&EPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention
(7101)
EPA712-C-011
January 2012
        Ecological Effects
        Test Guidelines

        OCSPP 850.4150:
        Vegetative Vigor

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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.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.

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OCSPP 850.4150: Vegetative vigor.

(a) Scope—

       (1) Applicability. This guideline is intended to be used to help develop data to submit to
       EPA under the Toxic Substances Control Act (TSCA)  (15  U.S.C.  2601, et  seq.), the
       Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C.  136, et  seq.), and
       the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).

       (2) Background.  The source materials used in developing this harmonized OCSPP test
       guideline include the OPP 122-1 Seed Germination/Seedling Emergence and Vegetative
       Vigor (Tier I) and 123-1 Seed Germination/Seedling Emergence and Vegetative Vigor
       (Tier II) guidelines (Pesticide Assessment Guidelines Subdivision J—Hazard Evaluation:
       Nontarget Plants); OPP  Standard  Evaluation  Procedure Nontarget Plants Vegetative
       Vigor (Tier I and  2); OECD 208 Terrestrial Plants, Growth Test; OECD 227 Terrestrial
       Plant Test: Vegetative Vigor Test, and ASTM E 1963-02, Standard Guide for Conducting
       Terrestrial Plant Toxicity Tests.  This guideline incorporates what were formerly Public
       Drafts OCSPP 850.4150 and OCSPP 850.4250 (April, 1996).

(b) Purpose. This guideline is intended for use in developing data on the toxicity of chemical
substances and mixtures ("test chemicals" or "test substances")  subject to environmental effects
test regulations.  The objective of the vegetative vigor test is to measure the  effect of a foliar
applied test substance upon terrestrial plants during the  vegetative growth period of their
development.  Procedures for testing a single exposure concentration (i.e. Tier I testing for
pesticides) as well as procedures for testing multiple exposure concentrations (i.e.  Tier II testing
for  pesticides)  are  described.  This guideline should  be  used in  conjunction  with  OCSPP
850.4000, which provides general information and overall guidance for the guidelines in OCSPP
Series 850, Group D.  The Environmental Protection Agency will use data from vegetative vigor
tests in assessing the hazard a test substance may present in the terrestrial environment.

(c) Definitions.  The definitions in OCSPP 850.4000  apply to this guideline.  In addition, the
more specific definitions in this paragraph also apply:

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

       Shoot height is defined as the length of the above-ground vegetation from the soil surface
       to the apical tip or highest aerial  part of the plant.

(d) General considerations—

       (1) Summary of the  test.  The vegetative vigor test is a test that evaluates  the affect of a
       test substance upon the growth of a number of plant species.  Effects measured include
       survival, plant height, and plant biomass.   In addition, qualitative phytotoxic effects are
       observed and evaluated.  The test is designed to determine the quantity of test  substance
       required to cause  a 25 percent (25%) inhibition in plant height and plant  biomass (IC25
       values and to determine the no observed effect concentration (NOEC) for these effect
       measures.   A number  of crop  and non-crop plant species may be used.  It may be

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performed  in  a growth  chamber, greenhouse, or in small field plots.  A natural  or
synthetic soil serves as the substrate and the test substance is sprayed on the foliage at test
initiation.  The results are used to establish toxicity levels, evaluate hazards or risks to
terrestrial plants, and to indicate if further testing  at a higher tier is  necessary.  Note
historically in OCSPP pesticide and industrial chemical guidelines the term ECX was used
to cover both the current OCSPP 850.4000 definition of ECX (concentration where x% of
the population exhibit the effect (e.g., survival)) and ICX (concentration resulting  in an x%
decrease or inhibition effect on an attribute of the population (e.g., plant yield)).

(2) General test guidance.  The general guidance in OCSPP 850.4000 applies to this
guideline, except as specifically noted herein.

(3) Range-finding test.  A range-finding test is usually  conducted  to  establish  the
appropriate test concentrations to be used for the definitive test. In the range-finding test,
the test organisms are exposed to a series  of widely-spaced  concentrations of the test
substance (e.g.,  1, 10, 100 pounds per  acre (Ibs/acre)). The details of the range-finding
test do not have to be the same as for definitive testing in that there are no replicates, and
the number of test organisms used, and the duration of exposure may be  less than in
definitive testing.  In addition, the types of observations made on test organisms are not
as detailed  or as frequently observed as that of a definitive test and results are  analyzed
using nominal concentrations. However,  the range-finding test will be more useful the
greater the similarity between the range-finding and the definitive test.

(4) Definitive test.  The goal of the definitive test is to determine for each measure of
effect (shoot height and shoot biomass) its concentration-response curve and NOEC (and
lowest observed effect concentration (LOEC)); the shoot height and weight IC25 values
(with 95%  confidence  intervals  and  standard errors).   If possible the  slopes of the
concentration-response  curves, associated  standard  errors,  and  the  95% confidence
intervals of the slopes should also be determined.  However, at a minimum, the full
concentration-response curve (concentration range covers 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 the most sensitive NOEC (or ICos) and IC25 values.
For a satisfactory test, the lowest  treatment concentration is below both the shoot height
and biomass IC25 values.  A summary of test conditions is  provided in Table 2 and
validity elements for an acceptable definitive test are listed  in Table 3.

