense
(Timothy)
Lifespan
and Habitat
Annuals
fields, roadsides,
arable land [16]
Perennials
fields, roadsides,
open habitats [16, 19]
Annuals
fields, turf,
open habitats [16]
Annuals
[16]
Perennials
riparian, disturbed sites
[16]
Perennials
fields, moist areas [16,
19]
Annuals
pastures, roadsides,
open habitats [16]
Perennials
pastures, arable fields,
disturbed sites [16, 19]
Seed
Weight
mg
0.45-2.28
[14, 29]
0.5-0.7
[14, 19,
29]
0.52-0.6
[14, 30]
1.5
[14]
4-5
[14, 30]
1.53-2.2
[16, 19]
3.28
[14]
0.45
[14, 19]
Photoperiod
for germination
or growth(a)
L=D
[14]
L=D
[14]
L=D
[14]
L = D[14]
L > D [3]
L=D
[11]
L = D[14]
L>D[10]
L>D
[10, 14]
Planting
Depth
mm(b)
3
[29]
0
[29]
10-20
[21]
10-20
[7,21]
1
[11]
20
[10]
0-10
[10, 19]
Time to
Germinate
days(c)
3 (50%)
[19]
7 (75%)
14 (94%)
[7]
14-28
[11]
9 (74%) [10]
2 (50%) [19]
2 (74%) [10]
8 (50%) [19]
Special
Treatments™
maturation period [1, 7, 32]
germination inhibited by light [1]
no special treatments [14]
germination not affected by
irradiance [19]
no special treatments [14, 29]
scarification, cold stratification, &
maturation [1, 7, 14, 32]
treat with 101 mg/L KNOs [14]
germination inhibited by darkness [1]
no special treatments [21]
scarification [7, 32]
germination not affected by
irradiance [1]
no special treatments [3, 14, 21]
no special treatments
[2,11]
no special treatments
[10, 19]
warm stratification [1]
germination not affected by
irradiance [1]
germination inhibited by darkness [19]
germination not affected
by irradiance [17]
no special treatments [10, 14, 17, 19]
Toxicity
Test16'
PRE&
POST [28,
31]
POST
[5]
PRE&
POST [18,
25, 31]
PRE&
POST [3, 21,
28, 31]
POST
[2]
POST
[10]
PRE
[31]
POST
[10]
Page 10 of 30
-------
FAMILY
Species Botanical Name
(English Common Name)
POLYGONACEAE
Polygonum convolvulus
(Black Bindweed)
Polygonum lapathifolium
(Pale Persicaria)
Polygonum
pennsylvanicum
(Pennsylvania
Smartweed)
Polygonum per/scar/a
(Smartweed)
Rumex crispus
(Curly Dock)
PRIMULACEAE
Anagallis arvensis
(Scarlett Pimpernel)
RANUNCULACEAE
Ranunculus acris
(Common Buttercup)
Lifespan
and Habitat
Annuals
open habitats,
roadsides [16]
Annuals
moist soil [16]
Annuals
Fields, open habitats
[16]
Annuals
disturbed areas,
arable land [16, 19]
Perennials
arable fields, roadsides
open areas [16, 19]
Annuals
arable fields, open
areas, disturbed sites
[16, 19]
Perennials
arable fields, roadsides,
Open areas [16, 19]
Seed
Weight
mg
5-8
[4, 14, 29]
1.8-2.5
[14]
3.6-7
[14, 29]
2.1 -2.3
[14, 19]
1.3-1.5
[4, 14, 19]
0.4-0.5
[4, 14, 19]
1.5-2
[14, 19,
29]
Photoperiod
for germination
or growth(a)
L=D
[20]
L>D
[6]
L>D
[13]
L=D
[14, 33]
L=D
[14]
L=D
[14]
Planting
Depth
mm(b)
0-2
[4, 29]
2
[29]
0
[19]
0
[4, 19, 33]
1
[29]
Time to
Germinate
days(c)
5 (94%)
[18]
<14[13]
2 (50%) [19]
3 (50%) [19]
6 (100%) [33]
1 (50%)
[19]
41 -56
[19,29]
Special
Treatments™
cold stratification for 4 - 8 weeks
[1,2,4,20,29]
germination not affected
by irradiance [1]
germination not affected
by irradiance [1]
germination inhibited by darkness [18]
cold stratification [1]
no special treatments [5]
cold stratification for 4 wks at 0 - 5 C
[1,29]
germination inhibited by darkness [1]
scarification, cold stratification, GA
treatment [14]
cold stratification, maturation [17-19]
germination inhibited by darkness [19]
no special treatments [13]
germination inhibited by
darkness [18, 19]
maturation may be necessary [18]
no special treatments [4, 14, 33]
cold stratification, GA treatment
[1,14, 18, 19, 32]
light required for germination [1]
no special treatments [2, 4]
no special treatments
[5, 14,22,24-26]
Toxicity
Test16'
PRE&
POST [1,2,
20,28, 31]
PRE&
POST [6]
PRE
[31]
POST
[13]
POST
[4, 33]
POST
[2,4]
POST
[5, 22, 24-26]
Page 11 of 30
-------
FAMILY
Species Botanical Name
(English Common Name)
ROSACEAE
Geum urbanum
(Yellow Avens)
RUBIACEAE
Galium aparine
(Cleavers)
Galium mollugo
(Hedge Bedstraw)
SCROPHULARIACEAE
Digitalis purpurea
(Foxglove)
Veronica persica
(Speedwell)
Lifespan
and Habitat
Perennials
hedgerows, moist areas
[16, 19]
Annuals
arable fields, moist
areas disturbed sites
[16, 19]
Perennials
hedge banks, open
areas [8]
Biennials, Perennials
Hedge rows, open
areas [16, 19]
Annuals
arable fields, open
areas, disturbed
sites [16, 19]
Seed
Weight
mg
0.8-1.5
[14, 19]
7-9
[14, 19]
7
[29]
0.1 -0.6
[4, 14, 19]
0.5-0.6
[14, 19]
Photoperiod
for germination
or growth(a)
L=D
[14]
L=D
[14]
L=D
[14]
L=D
[14]
L=D
[14]
Planting
Depth
mm(b)
0
[19]
2
[29]
0
[4, 19]
0
[19]
Time to
Germinate
days(c)
5 (50%) [19]
16 (79%) [18]
5 (50%) [19]
6 (100%) [18]
6 (50%) [19]
8 (99%) [18]
3 [19]
5 (96%) [18]
Special
Treatments™
germination inhibited by
darkness [18, 19]
warm stratification [1]
no special treatments [5, 14, 22, 25,
26]
cold stratification [1, 18, 19]
germination not affected by
irradiance [18, 19]
light inhibits germination [1]
no special treatments [6, 14]
no special treatments
[5, 14, 22, 24, 26, 29]
germination inhibited by darkness
[1, 17-19]
no special treatments [4, 22-26]
germination inhibited by
darkness [18, 19]
cold stratification [18]
no special treatments [14]
Toxicity
Test16'
POST
[5, 22, 25,
26]
PRE&
POST [6, 28]
POST
[5, 22, 24,
26]
POST
[4, 22 - 26]
PRE&
POST [28]
(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 12 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 non-
pesticide 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,
ECos), test substance concentrations should immediately bracket the point
estimate(s). The concentrations should be a geometric progression of
Page 13 of 30
-------
twofold (e.g., 0.1, 0.2, 0.4, 0.8, and 1.6 Ib/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 (ECos, ICos,
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 ECos or ICos) 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 14 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 15 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 16 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 °C while
temperature during the night should be 20 ± 6 °C.
(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 |imol/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 17 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 18 of 30
-------
plant. After shoot lengths are measured, plants are dried (constant weight
at 70 °C) 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 19 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 .
