&EPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention
(7101)
EPA712-C-010
January 2012
        Ecological Effects
        Test Guidelines

        OCSPP 850.4230:
        Early Seedling Growth
        Toxicity Test

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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.gov/ocspp and select "Test Methods &
Guidelines" on the left side navigation menu.  You may also access the guidelines in
http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-
0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.

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OCSPP 850.4230: Early seedling growth toxicity test.

(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 OPPT guideline under 40 CFR 797.2800 Early Seedling Growth
       Toxicity Test, and OECD guideline 227, Terrestrial Plant Test: Vegetative Vigour Test.

(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.  This guideline describes tests mainly using commercially important terrestrial
plants  (crop species) to develop data on the phytotoxicity of test substances.  Either a  foliar
exposure or a root exposure scenario is employed. If the anticipated fate of the test substance is
occurrence in soil or pore water, and the mechanism of concern is root uptake, the test substance
should be applied in nutrient solution to the root support media (or by coating onto sand or glass
beads). With a test substance whose anticipated mode of exposure to plants is surface deposition
by atmospheric transport, or irrigation water, the appropriate  testing method is foliar application
allowing subsequent movement into the rooting zone with watering. This guideline  should be
used in conjunction  with  the OCSPP 850.4000 guideline, which provides general information
and overall guidance for plant test guidelines. The Environmental Protection Agency will use
data from  these  tests  to assess  the hazard  a test  substance  may present  in  the  terrestrial
environment.

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

       Shoot length  is  defined as the above-ground vegetation from the support media surface
       (or water surface for hydroponic solutions) to the  apical tip or highest aerial part of the
       shoot.

       Root length (tap root system) is  defined as the below-ground vegetation from the support
       media surface (or water surface  for hydroponic solutions) to the apical tip of the primary
       root (tap root) and from the primary root to the apical tip of a first order lateral root.

       Root length  (fibrous root  system)  is defined as the  below-ground vegetation  from  the
       support media surface (or water surface for hydroponic  solutions) to the apical tip of a
       fibrous or first order lateral root.

(d) General considerations—

       (1) Summary of the test.  This  test evaluates the effect of a test substance applied to the
       roots or the  leaves of a number of terrestrial plant species.  In preparation for the test,
       seeds are planted in the potting containers (or in cotton or glass-wool plugs supported in

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hydroponic solution) and, after germination, seedlings are thinned by pinching the stem at
the support medium surface to the  10 most uniform seedlings per pot.  This marks the
start of the test and the time of first application of test substance.  Seedlings emerging
after this time are also pinched off at the surface. Potting mixtures of sand or glass beads
are sub-irrigated with nutrient solution.  The test substance is applied to the plants via
nutrient solution or sorbed to the support media (to produce a root exposure scenario) or
applied to the plants by either spraying or dusting the foliage or by exposing the plants to
gas in a fumigation chamber (to produce a foliar exposure scenario).  Plants are harvested
after 14 days and analyzed for growth.  Parameters  that may be  measured include
seedling survival, length and weight of whole plants, shoot length, shoot dry weight, root
length, and root dry weight, and observed phytotoxicity.  Results are reported as ECio and
ECso values  for seedling  survival  and ICio and ICso values for weight  and  length  of
shoots,  roots and  entire  plants  for a multiple-concentration test.   Also, for  industrial
chemicals, the  no  observed  effect  concentration (NOEC) and  lowest observed effect
concentration (LOEC) for the measures of effect (survival; lengths of roots, shoots, and
entire plant; weights of roots, shoots, and entire plant) may be determined. For a limit
test, results are  reported simply as percent effect  at the test treatment relative to the
control(s).   At a  minimum,  these  endpoints  should be calculated for survival, shoot
length,  and shoot  dry weight.   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 percent (x%) of the population exhibit the effect
(e.g., immobility)) and ICX (concentration resulting in an x% decrease or inhibition effect
on an attribute of the population (e.g., growth rate)).

(2) General test guidance. The general guidance in OCSPP 850.4000 and the references
in paragraphs (j)(2), G)(7) and 0)00) of this guideline applies to this guideline, except as
specifically noted herein. The protocol to examine seedling emergence and growth under
FIFRA requirements is the OCSPP 850.4100 guideline.

(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 (i.e., 0.01, 0.1, 1.0, 10, 100, 1000 milligrams per liter (mg/L)).  The details of
the range-finding test do not have to be the same as the definitive testing in that there are
not replicates, and the number of test organisms used, and  duration of exposure may be
less than in definitive testing. In addition, the types and frequency 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  survival; length, and weight  of the shoots,  roots and entire plant) its
concentration-response curve; the ECso and ECio values (with 95%  confidence intervals
and standard errors) for seedling  survival; and the ICso  and ICio values (with 95%
confidence intervals and standard errors) for length and weight of shoots, roots and entire
plant.  The slope of the concentration-response curve, its associated standard error, and


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       95% confidence interval should be determined for each measure of effect, if possible.
       However, at a minimum, the full concentration-response curve (curve between the ECio
       and ECgo (or ICio and ICgo)) is determined for the most sensitive measure of effect using
       at a minimum  five treatment levels, plus appropriate controls. For a satisfactory test, the
       lowest treatment concentration is below the ECio (seedling survival) and ICio (length and
       weight of shoots,  roots and entire plant) values for all measures  of effect.  The test
       substance should be added to the hydroponic or nutrient solution or coated on the support
       medium for the root exposure test, or sprayed, dusted, or gassed directly on the foliage in
       the  foliage  exposure  tests.   Analytical confirmation  of the test  concentrations is
       performed as described in OCSPP 850.4000.  A summary of test conditions is provided in
       Table 3 and validity elements for an acceptable definitive test are given in Table 4.