(5) Limit test.  In some situations, it is only necessary to ascertain that the EC25  value
and the shoot height and shoot biomass IC25 values for a given plant species occur above
a certain limit concentration, and that  at this limit concentration there is no observable
adverse effect.  In a vegetative vigor limit test, at least 40 seedlings (divided into at least
4 replicates of 10 plants each) are exposed to a single "limit concentration,"  with the
same number  of replicates  and  organisms in appropriate  controls.   The  multiple-
concentration definitive test may be waived for a given test species if the following two
conditions are met for survival, shoot height, shoot weight, and phytotoxic ranking.  First,
the "limit" treatment response is both statistically less than a 25% decrease  from  the


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       control response (i.e., ECzs and IC25 values > limit concentration), and second, the limit
       treatment responses are not significantly reduced (or inhibited) as compared to the control
       responses  (i.e.,  NOEC > limit concentration).  The  limit concentration is  3 times the
       estimated environmental concentration for industrial chemicals or for pesticides the limit
       concentration is based  upon the maximum label rate, e.g. the maximum recommended
       amount of active ingredient (a.i.), in the recommended minimum quantity of carrier (such
       as water),  to be used per land area. Results are reported in grams or pounds of a.i. per
       acre.  Except for the number of treatment groups, an acceptable limit test follows the
       same test procedures, is the same duration, and has the same number of controls as the
       definitive test. Acceptable limit tests like definitive tests include analytical confirmation
       of the applied dose.

(e) Test standards—

       (1)  Test substance.   For pesticides the substance to be tested is  the typical  end-use
       product (TEP).   If there is more than one TEP with  the same inert  substances, the  one
       with the highest percent a.i. and/or the  one most commonly  used should be tested.  If
       there is more than one TEP with different inert substances, a TEP representative of each
       different inert substance should be tested in the range-finding  test and at a minimum the
       most sensitive one tested in the definitive  or limit test.  In addition, if an  adjuvant is
       recommended for use on a TEP label, the adjuvant is added with the TEP at the label rate
       to constitute  the test  substance.   If a  TEP is not  available (i.e.  new pesticide),  the
       technical grade  a.i. is used with a representative formulation, which should include  any
       adjuvant that will be recommended for use on the label. If products are applied in a tank
       mixture, dosages of each a.i. should be reported with identification  and formulation for
       each product in the tank mix.  For industrial chemicals the substance to be tested should
       be technical  grade, unless the test is designed to test a specific formulation  or mixture.
       OCSPP 850.4000 lists  the type of information that should  be known about  the  test
       substance before testing.

       (2)  Test duration.  The limit and definitive tests last for 21 days  after test substance
       application.  If phytotoxic symptoms first start to appear between day  14 and 21 post-
       application, the test is extended to 28 days post-application.

       (3) Test organisms—

              (i)  Species.  The test is performed using  crop and/or non-crop terrestrial plant
              species selected from a cross-section of the terrestrial plant species that have been
              historically used for this type of testing.  A list of crop  and  non-crop plant taxa
              that have been used in toxicity tests can be found in Table 1 of this guideline  and
              Table  2  of the OCSPP 850.4100  guideline.  Endangered  or threatened species as
              determined by the Endangered Species Act of 1973 (Public Law 93-205) may not
              be used without permission from the Fish and Wildlife Service.

                    (A) Number of species  tested.   For testing industrial chemicals,  the
                    specific plant(s) used are  selected on a case-by-case basis. For pesticide
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                     testing, at a minimum ten plant species from the plant groups and families
                     identified in paragraphs (e)(3)(i)(A)(7y) through (e)(3)(i)(A)(%) are tested:

                            (1) Dicotyledoneae:  Six species of at least four families, one
                            species of which is soybean (Glycine max).

                            (2) Monocotyledoneae: Four species of at least two families, one
                            species of which is corn (Zea mays).

                            (3) At least one test species  should be a root crop (either a monocot
                            such as onion or dicot such as carrot, table beet or sugar beet).

                     (B) Crop  species.   A  representative  list of crop  species  which  are
                     acceptable test species are listed in Table 1.

       Table 1.—Crop species and families acceptable for use in the vegetative vigor test
              Family
                     Species
                              Common name
Dicots
Monocots
Solanaceae

Cucurbitaceae
Asteraceae
Fabaceae1
Brassicaceae
Apiaceae
Chenopodiaceae
Asteraceae
Fabaceae
Brassicaceae
Brassicaceae
Fabaceae
Malvaceae
Polygonaceae

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 Rhizobiumjaponicum is unnecessary.
                     (C) Non-crop plant species.  When selecting plant species other than the
                     three crop species (soybean, corn, and a root crop (onion, carrot, table beet
                     or  sugar beet)), which are tested at a minimum for pesticide testing, the
                     use of sensitive non-crop plant species is recommended.   Table 2  of
                     OCSPP  850.4100, which is based  on  a table from the  reference  in
                     paragraph (j)(5)  of this  guideline,  provides a list of recommended non-
                     crop species.
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       (ii) Source. Within a given test, all seedlings of a given species, including the
       controls, should be from the same source and lot number, and same  cultivation
       group.

       (iii) Age or growth stage and size.  Seedlings used in testing are selected from
       groups of plants grown from seed to the 2 to 4 true leaf stage. Selected seedlings
       should also be of uniform size.

       (iv) Condition.  Healthy plants should be used. For cultivation of seedlings from
       seed, healthy seeds should be used with reported germination rate for a seed lot of
       70% or better.  Seeds should be examined before use and sorted to remove broken
       or damaged seeds.