Equation1
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) ECis and ICis values. To ascertain that the survival EC 25 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 £€25 or
IC25, compared to controls, is operationally defined as a 25% reduction from the
control sample mean (Equation 2). The null hypothesis (Ho) stated in terms of
true population parameters is that the difference of the 'limit' treatment mean
response (|iiimit) from the control mean response (|iCOntroi) is greater than or equal to
a 25% reduction, compared to the control (i.e., HQ: |icontroi - Hiimit > So). The
alternative hypothesis (HA) is that this difference is less than a 25% reduction,
compared to the control (i.e., HA: ^control - Hiimit < So). 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).
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Equation 2
where:
5o = difference between two parameters, defined in this case as a p%
reduction from the control sample mean;
^control = control sample mean response (e.g., emergence, shoot length,
shoot weight, survival); and
p = percent reduction from the control sample mean, which is 25 in the
case of the £€25 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 ICis and ECis 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
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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 £€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
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 £€25 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 ICos value for shoot length or shoot weight) or the dose
at which 5% of the population is effected (i.e., the ECos 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 ICos and ECos 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.
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Table 3.—Summary of Test Conditions for Seedling Emergence and Seedling
Growth Test
Test type
Test duration
Substrate
Nutrients
Temperature
Relative humidity
Light intensity
Photoperiod
Watering
Test chamber (pot) size
Number of seeds per test chamber (pot)
Number of replicates per test treatment
Number of seeds per test treatment
Test treatment levels
Test substance application method
Measures of effect or measurement
endpoints
Seedling emergence and seedling growth
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)
Natural or synthetic soil with at most 3% organic matter and
pH 6.0 -7.5
As naturally available, or supplemented with either a soil
fertilizer or watered with nutrient solution
25/20 °C (daytime/nighttime) ± 6 °C
70% (daytime)± 15%
350 ± 50 umol/m2/sec at the soil surface
16 hours light: 8 hours dark (for non-crop species see Table
2 of this guideline)
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.
Varies with plant species selected. Six-inch diameter plastic
pots are typical. Flats are also encouraged
Varies with species and test chamber (pot) size
4 (minimum)
40 (minimum)
Unless performing limit test, minimum of 5 treatment levels
plus appropriate controls
Applied to soil surface immediately after planting seeds (i.e.
within the day)
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.
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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)0) 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.
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(4) Plant test species.
(i) Scientific and common name, plant family, and variety.
(ii) Test date of germination rating and germination percentage.
(iii) History of the seed: source, name of supplier, seed year or growing season
collected, batch or lot number, seed treatment(s), and storage conditions.
(iv) Seed size class.
(v) Description of handling and processing of seeds 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.
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(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.
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(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 ECos or ICos 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 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 ECos or ICos value is used in place of a NOEC provide a
description of statistical method(s) used for point estimates, including software
package, for determining ECos and ICos 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 DJ. Versteeg. 1992. A statistical procedure for modeling continuous
toxicity data. Environmental Toxicology and Chemistry 11:1485-1494.
(3) Gulley, D.D. etal., 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 27 of 30
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(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-UpD). Ecotoxicology,
12:271-285.
(3) Boutin, C., Lee, H-B., Peart, T., Batchelor, P.S., & Maguire, RJ. 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
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(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.
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(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
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chlorpropham and ethofumesate on wild plant species. Environmental Pollution, 114:21-
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(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.
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(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, AJ. 1997. A microcosm approach to detection of the effects
of herbicide spray drift in plant communities. Journal of Environmental Management,
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(27) Marshall, E.J.P. & Bernie, I.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,
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(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
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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
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(33) White, A. & Boutin, C. (National Wildlife Research Centre, Environment Canada).
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(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,
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