       (5) Limit test.  In some situations, it is only necessary to ascertain that the 14 day post-
       germination ECio,  ECso, ICio and ICso values are above  a certain limit concentration. In
       an early 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 for all measures of effect (seedling survival, length, and
       weight of the shoots, roots and entire plant), the "limit"  concentration has not caused an
       effect greater than 10% on  a plant species  (e.g.  ICio >limit concentration).  For most
       industrial  chemicals, 1,000 mg/L is considered appropriate as the "limit" concentration.
       Alternatively,  the  "limit" concentration, for water-soluble compounds,  should be the
       saturation concentration.  Except for the number  of  test concentrations, number of
       organisms used, an acceptable limit test follows  the same test procedures, is the same
       duration, and has the same number of controls as  the multi-concentration definitive tests
       (see Table 3).  Acceptable limit tests like definitive tests include analytical confirmation
       of the test concentration.

(e) Test standards—

       (1) Test substance. 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,
       and discusses methods for preparation on test substances.

       (2) Test  duration. The test lasts for at least  14 days from the time that  50%  of the
       control seeds have germinated.

       (3) Test organisms—

              (i) Species.

                     (A) The test is performed on species from a cross-section of the terrestrial
                     plant population that have been historically used for this type of testing,
                     and, therefore, have known types of responses (see reference in paragraph
                     (j)(l) of this guideline). At a minimum ten plant species  are tested, and
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                    include the six dicot species and the four monocot species listed in Table
                    1.
Table 1.—Species and families tested at a minimum for the early seedling growth toxicity
test

Dicots





Monocots



Family
Solanaceae
Cucurbitaceae
Asteraceae
Fabaceae1
Brassicaceae
Apiaceae
Poaceae
Poaceae
Poaceae
Liliaceae
Species
Lycopersicon esculentum or
Solarium lycopersicum
Cucumis sativus
Lactuca sativa
Glycine max
Brassica oleracea
Caucus carota
Avena sativa
Lolium perenne
Zea mays
Allium cepa
Common name
Tomato
Cucumber
Lettuce
Soybean
Cabbage
Carrot
Oat
Perennial ryegrass
Corn
Onion
 Inoculation with Rhizobium japonicum is unnecessary.

                     (B) Eight additional dicot species are listed in Table 2.  Some or all of
                     these may be tested in addition to the minimal data set of species (see
                     paragraph (e)(3)(i) of this guideline).  For example, if cabbage is initially
                     found to be sensitive to the test substance, it may be important to then test
                     turnips and canola plants, which are additional species  in the same plant
                     family  (Brassicaceae).   Other economically or ecologically  important
                     species (e.g.,  see Table 2  species  list in the OCSPP 850.4100 guideline
                     (see reference in paragraph (j)(8)  of this guideline) at  a specific site or
                     region  of impact  (from  production,  use  or  disposal)  may  also  be
                     appropriate and can be selected for  testing on a case-by-case  basis, in
                     addition to those listed in Tables 1  and 2 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.

       Table 2.—Dicot species for additional consideration in the early seedling growth
toxicity test
Family
Chenopodiaceae
Asteraceae
Fabaceae
Brassicaceae
Brassicaceae
Fabaceae
Malvaceae
Polygonaceae
Species
Beta vulgaris
Helianthus annuus
Pisum sativum
Brassica rapa
Brassica napus
Phaseolus vulgaris
Gossypium spp.
Fagopyrum esculentum
Common name
Sugar beet or table beet
Sunflower
Pea
Field mustard, Canola
Rape, Turnip
Garden bean
Cotton
Buckwheat
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(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) Growth stage. Newly germinated plants are used. Seedlings used in testing
are produced by planting seeds of the desired  species in the selected support
medium and allowing them to germinate, at which time they are thinned to 10
plants of similar size and condition.

(iv) Condition.  For a satisfactory test, healthy seeds should be used with reported
germination rate  for  a lot  number  which met or  exceed minimum  seed
germination standards (see paragraph (e)(5)(i) of this guideline). 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.
Seeds should be selected from a single class size within each  species.  The seeds
of most test species are sorted by the  supplier according to size.  However, if
necessary,  the seeds  should  be further sorted  prior  to use  to provide a more
uniform size class that reduces the potential for bias from testing different sizes of
seed.   In  addition broken or damaged seeds should be separated and removed
from those used in the test.

(v) Care and handling.

       (A)  Seeds  should be  stored in  a  dessicator and refrigerated until used.
       Pesticide treated seeds should be avoided.

       (B) If plants are to be grown hydroponically,  seeds should be planted in
       plugs of cotton or glass wool  supported in the tops of the containers.
       When sand or glass beads are used, the recommended planting procedure
       is to fill  the potting containers to within 2.5 centimeters (cm) of the top
       and to sow seeds directly on the support medium.  A sufficient number of
       seeds (e.g., 30) should be planted  to  provide an  excess number of
       seedlings.  After 50% of the seeds have  germinated, the seedlings should
       be thinned to the 10 most uniform per pot.  The condition and length of
       each of  the 10 seedlings  used in  the  test  is recorded.   Any  plants
       subsequently emerging are also pinched off.