       (v) Care and handling. Seeds, used to cultivate seedlings for this test, should be
       stored in a desiccator and refrigerated until needed.  Pesticide treated seeds should
       be avoided except for approved seed treatments. The Agency should be consulted
       prior to test initiation if seed treatments other than a weak hypochlorite solution
       (recommended by Environment Canada), captan, or thiram are used.  Captan and
       thiram seed treatments (nonsystemic mode of action; see paragraph (j)(4) of this
       guideline)  are  the  only Agency-approved pesticide seed treatments.   When
       unapproved pesticide seed treatments  are used in a study,  the test should be
       designed to demonstrate no synergistic or antagonistic interactions occur between
       or among the  seed treatment  and test  substance.  Steam sterilization of  soil  is
       recommended  as a non-pesticide alternative  for killing pathogens,  fungi, and
       insects in soil media.

(4) Administration of test substance.  Within 48 hours prior to the application of the
test substance, seedlings of similar size and condition (morphological symptoms) are
selected for use in the test, and the height and condition of each plant is determined and
recorded.  At test initiation, test substance is applied to the seedling foliage as a spray.

       (i) Preparation  of spray solutions.  The amount of water used in the spray as a
       carrier is equivalent to the recommendation on the pesticide label.  For example,
       vegetation within a given pot (test container) is sprayed with x milligrams of TEP
       mixed  into 10 milliliters of water, where the  10 milliliters of water per the pot
       area is equivalent to the minimum number of gallons per  acre specified on the
       label.  For a satisfactory test, all pots in all treatment levels and controls have the
       same equivalent volume of water applied (e.g., in the example just given this is 10
       milliliters).

       (ii) Treatment levels.

              (A) For  a  given  plant species,  five  treatment  levels  are tested at  a
              minimum.  A range-finding test can be used to establish the  appropriate
              test doses for the definitive test (see paragraph (d)(3) of this  guideline).
              For scientifically sound estimates of a given point estimate (e.g., IC25,
              ICos), test substance concentrations should immediately bracket the point
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              estimate(s).   The concentrations should be  a geometric progression of
              twofold at a minimum (e.g., 0.1, 0.2, 0.4, 0.8, and 1.6 Ib/acre). While a
              twofold progression is preferred, threefold and fourfold  progressions are
              acceptable. If a fourfold series progression is used, the rationale for using
              this  large an interval  between  concentrations  and the  effect on the
              accuracy and reproducibility of the point  estimate or NOEC  should be
              provided.  For an acceptable study for a given plant species, the lowest test
              treatment level should be lower than the IC25 values for  shoot height and
              biomass for that  plant  species.   The NOEC  should  be determined  by
              hypothesis testing for each effect measure.  The lack of a NOEC for an
              effect measure is not  critical  as  long as the response-curve for the  effect
              measure is acceptable for calculation of the  5% inhibition concentration
              (ICos). It is recommended that one or two additional test concentrations in
              the  lower tail of the  concentration-response  curve of the  most sensitive
              endpoint be added to insure  bracketing of both the most sensitive IC25
              value and the most sensitive NOEC (or ICos) value.

              (B)  For pesticides dosages should be expressed as mass  of test substance
              per  unit of  soil  surface  area  in  the  pot  or container (i.e. Ib/acre).
              Additionally for pesticides, dosages should be expressed in unit mass of
              a.i. or acid equivalent per unit of land area treated, as appropriate.

              (C)  The use of pesticide treatments  to control pests during  the test should
              be avoided. Mechanical, cultural, and biological pest control  methods are
              suggested.  If other pesticides are used in  the test for pest control,  for a
              satisfactory test a demonstration should be  performed (i.e., additional test
              data) documenting that the pesticide is not toxic to the test species and that
              no synergistic or antagonistic interactions with the test substance  exist
              (additional test data).
(5) Controls.
       (i)  Every  test  includes  controls  consisting  of the  same  support  medium,
       conditions, procedures,  seedling source, seed source and lot, except that no test
       substance is added.  In  addition, vehicle (solvent) controls are also included if a
       solvent is used.  However for pesticides, because TEP is typically tested, solvents
       are generally not necessary.

       (ii) If the negative control is contaminated with the test substance, the study is not
       acceptable and should be repeated.

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

(6) Number of test organisms  and replicates.

       (i) For each species, the minimum number of test organisms is  30  seedlings per
       dose level (a minimum of six replicates,  each with a minimum of 5 seedlings).

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       Alternatively, the number of test organisms  is 40  seedlings per dose level  (a
       minimum of four replicates, each replicate with a minimum of 10 seeds). A test is
       not acceptable  if a container is too  small, resulting  in  overcrowding  and
       competition among plants in the container.  The recommended loading in a 6 inch
       container for corn,  soybean,  tomato, cucumber or sugar beets is  one to  two
       seedlings; the loading for rape, onion, wheat, or other small seed species is three
       to four seedlings.  To prevent bias, seedlings are impartially or randomly assigned
       to the containers (pots, flats) or plots. Within  a given test, all test organisms of a
       given species,  including the  controls,  should  be from  the same  source  and
       cultivation group.  Note that a replicate in this case will  consist of  several pots
       and/or flats and the integrity of the replicate should be maintained throughout the
       duration of the study (i.e. pots/flats from one replicate are not moved to or mixed
       with another replicate and if the replicate is moved all pots/flats composing the
       replicate are moved as a unit).  Do not mix species within a replicate.

       (ii) Randomization.

              (A) Placement of the flats or pots within the greenhouse or arrangement of
              the field plots should follow a random or  randomized block  design.   To
              minimize spatial  differences,  which can have a significant impact upon
              plant growth, the placement of replicates should be randomized during the
              test (at least every three to five days).

              (B) Alternative placement  should be used with volatile test substances to
              prevent cross contamination.  For example,  in a greenhouse setting use
              positive  air flow  throughout the  duration of the study with placement  of
              controls  and treatment levels such that air  flows first  across controls and
              then from the lowest  treatment to the highest treatment.   In  addition,
              include another set of controls which are placed in a separate greenhouse
              for comparison with control results from the greenhouse containing the
              treatments.   Where tests are conducted using growth chambers where
              positive air flow can not be achieved, the controls and each treatment level
              should be placed in separate growth chambers.

(7) Facilities, apparatus and supplies—

       (i) Reliability.  All equipment used in  conducting the test, including equipment
       used to prepare  and administer the test substance, and equipment to maintain and
       record environmental conditions, should be of such design and capacity that tests
       involving this equipment can be conducted in a reliable  and scientific manner.
       Equipment  should be  inspected, cleaned, and  maintained regularly,  and  be
       properly calibrated.

       (ii) Application equipment. The application  equipment used in testing products
       in  small field plot  studies should be  designed to  simulate conventional farm
       equipment using the basic components of commercial application equipment in
       the design of the small-plot equipment. For  example, nozzle types, sizes,  and
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arrangements on small plot sprayers should be identical to those used by growers
on commercial ground sprayers.  The application equipment used in greenhouse
or growth chamber tests (such as a spray chamber or computerized belt sprayer)
should provide uniform coverage and should be calibrated at spray volumes (to
the extent practical) that are representative of field conditions.  Acceptable foliar
spray methods are found  in the reference in paragraph (j)(8) of this guideline.
The  amount of water used  in  spray  as a carrier  is to be equivalent to the
recommendation on the label.

(iii)  Facilities.  Vegetative vigor tests  should  be conducted under controlled
conditions in greenhouses and growth chambers, or under ambient conditions in
small field plots.

(iv)  Test containers.  Test containers  should  be  nonporous  so that the test
substance is not absorbed  or does not react in any way with the container.  Glass
or stainless steel  containers with drainage holes  can  be used  as  plant  pots.
Polyethylene  plastic  pots or flats  that  have  not  been  previously  used are
acceptable test containers, provided they are free of toxic materials.  Do not use
clay  or peat containers.  Containers should be thoroughly cleaned prior to use. A
dichromate solution should not be used to clean containers. The volume of the
pot should be large enough so that seedling growth is not restricted during the
test,  see paragraph (e)(6)(i) of this guideline.  Containers should be consistent
within species.

(v) Support medium. Plants may be  grown in a natural soil (free  of pesticide
contamination) or synthetic  soil.   Growth chamber and  greenhouse tests are
performed using a sterilized standardized soil that  consists primarily of sandy
loam, loamy sand, loamy  clay, or clay loam soil that contains up to  3% organic
matter (up to  1.5% organic carbon).  Commercial potting soil  or synthetic soil
mixes may be used as the soil medium provided  that the organic matter does not
exceed 3%.  Clay soils should not be used if the test substance is known to have a
high affinity for clay.  Field soils should be sieved to remove coarse (greater than
2 millimeters) particles and recommend pasteurizing or heat treating the soil to
reduce the effect of soil pathogens. The soil pH may be adjusted to the optimum
growing range of 6.0 to  7.5 by the  addition  of a basic substance (calcium
carbonate) or an acidic substance (gypsum, ammonium sulfate, or sulfuric acid).
A slow-release fertilizer may be added to the soil to provide nutrients for  plant
growth.

(vi)  Plant supply.  Seeds are planted and cultivated ahead of time to provide
sufficient seedlings at the  2- to 4-true leaf stage  of growth (typically one to four
weeks post-emergence) and of uniform size to initiate the test for a given test
species  on the same day.   Thinning the  seedlings to uniform size and  condition is
performed before use.  For non-crop species guidance on the time to germination
may be  found in Table 2 of OCSPP 850.4100.
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(8) Environmental conditions.  For greenhouse and growth chamber studies, general
conditions in paragraphs (e)(8)(i) through (e)(8)(v) of this guideline are recommended;
however, excursions outside these recommendations do not invalidate the study if other
acceptance criteria  are met.  Additional guidance for non-crop species is provided in
Table 2 of OCSPP 850.4100.

       (i) Temperature. Air temperature should be uniform throughout the greenhouse
       or growth chamber.  Air temperature during the day should be 25  ±  6 degrees
       Celsius (°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 top of the canopy, is desirable, on a photoperiod  of 16 hours light and 8
       hours darkness.  Artificial lighting may be used to lengthen short-day periods or
       to supplement natural sunlight on overcast days.  Care should be taken to ensure
       that plants are not affected from the heat generated from supplemental lighting.

       (iv) Watering.  For greenhouse and growth chambers,  top watering below the
       canopy is used at the first watering after the test substance has been applied to
       initiate the capillary movement of water for bottom watering. Bottom watering of
       test containers is used for the duration of the study in order to prevent washing the
       chemical off the foliage and through the soil during watering.  The  watering
       method should prevent the test substance from washing off the foliage or leaching
       out of the soil or the pots/flats.