       (C) Alternative planting methods  are used when the test  substance is
       highly  volatile.    An impervious  barrier  of  polyethylene  film,  a
       modification of the double pot method, a glass plate, or other appropriate
       apparatus should be used to prevent volatilization from the  root zone.
       Seeds should be germinated in the dark at 25 degrees Celsius (°C) and
       seedlings with radicle  lengths in the median range transplanted into  the
       potting containers.  The seedlings  should  be  positioned such  that their
       roots are exposed to the support media while the shoots pass through holes
       in  the barrier.  A ring  of nontoxic, inert, pliable putty should be used to
       seal  the  holes around the stems.   Control pots should  be handled
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              identically to the test pots except there is no exposure to the test substance.
              This transplanting procedure, without the volatilization  barrier, is also
              recommended when the test substance is adsorbed to the support medium.

(4) Administration of test substance—

       (i) Methods.  The choice of method for test substance  application to newly
       germinated seedlings is dependent upon the expected route of exposure of plants
       in the environment and the properties of the test substance.

              (A) Root exposure.  Root exposure  is accomplished by  adding the test
              substance  to the nutrient medium used  to  subirrigate  the plants,  as
              described in paragraphs (e)(4)(i)(A)(7y) and (e)(4)(i)(A)f2y) of this guideline
              for soluble and insoluble test substances, respectively, or by sorbing the
              test substance to the  sand or glass beads used as support  medium,  as
              described in paragraph (e)(4)(i)(A)(%) of this guideline.  For hydroponic
              solutions the entire test solution should be replaced weekly or  earlier if the
              concentration of test substance in the test or nutrient solution varies by
              more than 20% of that specified. The volume of solution added should be
              recorded.  An  automated system design is recommended.  Sand or glass
              filled potting containers should be periodically (dependent on the rate of
              loss of the nutrient solution) filled with nutrient solution and drained to
              provide aeration.

                     (1) Test substances that are soluble in water should be  dissolved in
                     the nutrient solution just prior to the beginning of the test. Reagent
                     water should  be used  in  making  stock  solutions  of the test
                     substance.  Sufficient quantities of each concentration should be
                     made up as needed to minimize storage time and disposal volume.
                     The OCSPP 850.4000 guideline contains additional information on
                     preparation of stock solutions.

                     (2) Test substances that are insoluble in water, but which can be
                     suspended in an aqueous solution by a vehicle, should be added to
                     the  nutrient solution.  The vehicle should  be soluble in water,
                     relatively nontoxic to plants, and should be used in the minimum
                     amount needed to dissolve or suspend the test substance.  There are
                     no preferred vehicles; however, acetone, gum arable, polyethylene
                     glycol,  ethanol, and others have been used extensively in testing
                     herbicides,  plant  growth  regulators,  fungicides,  and  other
                     chemicals that affect plants.  Vehicle controls should  be included
                     in the experimental design of the test  and tested  simultaneously.
                     OCSPP  850.4000 contains additional information on preparation of
                     stock solutions using  a vehicle.

                     (3) Water-insoluble test substances for which no nontoxic, water-
                     soluble vehicle is available should be dissolved  in an appropriate
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       volatile solvent.  The solution should be mixed with the sand or
       glass beads which are then placed in a rotary vacuum apparatus
       and evaporated, leaving a uniform coating of test substance on the
       sand or beads.  A weighed portion  of sand  or beads  should be
       extracted with the same organic  solvent and the concentration of
       test substance determined before the  potting containers are filled.
       Solvent controls are included in the experimental design and tested
       simultaneously.

(B) Foliar exposure.  Foliar exposure is accomplished by spraying the
plants, as  described  in  paragraph (e)(4)(i)(B)(7)  of this guideline,  by
dusting the plants, as  described  in paragraph (e)(4)(i)(B)0 of this
guideline  or by exposing the plants to gas in a fumigation  chamber, as
described in paragraph (e)(4)(i)(B)(3y) of this guideline.

       (1) Water-soluble test substances should be dissolved  in reagent
       water just prior to use. Test substances that can be suspended in an
       aqueous solution by a vehicle should be prepared as  described in
       paragraph  (e)(4)(i)(A)f2y) of this  guideline except using reagent
       water not  nutrient solution.   Sufficient quantities of each test
       concentration  should be made up as  needed.  These solutions
       should be applied daily (during a normal 5-day work week) to the
       foliage. Plants should be placed in an exhaust hood and the test
       substance applied to the foliage.  A plastic sleeve may be  fitted
       over the top of  the  pot, and the foliage  sprayed with specific
       quantities of test solution at known  concentrations.  The plastic
       sleeve, confining the test substance  to plant and pot, facilitates
       expression of test substance dosage to  quantity per pot area (i.e.,
       micrograms per square  meter).  Shoots of control plants are also
       sprayed, using reagent water.  In addition, a vehicle control group
       is  included if a vehicle was  used to prepare  the test  substance.
       Alternatively, a miniature compressed-air sprayer may be mounted
       on  a pendulum  and equipped  to  automatically spray  a  plant
       positioned directly beneath the center of its arc of swing. Track or
       belt  sprayers may also  be  used (see the OCSPP 850.4000 and
       OCSPP 850.4100 guidelines for additional information).   When
       radioisotope-labeled test substances are applied, health  and safety
       considerations  prohibit spray   application.    Instead,  specific
       quantities of labeled chemical should be applied directly to plants
       in  single drops.