       (v) Nutrients.  Nutrients may be supplied during the study by using  a nutrient
       solution  of  defined chemical  composition,  such  as  half-strength  modified
       Hoagland nutrient solution, to water the plants.  Alternatively, nutrients may  be
       supplied by  amending the test soil at the start of the test with fertilizer including
       standard nutrients for the species tested.

(9) Observations—

       (i) Measurement of test substance.  The dosing  solution (i.e. spray  tank) is
       sampled at the start of and end of application and analyzed for the test substance
       concentration (in a.i. or acid equivalent units for pesticides). The total volume of
       dosing solution used, and the total soil  surface area per  pot or container in a
       replicate  and treatment level should be recorded.  The analytical methods used to
       measure the  amount of test substance in a sample are validated before beginning
       the test, as described in OCSPP 850.4000.

       (ii)  Support medium.   Characteristics of a batch  of soil representative of that
       used in the study or the native soil (for tests done in field plots) are determined,
       including soil type and texture, pH, particle size distribution, and organic matter
       content.
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(iii) Environmental conditions.  Environmental conditions to be  monitored in
greenhouses and growth chambers include air temperature,  humidity, and light
intensity.  For field plots, environmental conditions should be monitored at the
field site during and after test substance application and daily  throughout the
duration of the study.  Environmental information to  be collected in the field
should include air temperature, precipitation, relative humidity, wind speed, light
intensity at the canopy, and cloud cover. Source of environmental field condition
data can be taken from a weather station that is within 5 miles of the field.

       (A) Air temperature and humidity.  The  air temperature and humidity
       during the study should be recorded at representative locations throughout
       the  area in which the test plants are growing.  Measurements are made
       preferably continuously,  but alternatively  as maximum  and minimum
       values over each 24-hour period.

       (B) Light intensity.  Light intensity at the  canopy should be  determined
       every  3 to 5 days at representative locations throughout the area in which
       the test plants are growing.  A photosynthetically active radiation (PAR)
       sensor should be  used to measure light quality.  For field  plots, ideally
       PAR measurements should be taken between eleven in the  morning and
       two in the  afternoon.  Additional  information on the use of lighting in
       plant  toxicity tests can be found in  the  references given  in  OCSPP
       850.4000.

       (C) Watering and  precipitation.   Frequency of watering should be
       recorded for greenhouse and growth chamber studies, and observations of
       moisture stress should be made and recorded daily. Frequency of watering
       and frequency and amount of precipitation should be recorded for field
       studies, and observations of moisture stress should be recorded daily.

       (D) Pests.  Daily observations should be made on pest pressure using an
       index  of the  extent  of infestation.    Pest infestation  may affect  the
       interpretation  of study  results and  therefore  should  be  adequately
       described.  Frequency, methods, and rates used for treating an insect or
       disease should be recorded.

(iv) Phytotoxic Effects

       (A)  Observations on the number of alive and dead plants, and visual
       morphological symptoms of phytotoxicity are made and recorded at least
       on days 7, 14 and 21 post-application, and if  the test is extended  (see
       paragraph (e)(2) of this guideline) on day  28.   Shoot  height for each
       individual surviving plant is recorded at a minimum on days 14 and 21
       post-application, and if the test is extended on day 28.  At test termination
       after shoot heights are measured, plants are dried (constant weight at 70
       °C) and the  total  plant biomass  (i.e.  dry  weight) in each replicate is
       measured and recorded.  Biomass measurements should be made for each
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                     replicate not each individual plant.

                     (B)  Seedling  condition is  determined using  a  standard  morphological
                     phytotoxicity  rating scale.  Observations  on morphological symptoms of
                     phytotoxicity  should include all variations, either inhibitory or stimulatory,
                     between the treated and the untreated organisms.  Such variations may be
                     phytotoxic symptoms (e.g.,  chlorosis, necrosis, pigmentation, leaf curling
                     and  wilting)  and formative (e.g., leaf and  stem deformation) effects.
                     Observations  should include the treatment level  and replicate, stage of
                     development  and  dates  when adverse  results  occurred, subsided or
                     recovered, and counts for each plant affected. Uniform scoring procedures
                     should  be used to evaluate the observable toxic responses.   Such data
                     should  include the actual values  used to determine any percentages of
                     effects. Any lack of effects by the test substance should also be reported.
(f) Treatment of results—

       (1) Summary statistics—
              (i) Environmental  conditions.  Air temperature, humidity,  and light intensity
              data  should  be summarized  in tabular form,  showing  the mean, standard
              deviation, and range during the test at each measurement location.   For field
              studies precipitation events and the amount of precipitation should be summarized
              in tabular form by date of occurrence, and total precipitation calculated.  Watering
              frequency and  duration  should be  summarized  in  tabular  form by date of
              occurrence.

              (ii) Test substance concentration.  Compare  the initial test substance dosing
              solution concentration with test substance concentration in the dosing solution at
              end of application.  If the substance was not stable calculate a rate of decline of
              the test substance.   The total volume of dosing solution used, total area treated,
              and the time application started and ended for a given treatment level summarized
              in tabular form.

              (iii) Shoot height.  Calculate and plot the average shoot height and standard
              deviation of shoot height for each replicate and the treatment level mean shoot
              height  and  standard   deviation  at  each  observation period  (including  test
              initiation).