       (2) Water-insoluble  test substances, existing  as  solids,  may be
       prepared for testing by grinding or other  reduction methods to
       particles smaller  than 200 microns (|im) in diameter.  Each day
       (during a normal 5-day work week) plants should be placed in an
       exhaust hood, a plastic sleeve fitted  over the top of the pot, and
       specific quantity of the test substance sprinkled uniformly over the


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              potted seedlings.  Prior to test substance application, plants should
              be misted  with water  to  promote foliar retention  of the  test
              substance.  Control plants  are  also misted with reagent water at
              each treatment date and dusted with an inert material of the same
              particle  size as the test  substance.  Applications are expressed as
              quantity per unit pot area (i.e., micrograms per square meter).

              (3) Test  substances existing  in gaseous  form at normal ambient
              temperatures  and  pressures can be generated as needed or stored
              under pressure.  The bottled  gas  may  be 100% test substance or
              may  be  mixed  with an  inert carrier, such as nitrogen, to known
              concentrations.   Controlled or measured concentrations of  test
              substance should be metered into the exposure chamber, uniformly
              mixed about the  plants, and exhausted  through  the  outlet port
              where the flow rate and  concentration are again measured. Use of
              this system design provides an  alternate method of analysis if the
              quantity  of test  substance sorbed  by  plants  is  less than  the
              sensitivity of the chemical analysis method.

(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.,  ECso,
       ECio), test substance concentrations should immediately bracket the point
       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 pounds per acre
       (lb/acre)). For  an acceptable study for a given  plant species, the lowest
       test treatment level  should  be lower  than  the  ECio value for seedling
       survival and the ICio values for weight  and length of shoots, roots  and
       entire plant  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 10% effect or inhibition
       concentrations (ECio or ICio).  It is recommend 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 the most
       sensitive ECio or ICio value.

       (B) 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).
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(5)  Controls.   Every test includes controls consisting of the same support medium,
conditions, procedures, and test population, except that no test substance is added.  In
addition, vehicle (solvent) controls are also included if a solvent is used.

       (i)  Seed germination standards.

              (A) For a satisfactory test, seed  germination percentage in each of the
              controls should be at least 65% or the percentages in minimum acceptable
              United  States  Department of Agriculture (USDA) seed  germination
              standards listed in paragraph  (e)(5)(i)(B) of this guideline.  For native
              species that do not have  germination standards, sufficient numbers of
              seeds should be used to insure the identified number of seedlings per pot
              for test initiation.

              (B) The minimum acceptable USDA seed germination (control) standards
              in this paragraph for vegetable  crops (as described in the Federal Seed Act
              Regulation, 7  CFR parts 201-202) and  other  available standards for
              agronomic crops will be used: Field corn (85%), pop corn (75%),  sweet
              corn (75%), carrot (55%), onion (70%), tomato (75%), field-garden bean
              (70%), pea (80%), pepper (55%), beet (65%), buckwheat (60%), cabbage
              (75%),  lettuce  (55%), mustard-all types (75%), soybean  (75%),  sugar
              beet (55%), small grains—wheat, oats, barley, rice (80%), ryegrass (75%),
              vetch (75%), alfalfa-clover (70%), rape (75%).  Refer  to regulation for
              additional vegetable crops.

       (ii) Control survival.  A test not acceptable if at test termination the mean control
       seedling survival is less than 90%.

(6) Number of test organisms and replicates.

       (i)  For each species, the  minimum number of test organisms  is 40 plants per
       concentration level (a minimum of four replicates each with 10 plants or five
       replicates each with 8  plants).  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 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.
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       (ii) Randomization.

              (A) For a satisfactory test, a system of impartial or random assignment of
              the seeds to the pots and the pots to  test and control groups is used to
              prevent bias.  For this  test a randomized complete block design with
              blocks delineated within the growth chambers or over greenhouse benches
              and  randomization  of  treatment  occurring  within  the  blocks  is
              recommended for placement of replicates.  If, because of the use of very
              large pots, there is inadequate space within chambers for blocking, total
              randomization of replicates within chambers is acceptable.  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 replicate 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) Environmental  chamber,  growth room or greenhouse.   Environmental
       chambers  and some greenhouses  or growth rooms  should provide  adequate
       environmental controls   to  meet  the carbon  dioxide,  humidity, luminance,
       photoperiod, and temperature specifications.  Chambers should be designed to
       prevent escape of internal air into  the external  environment other than through
       appropriate filtering material or media to prevent contamination of the external
       environment with radioactivity and/or test substances.  Growth  chambers  and
       growth  rooms  are preferred  over  greenhouses, because  in  the  first two,
       environmental conditions are more easily controlled.

       (ii)  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  pots  that   have  not been  previously  used  are  acceptable  test
       containers, provided they are free  of toxic materials.   Do  not use clay or peat
       containers. For  a satisfactory  test, pots sufficiently large to grow (e.g., large
       enough to effectively not result in  competition among the plants  for space,
       lighting, or nutrition) at least 10 plants up to 14 days should be used for each
       species. It is equally acceptable to use small, individual containers if plants are
       grown in hydroponic solution. Potting containers used in each experiment should
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       be of equal size and volume and possess the same configuration.  When sand,
       glass beads, or soil are used, the potting containers should be filled to within 2.5
       cm of their tops.

       (iii) Cleaning and disposal.

              (A) Potting and receiving containers, nutrient storage containers, and root
              support medium should be cleaned before use.  All equipment should be
              washed to remove any residues remaining from manufacturing or prior
              use. Bichromate solution should not be used for cleaning beads or pots.

              (B) Rooting media other than glass beads should be discarded at the end of
              the experiment. Disposal should conform  to existing regulations.  Glass
              beads can be re-used provided they are not contaminated  with  previous
              test substances.

       (iv) Support medium. An artificial support medium, consisting of quartz sand or
       glass beads, is used.  Alternatively, a hydroponic system may be used, in which
       the seeds are planted in plugs of cotton or glass wool supported at the top of the
       solution.   Perlite,  vermiculite,  natural (native) soils, or any formulated  soils
       should not be used as a root support medium in this test.

       (v) Nutrient media. A nutrient solution of defined chemical composition, such
       as half-strength modified Hoagland  nutrient solution (see the reference in
       paragraph (j)(5) of this guideline), should be utilized as nutrient medium for this
       test.

(8) Environmental  conditions.   Controlled environmental  conditions are  used to
maintain uniform growth  and ensure reliable  data.   Standardization of environmental
conditions is essential because it has been demonstrated that differences in environmental
conditions influence the response of plants exposed to chemicals.  For growth chamber
studies, the general conditions  listed in  paragraphs  (e)(8)(i) through (e)(8)(v) of this
guideline should be maintained.   If greenhouses  or large  growth rooms  are used,
comparability of the environment between  small groups of plants is more difficult to
achieve and therefore not as critical.  Environmental variability specifications may be
relaxed on a case-by-case basis.

       (i) Temperature. Air temperature should be uniform throughout the greenhouse
       or growth chamber or greenhouse.  In growth chambers air temperature during the
       day should be 25 ± 3 °C while temperature during the night should be 20 ± 3 °C.
       Temperature  in greenhouses is more  difficult to  control.    Under these
       circumstances, temperature during the day should be 25 ± 6 °C while temperature
       during the night should be 20 ± 6 °C.  For other very warm or cool crop species
       the test conditions should approximate those  optimal conditions for the species
       and varieties tested.

       (ii) Humidity.  Humidity  should be uniform throughout the greenhouse or growth
       chamber  or greenhouse.   In growth chambers  relative humidity should approach

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       70 ± 5% during light periods and  90 ± 5% during  dark periods.  Humidity in
       greenhouses is  more difficult to control.  Under these circumstances, relative
       humidity should approach 70 ± 15% during light periods.

       (iii) Lighting and photoperiod.  Luminance of 350 ± 50 micromoles per square
       meter per second (|imol/m2/s), measured at the top of the canopy, is desirable, on
       a photoperiod of 16 hours light and  8  hours  darkness  assuming an average
       wavelength of 400 to 700 nanometers (nm).  For greenhouses, 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 and nutrients. Nutrient media (containing test substance, if this is
       the  selected application route)  is used to subirrigate the plants  as needed, i.e.,
       frequent enough to maintain good health  (should not see nutrition deficiency in
       controls) and moisture.   Pots  should  be irrigated  regularly (for example,  30
       minutes  per hour (min/h)) with nutrient solution, preferably using a  system in
       which water flows from the bottom  of the pot upward (level should reach at least
       half way up the pot). An automatic system  design is recommended such as that
       described in OCSPP 850.4600.

       (v) Carbon dioxide.  Carbon dioxide concentration at 350 ± 50 parts per million
       (ppm) (678 ±97 milligrams per cubic meter (mg/m3)) of air.
(9) Observations—
       (i) Measurement of  test  substance.   For  a  satisfactory  test,  analytical
       confirmation of the concentration of test substance in the vehicle used to expose
       the plants (nutrient medium, glass beads or sand,  spray, dust  or gas) should be
       performed. The analytical methods used to measure the amount of test substance
       in a sample are validated before beginning the test,  as described in the OCSPP
       850.4000 guideline.

             (A) Nutrient solution.  Where test substance was introduced by addition
             to  the nutrient  medium, chemical  analysis  should be  conducted  at a
             minimum for each treatment level and controls at test initiation (0-hour) in
             the bulk  solutions and test termination  in at least three replicates per
             treatment and controls at test termination when nutrient solutions are not
             renewed.  Where nutrient solution is renewed during the study, chemical
             analysis  should be conducted at a minimum for each treatment level and
             controls  at test initiation (0-hour) in the bulk solutions and at the end of
             this first cycle  in the  used  nutrient solution, at  the beginning (fresh
             solution) and end (used solution) of the longest renewal cycle and the last
             renewal cycle.  It is also recommended that measurements be made at the
             beginning of each renewal cycle.
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(B) Quartz sand or glass bead support medium. Where test substance
was introduced  by addition  to the  quartz  sand  or  glass bead support
medium, chemical analysis should be conducted at a minimum in the bulk
support medium for each treatment level and control  at test initiation and
from  at  least three  pots  for each treatment  level  and control  at  test
termination.