              (iv) Shoot biomass. Calculate and plot the average plant  dry weight  (total dry
              weight biomass divided by number of surviving plants) for each replicate and the
              treatment level mean shoot weight and standard deviation.

              (v) Survival.   Calculate and plot  the percent survival (number of  surviving
              seedlings divided by the number of seedlings at test initiation) for each replicate
              and the treatment level mean and standard deviation percent survival at each
              observation time. For  each treatment level calculate mean percent survival  as a
              percentage of the control mean percent survival at test termination.

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       (vi) Phytotoxic  effects.   Morphological symptoms  of plant injury should be
       summarized in tabular form  by time of observation, treatment, and replicate.
       Definition of any index values used for morphological symptoms, indicating the
       severity of the symptom(s), should be provided.

(2) Percent inhibition.   For shoot height and  shoot  biomass calculate  the percent
inhibition (%I) at each treatment level using Equation 1 .
                             0/T
                             %/ = -i - ii — L                         Equation 1


       where:

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

       X = the treatment mean response value (shoot height or shoot biomass,
       respectively). Stimulation is reported  as negative %I.
(3) Limit test—
       (i) ECis and ICis values.  To ascertain that the survival £€25 value and shoot
       height and shoot biomass IC25 values for a given plant species occur above the
       "limit" concentration, a one-sided test  which compares the difference between
       two sample groups to a fixed value (or difference) is performed for each of these
       response measures.  For a comparison of sample means, the difference defining
       the EC25 and IC25 compared to controls  is operationally defined as  a 25%
       reduction or inhibition from the control  sample mean (Equation 2).  The null
       hypothesis (Ho) stated in terms of true population parameters is that the difference
       of the "limit"  treatment mean response (niimit) from the control mean response
       (^control) is greater than or equal to a 25% inhibition or reduction, compared to the
       control (i.e.., H0: |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., survival, shoot height and biomass) in
       the limit treatment as compared  to the control is declared to be less than 25%
       (e.g., IC25 > limit dose). If the null hypothesis is not rejected, the effect level or
       inhibition level in the  limit treatment  as compared to  the control  response  is
       declared to be 25% or greater (e.g., IC25 < limit dose).

                           S0=(xamtml)^{p/I00)                        Equation 2

             where:

             So = difference between two parameters, defined in this  case as ap percent
             reduction from the control sample mean;
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              ^control = control sample mean response (e.g., survival, shoot height and
              biomass); and

             p = percent reduction from the control sample mean, which is 25 in the
              case of the EC25 and IC25.

       (ii) NOEC. To ascertain that there is no observable effect at the limit treatment
       (i.e., NOEC > limit dose) for a given response measure (survival, shoot height and
       biomass), the  limit treatment response  is compared to the  control treatment
       response using a one-sided  two-sample  parametric or nonparametric  test, as
       appropriate  (see  OCSPP  850.4000).    The  minimum significant difference
       detectable by the test or a  similar estimate of the sensitivity of the test should be
       determined and reported.

       (iii) Multiple-dose definitive testing.

              (A) A multiple-dose definitive test is performed for a given test species if
              either the effect or inhibition level for one or more response measures (i.e.,
              survival, shoot height and biomass) in the limit treatment as compared to
              the  control response at test termination are declared to be 25% or greater
              effect (i.e., the null  hypothesis is not rejected) or the NOEC is less than the
              limit concentration.

              (B) Multiple-dose definitive testing may be waived for a given test species
              if at test termination the "limit" treatment response is both statistically less
              than a 25% decrease from the control response and there is no observable
              adverse  effect  from  the  control  response  for  all measures of  effect
              (survival, shoot height and biomass).

(4) Multiple-dose definitive test —

       (i) Dose-response curves,  slopes and ICis and EQs values —
              (A) Shoot height and shoot biomass. For dose-response-response tests
              the IC25 value (standard error and 95% confidence interval) is calculated
              for each of shoot height and shoot biomass (see OCSPP 850.4000 and
              references in  paragraphs (j)(2) and (j)(9) of this guideline for statistical
              guidance).  If a dose-response curve was fit to the data to determine the
              IC25, the model parameters (e.g., slope)  and their uncertainty estimates
              (e.g., standard error) should be recorded.  Where the dose-response range
              tested does not result in the determination of a definitive IC25 value for a
              given response measure, test and document that the IC25 value is above the
              highest treatment level  tested (see the  statistical guidance  in  OCSPP
              850.4000 and in paragraph (f)(3) of this guideline).  Such an event may
              arise if one of the other response measures is  much  more sensitive, and
              while the full response  curve for that response measure  is captured too
              many additional treatments would be needed to capture the full response
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                    relationship for the  other less  sensitive response measure(s).  Methods,
                    assumptions, and results of the statistical approaches  used should be
                    recorded.

                    (B)  Percent  survival.    The  EC25  value (standard  error and 95%
                    confidence  interval) is  calculated  for  percent survival (see  OCSPP
                    850.4000 for statistical guidance).  If a dose-response curve was fit to the
                    data to determine the EC25 the model parameters (e.g.,  slope) and their
                    uncertainty estimates (e.g., standard error) should be recorded. Where the
                    dose-response  range tested does  not result in the  determination of a
                    definitive £€25 value, test and document that the EC25 value  is above the
                    highest treatment level tested (see statistical guidance in OCSPP 850.4000
                    and in paragraph (f)(3) of this guideline).  Such an event may arise if one
                    of the other response measures is much more sensitive, and while the full
                    response curve for that response measure is captured too many additional
                    treatments would be needed to capture the full response  relationship or a
                    definitive EC25 for  survival.  Methods,  assumptions, and results of the
                    statistical approaches used should be recorded.

              (ii) NOEC.  The NOEC (and LOEC) for each response measure (survival, shoot
              height and shoot weight) is determined (see OCSPP 850.4000 and the reference in
              paragraph (j)(3) of this guideline). If a NOEC value can not be  determined for a
              given response measure, as appropriate, the dose at which there is a 5% inhibition
              (i.e.,  an ICos value for shoot height and shoot weight) or the dose at which there is
              a 5% reduction in survival of the exposed population (i.e., an ECos value) is
              estimated and used in place of the given NOEC.  The standard error and 95%
              confidence interval  should also be calculated  for the ICos and ECos  values.
              Methods, assumptions, and results of the  statistical approaches used should be
              recorded.

(g) Tabular summary of test conditions.  Table 2 lists the important conditions that should
prevail during the definitive test. Except for the number of test treatments,  Table 2 also  lists the
important conditions that should prevail during a limit test.  Meeting these conditions will greatly
increase the likelihood that the completed test will be acceptable or valid.
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       Table 2.—Summary of Test Conditions for Vegetative Vigor Test
Test type
Test duration
Substrate
Nutrients
Temperature
Relative humidity
Light intensity
Photoperiod
Watering
Test chamber (pot) size
Number of plants per test chamber
Number of replicates per test treatment
Number of plants per test treatment
Test treatment levels
Test substance application method
Measures of effect or measurement endpoints
Vegetative vigor test
Minimum of 21 days after test substance application
(extended to 28 days post-application if phytotoxic
symptoms initially show between days 14 and 21 post-
application).
Natural or synthetic soil with 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 top of the canopy
16 hours light: 8 hours dark (for non-crop species see
Appendix 1 of this guideline)
For greenhouse and growth chamber, the initial
watering event is by top watering below the canopy and
bottom watering is used throughout the remainder of
the study. In field plots top watering below the canopy
is used.
Varies with plant species selected. Six-inch diameter
plastic pots are typical. Flats are also encouraged.
Varies with species and test chamber (pot) size.
6 (a minimum of 4 if increase number of seedlings per
replicate to 1 0)
30 plants (a minimum of 40 seedlings if there is only 4
replicates)
Unless performing limit test, minimum of 5 treatment
levels plus appropriate controls
Applied to foliage
IC25 and NOEC (or IC05) for each of shoot height and
shoot weight
EC25 and NOEC (or EC05) for survival
(h) Test validity elements. This test would be considered to be unacceptable or invalid if one or
more of the conditions in Table 3 did not occur or one or more performance objectives in Table 3
were not met.  This list should not be misconstrued as limiting the reason(s) that a test could be
found unacceptable or invalid.   However, except for the  conditions listed in Table 3 and in
OCSPP 850.4000, it is unlikely that a study will be rejected when there are slight variations from
guideline  environmental  conditions  and study  design  unless  the  control  organisms are
significantly affected, the precision of the test is reduced, the power of a test to detect differences
is  reduced, and/or  significant biases  are introduced in defining the  magnitude of  effect on
measurement endpoints as compared  to guideline conditions.   Before departing significantly
from this guideline, the investigator should  contact the Agency to discuss the reason  for the
departure and the  effect the change(s)  will  have on test  acceptability.  In the  test report, all
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departures from the guideline should be identified,  reasons for these changes given, and any
resulting effects on test endpoints noted and discussed.

       Table 3.—Test Validity Elements for the Vegetative Vigor Test
1.  Mean control plant survival is at least 90% at test termination.

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

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

4. All test chambers used for a particular species should be identical and should contain the same
amount of soil from the same source.

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

6. For a given plant species, the lowest test concentration level was below both the shoot height and
biomass IC25 values for the plant species.

7. If pesticides are used for pest control during the test, additional test data was submitted to document
that the pesticide used was not toxic to the test species and that there were no synergistic or antagonistic
interactions with the test  substance.

8. The water carrier used at the time of test substance application did not  excessively exceed the amount
of water on the label (gallon per acre).
(i) Reporting—

       (1) Background information.  Background information to be  supplied  in  the report
       consists at a minimum of those background information items listed in paragraph (j)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.


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       (iv) If a vehicle (e.g., solvent, dust) is used to prepare  stock or test substance
       provide: the name and source of the vehicle, the nominal concentration(s) of the
       test substance in the vehicle in stock solutions or mixtures, and the vehicle dose(s)
       used in the treatments.

(4) Plant test species.

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

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

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

       (iv) Date of planting.

       (v) Pre-test planting and seedling cultivation conditions, grow out conditions and
       handling including pest and disease treatments, for the young plants before use in
       the test.

       (vi) Height and morphological condition (i.e.,  any  abnormalities, pigmentation,
       wilting, etc.) of seedlings selected for the test.

(5) Test system and conditions. Description of the test system and conditions used in
the definitive or limit test, and any preliminary  range-finding tests.

       (i) Location of testing, and if not a field plot study the type of growth chamber or
       greenhouse.