(C) Foliar spray. Where test substance is applied by foliar spray and a
single batch of test solution for each treatment level and control is used
throughout the study, chemical analysis in the spray solutions should be
conducted  at  a minimum at test  initiation and  termination  for  each
treatment level  and control batch.  If one or more spray solution batches
are prepared during the study, chemical analysis of spray solutions should
be conducted at a minimum for each treatment level and controls at test
initiation (0-hour) and at the end of the use of this nutrient solution batch,
at the beginning  (fresh solution) and end (used solution) of the longest
time between batches and the last batch.  It is also recommended  that
measurements be made at the beginning of each batch.

(D) Foliar dusting. Where test substance is applied as a dust and a single
batch of test substance  for  each treatment level and control is  used
throughout the study, chemical analysis of the dust (to determine  test
substance exposure)  should be conducted at a minimum at test initiation
(0- hour) and at test termination for each treatment level  and control batch.
If one or more batches of dust are prepared and used  during the study,
chemical analysis of each dust batch should be conducted at a minimum
for each treatment level and control at test initiation  (0- hour) and at the
end of the use of a particular dust batch, at the beginning (using fresh dust)
and end (used dust) of the longest time between batches and the  final
batch.   It is also recommended  that measurements  be made  at the
beginning of each batch. When dusting the plants, care should be taken to
uniformly dust all plant  surfaces,  to reduce exposure  variability of the
different areas of the plant.

(E) Gas application. Where test substance is applied as a gas in a growth
chamber, and a single source of the gas is used for each treatment level
and control throughout the study (recommended), chemical analysis of the
gas  should be  conducted at  a  minimum at test  initiation  and  test
termination for each treatment level and control.  Use of the gas only in a
growth chamber will help minimize variability in exposure concentrations
over the test period. For the gas exposure system, at the time of fumigation
the air temperature,  dew  point temperature or water vapor pressure of
incoming and outgoing air streams, air speed within chamber,  carbon
dioxide concentration at inflow and outflow  ports, and  gas flow rate into
and out of exposure system are measured and recorded.
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(ii) Environmental conditions—

       (A)  Carbon  dioxide.   In  environmental chambers,  carbon  dioxide
       measurements should be  made at the top of the plant canopy, preferably
       on a continuous basis.

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

       (C) Light intensity.   Light  intensity  should be  determined daily 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.   Additional information  on  the  use of
       lighting in plant toxicity tests can be found in the reference in paragraph
       (j)(2) of this  guideline and the references  given in the OCSPP 850.4000
       guideline.

       (D) Watering. Records  should be kept noting the days upon which the
       plants are watered and observations of possible moisture stress should be
       made and recorded daily.

       (E) 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.

       (F) Nutrient  media pH.  The pH of nutrient solutions are measured prior
       to  use in bulk solutions  and in used solutions at test termination in each
       replicate.  If more than one batch of nutrient solution is used throughout
       the study  the pH of each nutrient solution  treatment concentration and
       control(s) are measured in the nutrient solution batch prior to use, and at
       replacement in the used solution.

(iii) Phytotoxic effects—

       (A) Survival and morphology.   Observations of plants should be made
       approximately every 3 to 4 days (e.g.,  day 3  or 4, 7, 10, and 14),  or more
       often if necessary, after treatment for mortality and visual symptoms of
       phytotoxicity. All abnormalities (visible effects of the test substance on
       plant growth  and morphology including stunting of growth, discoloration,
       chlorosis and/or necrosis of the  leaves,  or morphological  abnormalities)
       should be recorded.   Observations on root  symptomology may also be
       useful. Seedling and root condition should be determined using a standard
       visual  phytotoxicty  rating  scale.    Observations  should  include  the

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                    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.

                    (B) Length of roots, shoots and entire plant.  Length of roots, shoots,
                    and entire plants (root and shoot) are measured at test termination for each
                    individual surviving plant, to the nearest millimeter (mm).  For fibrous
                    root systems with a high root density where  it is not practical to measure
                    all fibrous or first order lateral roots,  take representative samples of the
                    root system and measure and record the length of each root in the samples
                    and calculate the mean and median lengths.  Where fibrous root density is
                    low and where it is practical  all fibrous or first order lateral roots are
                    measured and the mean and median lengths calculated.  For  a tap root
                    system measure and record the length  of the tap root and each first order
                    lateral root and calculate the mean and median lengths of the lateral roots.

                    (C) Weight of roots,  shoots  and entire  plant    After lengths are
                    determined at test termination, the mass  of roots, shoots, and entire plants
                    (root and shoot) are measured.  Dry weight (to constant weight at 70 °C) is
                    preferred because it  is  less  variable than  wet  weight.   Biomass
                    measurements should be made for each replicate not each individual plant.
(f) Treatment of results—

       (1) Summary statistics—
              (i) Environmental conditions.  Air carbon dioxide, temperature, humidity, and
              light intensity data should  be summarized in tabular form, showing the mean,
              standard deviation, and range during the test.  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) Seedling survival. Means and standard deviations are calculated and plotted
              for each treatment and control. For each treatment level calculate mean, relative
              to control mean survival.

              (iv) Length of roots, shoots and entire plant.  Means and standard deviations
              and medians are calculated and plotted for each treatment and control.
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       (v) Weight of roots, shoots and entire plant.  Means and standard deviations are
       calculated and plotted for each treatment and control.

       (vi) Morphological conditions.  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 length of roots, shoots, and entire plant and weight of roots,
shoots and  entire plant calculate the % inhibition (%I) at each treatment level using
Equation 1.
                                                                        Equation1
       where:
       C = the control mean response value (length of roots, shoots, or entire plant;
       weight of roots, shoots, or entire plant); and

       X = the treatment mean response value (length of roots, shoots, or entire plant;
       weight of roots,  shoots, or entire plant, respectively).  Stimulation is reported as
       negative %I.

(3) Limit test.  For a limit test, calculate  and report the percent effect at the limit
treatment, relative to the control, for each  response measure.  To  ascertain that  the
survival ECio and the length and weight ICio 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 of these response measures.
For a comparison of sample means, the difference defining the ICio or ECio, compared to
the controls, is operationally defined as a 10% 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 10  reduction, compared to the control (i.e.,
HQ: ^control - Hiimit > So).  The alternative hypothesis (HA) is that this difference is less than
a 10% reduction, compared to the control (HA:  ^control - Hiimit < 5o).  An example of a
parametric two-sample comparison test of this is the Student's t-test. If the effect level or
inhibition level for all response measures (i.e., survival  and length and weight of roots,
shoots and entire plant)  in the limit treatment as  compared to the control response at test
termination are declared to be less than  10% (i.e., the null hypothesis is rejected),  the
multiple-dose definitive test may be waived.  A multiple-dose definitive test is performed
if the effect or inhibition level for one or more response measures  (i.e., survival and
length and weight of roots, shoots, and entire  plant) in the limit treatment as compared to
the control response at test termination are declared to be not distinguishable from a 10%
or greater effect (i.e., the null hypothesis is not rejected).

                           80=(xcontml)x(p/lOO)                        Equation 2


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              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., survival and length and weight of
              roots, shoots, and entire plant); and

              p =  percent reduction from the control sample mean, which is 10 in the case of the
                   and ICio.
       (4) Multiple-dose definitive test —

              (i) Seedling  survival.   For dose-response  tests,  statistical  procedures (see
              references in paragraphs (j)(3)  and (j)(9) of this  guideline) are employed to
              calculate the ECio and ECso values based upon number of surviving plants at test
              termination.   Standard error and 95%  confidence  interval  (CI)  values  for the
              calculated EC values are to be included.  The slope of the concentration-response
              curve, its  standard  error,  and 95%  CI  should also be recorded.  Appropriate
              statistical  analyses  should  provide  a  goodness-of-fit  determination  for the
              concentration-response curves.   Hypothesis testing procedures can  be used to
              determine NOEC (and LOEC) values.

              (ii) Length and weight of roots, shoots  and entire  plant.  For dose-response
              tests, statistical procedures are employed to calculate the ICio and  ICso values for
              each of shoot, root and entire plant length and weight at test termination (see
              references in paragraph (j)(4) and G)(H) of this guideline).   Standard error and
              95% confidence interval (CI) values for the calculated 1C values are to  be
              included.  The slope of the concentration-response curve, its standard error, and
              95% CI  should also be recorded.  Appropriate statistical analyses should provide a
              goodness-of-fit determination for the concentration-response curves.  Hypothesis
              testing procedures can be used  to determine NOEC (and  LOEC)  values (see
              reference in paragraph (j)(6) of this guideline).

(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 test conditions will
greatly increase the  likelihood that the  completed test will be acceptable  or valid. These test
conditions  apply mainly to  growth chambers  and  growth  rooms;  they are preferred  to
greenhouses where for the latter it may be more difficult to meet or control these conditions.
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       Table 3.—Summary of Test Conditions for Early Seedling Growth Toxicity Test1
Test duration
Substrate
Nutrients
Temperature
Relative humidity
Carbon dioxide
Light quality
Light intensity
Photoperiod
Watering
Test chamber (pot) size
Number of organisms per test
chamber
Number of replicate chambers
per test treatment
Number of organisms per test
treatment
Test treatment levels
Test substance application
method
Measures of effect
(measurement endpoints)
At least 14 days after 50% of the control plants have germinated
Quartz sand or glass beads. Hydroponic system may also be used.
Watered with nutrient solution (e.g., half-strength modified Hoagland's
medium)
25/20 °C (day/night) ± 3 °C
70/90% (day/night) ± 5%
350 ± 50 ppm at the top of the canopy
Fluorescent or representative of natural sunlight
350 ± 50 umol/m^/sec at the top of the canopy
16 hours light: 8 hours dark
Bottom watering as needed, using nutrient solution
Varies with plant species selected. Six-inch diameter plastic pots are
typical.
8-10 seedlings of one species per pot
4-5
40 (minimum)
Unless performing limit test, minimum of 5 treatment levels plus
appropriate controls
Root exposure (via watering with nutrient solution or sorbed onto the
substrate) or foliar exposure (via spray, dust or gas)
EC-io, EC50, NOEC and LOEC based upon survival
IC-io, IC50, NOEC and LOEC for each of roots, shoots and entire plant
length
IC-io, IC50, NOEC and LOEC for each of roots, shoots and entire plant
weight
Visual phytotoxicity reported
 These environmental conditions are mainly applicable to growth chamber or growth room studies.

(h) Test validity elements. This test would likely 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 4 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 the OCSPP 850.4000 guideline, it is unlikely 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.  It is preferred that this test be conducted
in growth chambers or growth rooms, instead  of in greenhouses (see paragraphs (e)(7)(i) and (g)
of this guideline).
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       Table 4.—Test validity elements for the early seedling growth toxicity test
1. Seed germination percentage in each of the controls is at least 65% for the recommended test species;
or the minimum acceptable USDA seed germination standards listed in paragraph (e)(5)(i)(B) of this
guideline. For native species that do not have germination standards, sufficient numbers of seeds, are
used to provide statistically valid measures of effect.