       (ii) For small field plot testing describe  the plot design: size of field plots, number
       of control and experiment plots, the number of plots per treatment and control, the
       plot lay-out, the number of plants in each plot.

       (iii) For greenhouse or growth chambers the description of the  containers,  pots
       and flats: type, material, dimensions,  and  the  soil  surface area per pot or test
       container.

       (iv) Number of seedlings per pot/flat.

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

       (vi)  Description  of the  support medium: source,  soil  type designation,  soil
       composition, pH, percent organic matter, type and amounts of soil amendments.

       (vii) Methods used  for  treatment randomization  and impartial  assignment of
       plants to test plots, pots, and/or flats.
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       (viii) Method of test  substance application: equipment type, equipment design,
       method for calibrating the application equipment, dose levels, volume of dosing
       solution applied.

       (ix) Date of test  substance application  and test duration (date plants  were
       harvested at test termination).

       (x) Culture practices during the test such as cultivation, pest control, and irrigation
       practices (type and watering schedule or regime).

       (xi) The photoperiod and light source.

       (xii) Methods and  frequency of environmental monitoring performed during the
       definitive or limit study for air temperature, humidity,  and light intensity and
       additionally for field plot tests rainfall and cloud cover.

       (xiii)  For the  definitive,  or  limit  test,  all analytical  procedures  should be
       described.  The accuracy of the method, method detection limit, and limit of
       quantification should be given.
(6) Results.
       (i) Environmental  monitoring data results (air temperature, humidity and light
       intensity) in tabular form (provide raw data for measurements not made on a
       continuous basis), and descriptive statistics (mean, standard deviation, minimum,
       maximum).

       (ii)  For preliminary range-finding tests, if conducted, the number of surviving
       plants, and  shoot  and  biomass,  if measured, at each  dose  level  and  in the
       control(s).  A  description and count of visual phytotoxic effects, if recorded,  at
       each dose level and in the control(s).

       (iii)  For a limit test, tabulate for the limit concentration and the  control by
       replicate, the number of seedlings exposed and their shoot height at test initiation,
       the  number of surviving and number of dead seedlings at each observation time,
       and the shoot height and biomass at test termination (provide the raw data).

       (iv) For the definitive test, tabulation by treatment and replicate the  number of
       seedlings exposed and their shoot height at test initiation, the number of surviving
       and number of dead seedlings at each observation time, and the shoot height for
       each individual plant and total plant biomass at test termination (provide the raw
       data).

       (v)  For the limit  and definitive  tests, tabulation by treatment of the  percent
       reduction in mean height, biomass, and survival as compared to control plants at
       test termination.
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              (vi) For the limit and definitive test, a description of visual (morphological) signs
              of phytotoxicity including:  time of onset,  duration,  severity (e.g. rank), and
              number affected at each dose level and control(s) (provide the raw data).   A
              description of the phytotoxicity rating system used should be included.

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

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

              (ix) For  the  limit test, provide a  description of the statistical methods used
              including software package, and the basis for the choice of method.

              (x)  For the  definitive  study and for those effect measures (shoot height and
              weight) with data sufficient to fit a concentration-response relationship, tabulation
              of the slope of the dose-response curve and its standard error  and 95% confidence
              limits and any goodness of fit results.

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

              (xii) For the definitive test, a tabulation of the NOEC and LOEC for each measure
              of effect (plant height, and plant biomass).   The ECos or  ICos, as applicable,
              should be reported for  effect measure data where  an  NOEC could not be
              determined.

              (xiii)  Description of statistical method(s)  used for point  estimates, including
              software package, for determining IC25  values, fitting  the dose-response model,
              and the basis for  the choice of method.  Provide results of any goodness-of-fit
              tests.

              (xiv)  Description  of  statistical   method(s)  used  for  NOEC  and  LOEC
              determination,  including software  package,  and the  basis for  the  choice  of
              method.  If an  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  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.
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(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 (OECD).  2006.  New Test
Guideline (Section 2- Effects On Biotic Systems), 227, Terrestrial Plant Test: Vegetative
Vigour  Test, 21pp.   Adopted on  19 July  2006  and published as  part  of the  17th
Addendum to the OECD Guidelines for the Testing of Chemicals, 972006131, ISBN 92-
64-01553.

(6) OECD.   2006.   Revised Test  Guideline  (Section 2), 208, Terrestrial Plant Test:
Seedling Emergence  and Seedling Growth Test, 21pp.  Adopted on 19  July 2006 and
published as  part of the  17th Addendum to the OECD Guidelines for  the Testing  of
Chemicals, 972006131, ISBN 92-64-01553-1.

(7) U.S. Environmental Protection Agency, 1982.  Pesticide Assessment Guidelines
Subdivision  J, Hazard Evaluation: Nontarget Plants.  Office of Pesticides and Toxics,
Washington, D.C. EPA 540/9-82-020, October 1982.

(8) U.S. Environmental Protection Agency, 1986.  Hazard Evaluation Division Standard
Evaluation Procedure, Nontarget Plants: Vegetative Vigor -Tiers 1 and 2.  Office  of
Pesticides Programs, Washington, D.C. EPA 540/9-86-133, June 1986.

(9) VanEwijk, P.H. and J.A. Hoekstra, 1993. Calculation of the EC50 and its confidence
interval  when a subtoxic stimulus is present.  Ecotoxicology and Environmental Safety
25:25-32.
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