2. Mean control seedling survival is at least 90% for the duration of the study.

3. Control seedling condition should not indicate any visible phytotoxic or developmental symptoms
during the test.

4. For a given test species, all seeds used in the test are from the same source and lot number.

5. All test chambers used for a particular species are identical and contain the same amount of substrate
from the same source.

6. An untreated (or negative) control [and solvent (or vehicle) control, when a solvent was used] is
included in the test.
(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
       the  OCSPP 850.4000 guideline.

       (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, 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.
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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, pre-test planting, production,
       and handling of test seedlings before use in test.

       (vi) Length and condition 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) A description of the test system, including type of growth  chamber, growth
       room, or greenhouse.

       (ii) The description of pots or test containers: type, material, dimensions.

       (iii) Number of seedling plants per pot or test container.

       (iv) Number of pots per replicate, and number of replicates per treatment level.

       (v) Description of the  support medium or hydroponic  system: source, type,
       material.

       (vi) Volume of support medium per pot or test container.

       (vii) Methods used for treatment randomization.

       (viii) Methods  for preparing the test treatments: exposure route (root  or foliar
       exposure),  application  methods (including  equipment  type  and method  for
       calibrating  the  application equipment), information about any solvent  used to
       dissolve and apply the test substance, and the concentrations (concentration of the
       test  substance in nutrient  solution  or in the foliar application vehicle, or  the
       quantity  of test substance  per unit weight of root support material  when it is
       coated on the support material, the concentration of test substance applied to the
       soil  surface; the concentration  of test  substance in  the  dust  or spray; or  the
       concentration of gaseous test substances at inflow and  outflow ports)  and  the
       amounts used.

       (ix) Number of applications and dates applied.
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         (x) Culture practices during the test such as 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.

         (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  (carbon  dioxide concentrations, 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 initial
         seedlings,  number of surviving seedlings,  and length and dry weight of roots,
         shoots, and entire plants, 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 seedlings exposed  at  test initiation, the  number  of
         surviving and number of dead seedlings at each observation time, and the length
         and dry weight of roots, shoots and entire plants  at test termination (provide the
         raw data).

         (iv) For the definitive test, tabulation by treatment and replicate of the number of
         seedlings exposed at test initiation, the number of surviving and number of  dead
         seedlings at each observation time, and the length and dry weight of roots,  shoots
         and entire  plants (provide the raw data).

         (v) For the limit and definitive  test, means and standard deviations for survival,
         length and weight of roots, shoots, and entire plants in each treatment and control.

         (vi) For the  limit and definitive  test,  tabulation by treatment of  the percent
         reduction in mean length and dry weight of roots,  shoots and entire plants relative
         to control mean length and dry weight of roots, shoots and entire plants.

         (vii) 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.
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             (viii) Graphs of the dose-response data for survival, and length and dry weight of
             roots, shoots, and entire plant at test termination.

             (ix) For the definitive study and  for those effect measures  (seedling survival,
             length and  dry weight of roots, shoots and  entire  plant) with sufficient data
             coverage 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.

             (x) For the definitive test, provide table of ECio and ECso values for survival and
             ICio and ICso values for length and dry weight of roots, shoots and entire plants.
             Also provide  95%  confidence intervals  and  standard  errors  for these point
             estimates.

(j) References.  The references in this paragraph 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) American Society for Testing and Materials.  ASTM E 1733-95.  Standard guide for
       the use of lighting in laboratory testing.   In Annual Book of ASTM Standards, Vol.
       11.05, ASTM, West Conshohocken, PA. Current edition approved September 10, 1995.

       (3) Boutin, C. et al, 1993. Proposed Guideline For Registration Of Chemical Pesticides:
       Nontarget plant testing and evaluation. Tech. Rpt. Series No.  145,  Canadian Wildlife
       Service, Environment Canada, pg. 1-91.

       (4) Bruce, R.D.  and DJ. Versteeg, 1992. A statistical procedure for modeling continuous
       toxicity data. Environmental Toxicology and Chemistry 11:1485-1494.

       (5) Downs, RJ. and Helmers, H.,  1975.   Environment and Environmental Control  of
       Plant Growth. Academic Press, NY.

       (6) Gulley, D.D. etal,  1989. Toxstat Release 3.0. University of Wyoming, Laramie, WY.

       (7) Nyholm, N., P.S. Sorenson, K.O. Kusk, and E.R. Christensen, 1992.   Statistical
       treatment of data from microbial toxicity tests. Environmental Toxicology and Chemistry
       11:157-167.

       (8)  Organization  for  Economic Cooperation and Development.    2006.   New Test
       Guideline (Section 2- Effects On Biotic Systems), 227, Terrestrial Plant Test: Vegetative
       Vigour Test, 21 pp.; 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.
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(9)  Stephan,  C.E., 1977. Methods for  calculating an  LC50. In F.L. Mayer and J.L.
Hamelink, eds., Aquatic Toxicology and Hazard Evaluation, STP 634, American Society
for Testing and Materials, Philadelphia, PA, pp. 65-84.

(10) Truelove,  B., (ed).,  1977. Research Methods in  Weed Science. Southern Weed
Science Society, Auburn Printing Inc., Auburn, AL 36830.

(11) 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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